Combinatorial Microarray Assay for Clade Variant Detection

Information

  • Patent Application
  • 20220251635
  • Publication Number
    20220251635
  • Date Filed
    May 27, 2021
    3 years ago
  • Date Published
    August 11, 2022
    2 years ago
Abstract
Provided herein is a method for detecting the presence of clade variants in the COVID-19 virus in a human sample and/or an environmental sample. Samples are processed to obtain total RNA. The RNA is used as a template in a combined reverse transcription and amplification reaction to obtain fluorescent COVID-19 virus amplicons. These amplicons are hybridized on a microarray with nucleic acid probes having sequences that discriminate among the various clade variants. The microarray is imaged to detect the clade variant. Also provided is a method of distinguishing each clade variant from others by generating an intensity distribution profile from the image, which is unique to each of the clade variants.
Description
BACKGROUND OF THE INVENTION
Field of the Invention

The present invention relates to the field of multiplex based viral pathogen detection and analysis. More particularly, the present invention relates to detecting the presence of clade variants of SARS-COVID-2 virus in patient and environmental samples.


Description of the Related Art

The COVID-19 pandemic has increased awareness that viral infection can be an existential threat to health, public safety and the US economy. More fundamentally, there is a recognition that the viral risks are exceedingly dangerous and complex and require new approaches to diagnostics and screening.


The next pandemic wave is expected to have more pronounced flu-like symptoms (seasonal influenza A and/or B) coupled with the COVID-19, or COVID-19 variants that will coexist with the Coronavirus already responsible for the common cold. These complexities are expected to pose significant challenges to public health and the healthcare system in diagnosing multi-symptom conditions accurately and efficiently.


The COVID-19 pandemic has also led to the realization of an additional level of complexity that the realization that human health and environmental contamination are linked in a fundamental way that affects collection efficiency and increases risk to the healthcare workers (1, 2). Alternatives to nasopharyngeal collection methods such as for example, saliva collection are needed to enable scalability among millions of individuals.


Q-RT-PCR technology has dominated COVID-19 diagnostics and public health screening. Independent of the test developer, Q-RT-PCR has been shown to have an unusually high false negative rate (15% up to 30%). As of May 2020, the CDC has recorded 613, 041 COVID-19 tests. With a 15% false negative rate, approximately 91, 956 people would thus be falsely classified as free of infection. Meta-analysis has shown that the false negative rate for Q-RT-PCR is high below day 7 of infection when viral load is still low. This renders Q-RT-PCR ineffective as a tool for early detection of weak symptomatic carriers while also lessening its value in epidemiology.


As for other organisms, genetic variations in SARS-COVID-2 are grouped into clades. There are over 52, 600 complete and high-coverage genomes available on the Global Initiative on Sharing Avian Influenza Data (GISAID). Presently, WHO has identified 10, 022 SARS-COVID-2 genomes from 68 different countries and detected 65, 776 variants and 5, 775 distinct variants that comprised missense mutations, synonymous mutations, mutations in non-coding regions, non-coding deletions, in-frame deletions, non-coding insertions, stop-gained variants, frameshift deletions and in-frame insertions among others. Identifying these clade variants in population and environmental samples while a daunting task, is critical for global public health management directed to controlling the pandemic.


When first identified, it was widely assumed that COVID-19 would mutate slowly, based on a relatively stable genome that would experience minimal genetic drift as the pandemic spread. Unfortunately, perhaps as a function of environmental selection pressure (crowding) physical selection pressure (PPE) and therapeutic selection pressure (vaccination) the original Wuhan clade has evolved into a very large number of clade variants. Consequently, in the past 3 months there has been an international effort to discover and track the full range of clade variant evolution.


Next Generation Sequencing (NGS), primarily Targeted Resequencing of the CoV-2 Spike gene, has been instrumental in elucidating the patterns of genetic variation which define the growing set of clade variants of present international concern (UK, South Africa, Brazil, India, US California, US NY, US Southern) with others emerging at an expanding rate. Whereas NGS is without equal as a discovery tool in genetic epidemiology, it is not ideally suited for field-deployed, public health screening at population scale due to complexities associated with purchasing and managing the kits supply chain, setting up and training personnel, especially when compared to Q-RT-PCR, which is the present standard for nucleic acid based COVID-19 screening. Conversely, while Q-RT-PCR (especially TaqMan) is now the clear standard in COVID-19 testing laboratories for simple positive/negative screening, its suitability for screening clade variants is limited. Deploying TaqMan for COVID-19 clade Identification requires running about 10-15 TaqMan kits on each sample to generate sequence content equivalent to Spike targeted NGS, thereby negating the benefits of costs and logistics with Q-RT-PCR.


Thus, there is a need in the art for superior tools to not only administer and stabilize sample collection for respiratory viruses from millions of samples in parallel obtained from diverse locations including, clinic, home, work, school and in transportation hubs, but also to detect and identify clade variants in the population at the highest levels of sensitivity and specificity. The present invention fulfills this longstanding need and desire in the art.


SUMMARY OF THE INVENTION

The present invention is directed to a method for detecting clade variants in the Coronavirus disease 2019 virus in a sample. The sample is obtained from which viruses are harvested. Total RNA is isolated from the harvested viruses. A combined reverse transcription and first amplification reaction is performed on the total RNA using at least one first primer pair selective for all COVID-19 viruses to generate COVID-19 virus cDNA amplicons. A second amplification is performed using the COVID-19 virus cDNA amplicons as template and at least one fluorescent labeled second primer pair selective for a target nucleotide sequence in the COVID-19 virus cDNA to generate at least one fluorescent labeled COVID-19 virus amplicon. The fluorescent labeled COVID-19 virus amplicons are hybridized to a plurality of nucleic acid probes. Each nucleic acid probe is attached to a solid microarray support, and has a sequence corresponding to a sequence determinant that discriminates among clade variants of the COVID-19 virus. After hybridization, the array is washed at least once and imaged to detect at least one fluorescent signal from the hybridized fluorescent labeled COVID-19 virus amplicons. The present invention is directed to a related method where prior to the harvesting step, the method further comprises mixing the sample with an RNA stabilizer.


The present invention is further directed to a method for detecting clade variants in the Coronavirus disease 2019 virus in a sample. The sample is obtained from which, viruses are harvested. Total RNA is isolated from the harvested viruses. A combined reverse transcription and first amplification reaction is performed on the total RNA using at least one fluorescent labeled primer pair comprising an unlabeled primer, and a fluorescently labeled primer, selective for a target sequence in all COVID-19 viruses to generate at least one fluorescent labeled COVID-19 virus amplicon. The fluorescent labeled COVID-19 virus amplicons are hybridized to a plurality of nucleic acid probes. Each nucleic acid probe is attached to a solid microarray support, and has a sequence corresponding to a sequence determinant that discriminates among clade variants of the COVID-19 virus. After hybridization, the array is washed at least once and imaged to detect at least one fluorescent signal from the hybridized fluorescent labeled COVID-19 virus amplicons. The present invention is directed to a related method where prior to the harvesting step, the method further comprises mixing the sample with an RNA stabilizer.


Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.





BRIEF DESCRIPTION OF THE FIGURES

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions and certain embodiments of the invention briefly summarized above are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.



FIG. 1 shows Clade Chip target site performance data for the Spike 69-70 deletion.



FIG. 2 shows Clade Chip target sites performance data normalized to a universal probe for the Spike D80A mutation.



FIG. 3 shows Clade Chip target sites performance data normalized to a universal probe for the Spike D138Y mutation.



FIG. 4 shows Clade Chip target sites performance data normalized to a universal probe for the Spike W152C mutation.



FIG. 5 shows Clade Chip target sites performance data normalized to a universal probe for the Spike N440K mutation.



FIG. 6 shows Clade Chip target sites performance data normalized to a universal probe for the Spike L452R mutation.



FIG. 7 shows Clade Chip target sites performance data normalized to a universal probe for the Spike E484K mutation.



FIG. 8 shows Clade Chip target sites performance data normalized to a universal probe for the Spike N501Y mutation.



FIG. 9 shows Clade Chip target sites performance data normalized to a universal probe for the Spike D614G mutation.



FIG. 10 shows Clade Chip target sites performance data normalized to a universal probe for the Spike P681 H mutation.



FIG. 11 shows Clade Chip target sites performance data normalized to a universal probe for the Spike A701V mutation.



FIGS. 12A-12B shows the results of DETECTX-Cv analysis using a multiplex of Amplimers 2, 3, 6, 8. FIG. 12A shows the multiplex analysis data normalized to Universal probe. FIG. 12B shows the multiplex analysis data normalized to Wild-type probe.



FIGS. 13A-13B shows the results of DETECTX-Cv analysis using a multiplex of Amplimers 2, 3, 5, 6, 8. FIG. 13A shows the multiplex analysis data normalized to Universal probe. FIG. 13B shows the multiplex analysis data normalized to Wild-type probe.



FIGS. 14A-14Y shows analytical LoD data for a series of synthetic G-block fragments, used as “synthetic clade variant standards”, corresponding to domains 2-8. FIG. 14A shows DETECTX-Cv analysis for the indicated variants corresponding to the Brazil region. FIG. 14B shows DETECTX-Cv analysis for the indicated variants corresponding to the Brazil region. FIG. 14C shows DETECTX-Cv analysis for the indicated variants corresponding to the Brazil region. FIG. 14D shows DETECTX-Cv analysis for the indicated variants corresponding to the Brazil region. FIG. 14E shows DETECTX-Cv analysis for the indicated variants corresponding to the Brazil region. FIG. 14F shows DETECTX-Cv analysis for the indicated variants corresponding to the Brazil region. FIG. 14G shows DETECTX-Cv analysis for the indicated variants corresponding to the California region. FIG. 14H shows DETECTX-Cv analysis for the indicated variants corresponding to the California region. FIG. 141 shows DETECTX-Cv analysis for the indicated variants corresponding to the California region. FIG. 14J shows DETECTX-Cv analysis for the indicated variants corresponding to the California region. FIG. 14K shows DETECTX-Cv analysis for the indicated variants corresponding to the Indian region. FIG. 14L shows DETECTX-Cv analysis for the indicated variants corresponding to the Indian region. FIG. 14M shows DETECTX-Cv analysis for the indicated variants corresponding to the South Africa region. FIG. 14N shows DETECTX-Cv analysis for the indicated variants corresponding to the South Africa region. FIG. 14O shows DETECTX-Cv analysis for the indicated variants corresponding to the South Africa region. FIG. 14P shows DETECTX-Cv analysis for the indicated variants corresponding to the South Africa region. FIG. 14Q shows DETECTX-Cv analysis for the indicated variants corresponding to the South Africa region. FIG. 14R shows DETECTX-Cv analysis for the indicated variants corresponding to the South Africa region. FIG. 14S shows DETECTX-Cv analysis for the indicated variants corresponding to the South Africa region. FIG. 14T shows DETECTX-Cv analysis for the indicated variants corresponding to the South Africa region. FIG. 14U shows DETECTX-Cv analysis for the indicated variants corresponding to the UK region. FIG. 14V shows DETECTX-Cv analysis for the indicated variants corresponding to the UK region. FIG. 14W shows DETECTX-Cv analysis for the indicated variants corresponding to the UK region. FIG. 14X shows DETECTX-Cv analysis for the indicated variants corresponding to the UK region. FIG. 14Y shows DETECTX-Cv analysis for the indicated variants corresponding to the UK region.



FIGS. 15A-15E shows DETECTX-Cv analysis using synthetic Clade Variant standards. FIG. 15A shows the analysis using synthetic Clade Variant standard corresponding to Brazil. FIG. 15B shows the analysis using synthetic Clade Variant standard corresponding to California 452 (CA 452). FIG. 15C shows the analysis using synthetic Clade Variant standard corresponding to India. FIG. 15D shows the analysis using synthetic Clade Variant standard corresponding to South Africa. FIG. 15E shows the analysis using synthetic Clade Variant standard corresponding to United Kingdom.



FIG. 16 shows LoD range finding DETECTX-Cv analysis for clinical samples processed using Zymo bead capture.



FIG. 17 shows LoD range finding DETECTX-Cv analysis for clinical negative saliva samples processed using Zymo bead capture.



FIGS. 18A-18E shows representative DETECTX-Cv analysis of synthetic Clade variant standards. FIG. 18A shows a histogram analysis for the South Africa synthetic cocktail, D80A-, E484K, N501Y, A701V. FIG. 18B shows a histogram analysis for the California synthetic cocktail, W152C, L452R. FIG. 18C shows a histogram analysis for the India synthetic cocktail, N440K. FIG. 18D shows a histogram analysis for the Brazil P.1 synthetic cocktail, D138Y, E484K, N501Y. FIG. 18E shows a histogram analysis for the UK (B.1.1.7) synthetic cocktail, 69-70 deletion, N501Y, P681 H.



FIGS. 19A-19K show representative data for DETECTX-Cv analysis of clinical positive samples performed at TriCore. FIG. 19A shows a histogram analysis for a sample comprising Wuhan/European progenitor variants. FIG. 19B shows a histogram analysis for a sample comprising California variants, W152C AND L452R. FIG. 19C shows a histogram analysis for a sample comprising California variants, W152C and L452R. FIG. 19D shows a histogram analysis for a sample comprising UK variants, 69-70 deletion, N501Y and P681 H. FIG. 19E shows a histogram analysis for a sample comprising UK variants, 69-70 deletion, N501Y and P681 H. FIG. 19F shows a histogram analysis for a sample comprising, 69-70 deletion, and P681 H. FIG. 19G shows a histogram analysis for a sample comprising variant P681 H. FIG. 19H shows a histogram analysis for a sample comprising variant P681 H. FIG. 19I shows a histogram analysis for a sample comprising California variants, W152C and L452R. FIG. 19J shows a histogram analysis for a sample that did not pass QA/QC. FIG. 19K shows a histogram analysis for a sample that did not pass QA/QC.



FIGS. 20A-20J show representative data for DETECTX-Cv analysis of clinical positive samples performed at PathogenDx. FIG. 20A shows a histogram analysis for a sample comprising California variants W152C and L452R. FIG. 20B shows a histogram analysis for a sample comprising likely California variant L452R. FIG. 20C shows a histogram analysis for a sample comprising California variants, W152C and L452R. FIG. 20D shows a histogram analysis for a sample that did not pass QA/QC. FIG. 20E shows a histogram analysis for a sample comprising California variants W152C and L452R. FIG. 20F shows a histogram analysis for a sample comprising California variant, W152C and L452R. FIG. 20G shows a histogram analysis for a sample comprising California variants, W152C and L452R. FIG. 20H shows a histogram analysis for a sample comprising variant P681H. FIG. 201 shows a histogram analysis for a sample comprising variant P681H. FIG. 20J shows a histogram analysis for a sample comprising Wuhan/European progenitor variants.





DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one, ” but it is also consistent with the meaning of “one or more, ” “at least one, ” and “one or more than one.” Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method described herein can be implemented with respect to any other method described herein.


As used herein, the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or”.


As used herein, “comprise” and its variations, such as “comprises” and “comprising” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the exclusion of any other item, element or step or group of items, elements or steps unless the context requires otherwise. Similarly, “another” or “other” may mean at least a second or more of the same or different claim element or components thereof.


In one embodiment of the present invention there is provided a method for detecting clade variants in a Coronavirus disease 2019 virus (COVID-19) in a sample, comprising obtaining the sample; harvesting viruses from the sample; isolating a total RNA from the harvested viruses; performing a combined reverse transcription and first amplification reaction on the total RNA using at least one first primer pair selective for all COVID-19 viruses to generate COVID-19 virus cDNA amplicons; performing a second amplification using the COVID-19 virus cDNA amplicons as template and at least one fluorescent labeled second primer pair selective for a target nucleotide sequence in the COVID-19 virus cDNA to generate at least one fluorescent labeled COVID-19 virus amplicon; hybridizing the fluorescent labeled COVID-19 virus amplicons to a plurality of nucleic acid probes, each having a sequence corresponding to a sequence determinant that discriminates among the clade variants of the COVID-19 virus, where the nucleic acid probes are attached to a solid microarray support; washing the microarray at least once; and imaging the microarray to detect at least one fluorescent signal from the hybridized fluorescent labeled COVID-19 virus amplicons, thereby detecting the clade variants of the COVID-19 virus in the sample.


A total RNA potentially comprising RNA from COVID-19 virus and other contaminating pathogens and human cells is isolated from the sample. Commercially available RNA isolation kits such as for example, a Quick-DNA/RNA Viral MagBead Kit from Zymo Research are used for this purpose. The total RNA thus isolated is used without further purification. Alternatively, intact virus may be captured with magnetic beads, using kits such as that from Ceres Nanosciences (e.g., CERES NANOTRAP technology), or by first precipitating the virus with polyethylene glycol (PEG), followed by lysis of the enriched virus by heating with a “PCR-Friendly” lysis solution such as 1% NP40 in Tris-EDTA buffer and then used without additional purification.


The COVID-19 virus RNA in the total RNA isolate is used as a template for amplifying a COVID-19 virus specific sequence. This comprises first performing a combined reverse transcriptase enzyme catalyzed reverse transcription reaction and a first amplification reaction using a first primer pair selective for the virus to generate COVID-19 virus cDNA amplicons. In this embodiment, the first primer pairs have forward (odd numbers) and reverse (even number) sequences shown in SEQ ID NO: 1 to SEQ ID NO: 8 (Table 1).). Commercially available reverse transcriptase enzyme and buffers are used in this step. Controls including, but not limited to a RNAse P control having first primer pair (forward primer SEQ ID NO: 130, reverse primer SEQ ID NO: 131) are also used herein (Table 1). The COVID-19 virus cDNA amplicons generated in the first amplification reaction are used as a template for a second amplification that employs at least one fluorescent labeled second primer pair selective for a target nucleotide sequence in the COVID-19 virus cDNA to generate at least one fluorescent labeled COVID-19 virus amplicon.


The fluorescent labeled COVID-19 virus amplicons hybridize to the nucleic acid probes, which are attached at specific positions on a microarray support, for example, a 3-dimensional lattice microarray support. After hybridization, the microarray is washed at least once to remove unhybridized amplicons. Washed microarrays are imaged to detect a fluorescent signal from the hybridized fluorescent labeled COVID-19 virus amplicons to detect the Clade variants of the COVID-19 virus in the sample.









TABLE 1







Primer sequences used for the first amplification reaction












Amplimer


Primer Sequence


SEQ ID NOS.
#
Target
Gene
(5′ to 3′)





SEQ ID NO: 1
1/2
AA 00-103
Spike
TTTAACAGAGTTGTTA






TTTCTAGTGATG





SEQ ID NO: 2
1/2
AA 00-103
Spike
TTTTCTAAAGTAGTAC






CAAAAATCCAGC





SEQ ID NO: 3
3/4
AA 124-262
Spike
TTTCCCTACTTATTGT






TAATAACGCTAC





SEQ ID NO: 4
3/4
AA 124-262
Spike
TTTAGATAACCCACAT






AATAAGCTGCAG





SEQ ID NO: 5
5/6
AA 397-517
Spike
TTTATCTCTGCTTTAC






TAATGTCTATGC





SEQ ID NO: 6
5/6
AA 397-517
Spike
TTTACAAACAGTTGCT






GGTGCATGTAGA





SEQ ID NO: 7
7/8
AA 601-726
Spike
TTTTGGTGTCAGTGTT






ATAACACCAGGA





SEQ ID NO: 8
7/8
AA 601-726
Spike
TTTTGTCTTGGTCATA






GACACTGGTAGA





SEQ ID: 130

RNAse P
RNAse P
TTTACTTCAGCATGG




control

CGGTGTTTGCAGA





SEQ ID: 131

RNAse P
RNAse P
TTTTGATAGCAACAAC




control

TGAATAGCCAAG









Further to this embodiment, prior to the harvesting step, the method comprises mixing the sample with an RNA stabilizer. A representative RNA stabilizer is a chemical stabilizer that protects the RNA from degradation during storage and transportation.


In both embodiments one or more of the at least one fluorescent labeled second primer pair is selective for a panel of target nucleotide sequences within a target region of a gene in the COVID-19 virus; and the nucleic acid probes are specific to the target region of the gene, whereby the at least one fluorescent labeled COVID-19 virus amplicon generated is hybridized to the nucleic acid probe thereby discriminating the clade variants of the COVID-19 virus in the sample. Further to this embodiment the method comprises detecting the at least one fluorescent signal from the hybridized at least one fluorescent labeled COVID-19 virus amplicons associated with the panel of target nucleotide sequences within the target region of the gene; and generating an intensity distribution profile unique to each of the clade variants, whereby each of the clade variants is distinguishable from others. Particularly, the gene may be a Spike gene.


In a non-limiting example, the target region may be in the Spike gene in the COVID-19 virus and the fluorescent labeled second primer pairs may have forward (odd numbers) and reverse (even number) sequences shown in SEQ ID NO: 9 to SEQ ID NO: 29 (Tables 2 and 11). Controls including, but not limited to a RNAse P control having primer pair (forward primer SEQ ID NO: 132, reverse primer SEQ ID NO: 133) are also used herein (Table 2).


Any fluorescent label may be used in the fluorescent labeled second primer pairs including, but not limited to, a CY3, a CY5, SYBR Green, a DYLIGHT™ DY647, a ALEXA FLUOR 647, a DYLIGHT™ DY547 and a ALEXA FLUOR 550.









TABLE 2







Fluorescent labeled primer sequences used for amplification reactions












Amplimer


Primer Sequence


SEQ ID NOS.
#
Target
Gene
(5′ to 3′)





SEQ ID NO: 9
2
AA64-80
Spike
ACCTTTCTTTTCCAATGT






TACTTGGTTC





SEQ ID NO: 10
2
AA64-80
Spike
Cy3-TTTTATGTTAGA






CTTCTCAGTGGAAGCA





SEQ ID NO: 11
3
AA126-157
Spike
TTTCTTATTGTTAATAAC






GCTACTAATG





SEQ ID NO: 12
3
AA126-157
Spike
Cy3-TTTCATTCGCACT






AGAATA AACTCTGAA





SEQ ID NO: 13
5
AA408-456
Spike
TGTAATTAGAGGTGATG






AAGTCAGA





SEQ ID NO: 14
5
AA408-456
Spike
Cy3-TTTAAAGGTTTGA






GATTAG ACTTCCTAA





SEQ ID NO: 15
6
AA475-505
Spike
TTTTATTTCAACTGAAAT






YTATCAGGCC





SEQ ID NO: 16
6
AA475-505
Spike
Cy3-TTTAAAGTACTAC






TACTCT GTATGGTTG





SEQ ID NO: 17
8
AA677-707
Spike
TTTTATATGCGCTAGTTA






TCAGACTCAG





SEQ ID NO: 18
8
AA677-707
Spike
Cy3-TTTTGGTATGGC






AATAGA GTTATTAGAG





SEQ ID NO: 19
1
AA11-33
Spike
TTTTTTTCTTGTTTTATTG






CCACTAGTC





SEQ ID NO: 20
1
AA11-33
Spike
Cy3-TTTTTGTCAGGG






TAATAAA CACCACGTG





SEQ ID NO: 21
4
AA213-260
Spike
TTTTAAGCACACGCCTA






TTAATTTAGTG





SEQ ID NO: 22
4
AA213-260
Spike
Cy3-TTTCCACATAAT






AAGCTGCAGCACCAGC





SEQ ID NO: 23
7
AA603-618
Spike
TTTAGTGTTATAACACCA






GGAACAAATA





SEQ ID NO: 24
7
AA603-618
Spike
Cy3-TTTTGCATGAAT






AGCAACAGGGACTTCT





SEQ ID NO: 132

RNAse P
RNAse P
TTTGTTTGCAGATTTGG




control

ACCTGCGAGCG





SEQ ID NO: 133

RNAse P
RNAse P
Cy3-TTTAAGGTGAG




control

CGGCTGTCTCCACAAGT









In all embodiments the clade variants of the COVID-19 virus may be Denmark, UK (B.1.1.7), South African (B.1.351), Brazil/Japan (P1), Brazil (B1.1.28), California USA, L452R (1.429), India (N440K), or Wuhan, or a combination thereof. The COVID-19 virus is a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV 2) or a mutated form thereof. A combination of these variants also may be detected simultaneously.


Also in all embodiments the first primer pair may comprise the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, or SEQ ID NO: 3 and SEQ ID NO: 4, or SEQ ID NO: 5 and SEQ ID NO: 6, or SEQ ID NO: 7 and SEQ ID NO: 8, or a combination thereof. Sequences of the first primer pairs are shown in Table 1.


In addition in all embodiments the fluorescent labeled second primer pair may comprise the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 10, or SEQ ID NO: 11 and SEQ ID NO: 12, or SEQ ID NO: 13 and SEQ ID NO: 14, or SEQ ID NO: 15 and SEQ ID NO: 16, or SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO:


27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 24, or a combination thereof. Sequences of the first primer pairs are shown in Table 2.


Furthermore, in all embodiments the nucleic acid probes may comprise at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 30-129. The nucleic acid probes may have a sequence corresponding to a sequence determinant that discriminates among the Clade variants of the COVID-19 virus. The nucleic acid probes are specific to the target region of the gene in the COVID-19 virus as discussed supra. This enables hybridization of the one fluorescent labeled COVID-19 virus amplicon generated to the Spike gene-specific nucleic acid probe thereby discriminating the Clade variants of the COVID-19 virus in the sample. In a non-limiting example, the target region is in a Spike gene in the COVID-19 virus and the nucleic acid probes have a sequence shown in SEQ ID NO: 30 to SEQ ID: 129 (Tables 3, 10 and 14). Controls including, but not limited to, a RNAse P control nucleic acid probe (SEQ ID NO: 68 and SEQ ID NO: 69) and a negative control nucleic acid probe (SEQ ID NO: 70) are also used herein (Table 3).









TABLE 3







Nucleic acid probe sequences used for hybridization











Amplimer

Probe Sequence


SEQ ID NOS.
#
Target
(5′ to 3′)





SEQ ID NO: 31
2
AA69-70 HV
TTTTTCCCATGCTATACATGTCTC





TGTTTTTT





SEQ ID NO: 32
2
AA69-70 DEL
TTTTTTTTTCCATGCTATCTCTGG





GATTTTTT





SEQ ID NO: 33
2
AA D80A
TTTTTCAGAGGTTTGMTAACCCTG





TCTTTTTT





SEQ ID NO: 34
2
AA D80_
TTTTTTTGGTTTGATAACCCTGCTT





TTTTT





SEQ ID NO: 35
2
AA_80A
TTTTTTTGGTTTGCTAACCCTGCT





TTTTTT





SEQ ID NO: 36
3
AA D138Y
TTTTATTTTGTAATKATCCATTTTT





GTTTT





SEQ ID NO: 37
3
AA D138_
TTTTTCTTTGTAATGATCCATTTTC





TTTTT





SEQ ID NO: 38
3
AA_138Y
TTTTTTTTTGTAATTATCCATTTTC





TTTTT





SEQ ID NO: 39
3
AA W1520
TTTTTAGTTGKATGGAAAGTGAGT





TCTTTT





SEQ ID NO: 40
3
AA W152_
TTTCTCTAAAAGTTGGATGGAAAC





TCTTCT





SEQ ID NO: 41
3
AA_152C
TTTCTTCAAAGTTGTATGGAAAGC





CTTCTT





SEQ ID NO: 42
5
AA 439K + N440K
TTTTTAATTCTAAMAAKCTTGATTC





TAATTTT





SEQ ID NO: 43
5
AA N439_ + N440_
TTTTTAATTCTAACAATCTTGATTT





CTTTT





SEQ ID NO: 44
5
AA N439_ + _440K
TTTTTTATTCTAACAAGCTTGATTT





TTTTT





SEQ ID NO: 45
5
AA_439K + N440
TTTTCTATTCTAAAAATCTTGATTT





CTTTT





SEQ ID NO: 46
5
AA L452R
TTTCTATAATTACCTGTATAGATTG





TCTTT





SEQ ID NO: 47
5
AA L452_
TTTTTTTAATTACCTGTATAGATTT





CTTTT





SEQ ID NO: 48
5
AA_452R
TTTTTCATAATTACTGGTATAGATC





TTTTT





SEQ ID NO: 49
6
AA S477_
TTTTTTCGCCGGTAGCACACCTCT





TTTTTT





SEQ ID NO: 50
6
AA_477N
TTTTCTTCCGGTAACACACCTTTT





TTTTTT





SEQ ID NO: 51
6
AA V483A + E484K
TTTTTTAATGGTGTTRAAGGTTTTA





ATTTTTT





SEQ ID NO: 52
6
AA V483 + E484_
TTTTTTCTGGTGTTGAAGGTTTTA





CTTTTT





SEQ ID NO: 53
6
AA V483_ + _484K
TTTTTTTATGGTGTTAAAGGTTTTC





TTTTT





SEQ ID NO: 54
6
AA 483A + E484_
TTTTTTTATGGTGCTGAAGGTTCT





TTTTTT





SEQ ID NO: 55
6
AA N501Y
TTTTTTTCCAACCCACTWATGGT





GTTTTTTTT





SEQ ID NO: 56
6
AA N501_
TTTTTTTTACCCACTAATGGTGTCT





TTTTT





SEQ ID NO: 57
6
AA N_501Y
TTTTTTTTACCCACTTATGGTGTCT





TTTTT





SEQ ID NO: 58
8
AA P681H
TTTTTCAGACTAATTCTCMTCGG





CTTTTT





SEQ ID NO: 59
8
AA P681_
TTTTTTTCTAATTCTCCTCGGCGTT





TTTTT





SEQ ID NO: 60
8
AA_681H
TTTTTTTTTAATTCTCATCGGCGTT





TTTTT





SEQ ID NO: 61
8
AA A701V
TTTCACTTGGTGYAGAAAATTCA





GTTTTT





SEQ ID NO: 62
8
AA A701_
TCTTCTTCTTGGTGCAGAAAATTA





TTCTTT





SEQ ID NO: 63
8
AA_701V
TCTTCTTCTTGGTGTAGAAAATTA





TTCTTT





SEQ ID NO: 134

RNAse P
TTTTTTTTCTGACCTGAAGGCTCT





GCGCGTTTTT





SEQ ID NO: 135

RNAse P
TTTTTCTTGACCTGAAGGCTCTGC





TTTTTT





SEQ ID NO: 136

Negative Control
TTTTTTCTACTACCTATGCTGATTC





ACTCTTTTT









Further still in all embodiments the sample may comprise at least one of a nasopharyngeal swab, a nasal swab, a mouth swab, a mouthwash, an aerosol, or a swab from a hard surface. In one aspect the sample may be any sample obtained from a subject including, but not limited to, a nasopharyngeal swab, a nasal swab, a mouth swab, and a mouthwash (sample obtained by rinsing the subject's buccal cavity). A pooled sample obtained by combining two or more of these samples or by combining samples from multiple subjects also may be used. In another aspect, the sample is an environmental sample obtain from inanimate sources including but is not limited to an aerosol and a hard surface. The aerosol samples may be obtained using commercial air samplers such as for example a Coriolis Micro Air Sampler. A sample from a hard surface may be obtained using a swab. In both aspects, the viruses from samples obtained on swabs are dispersed in a liquid such as phosphate buffered saline. Aerosol samples are transferred into a volume of a liquid such as phosphate buffered saline.


In another embodiment of the present invention, there is provided a method for detecting Clade variants in the Coronavirus disease 2019 virus (COVID-19) in a sample, comprising obtaining the sample; harvesting viruses from the sample; isolating total RNA from the harvested viruses; performing a combined reverse transcription and asymmetric PCR amplification reaction on the total RNA using at least one fluorescent labeled primer pair comprising an unlabeled primer, and a fluorescently labeled primer, selective for a target sequence in all COVID-19 viruses to generate at least one fluorescent labeled COVID-19 virus amplicon; hybridizing the fluorescent labeled COVID-19 virus amplicons to a plurality of nucleic acid probes, each having a sequence corresponding to a sequence determinant that discriminates among the clade variants of the COVID-19 virus, where the nucleic acid probes are attached to a solid microarray support; washing the microarray at least once; and imaging the microarray to detect at least one fluorescent signal from the hybridized fluorescent labeled COVID-19 virus amplicons, thereby detecting the clade variants of the COVID-19 virus in the sample.


The total RNA is isolated as described supra and any COVID-19 virus RNA in the total RNA isolate is used as a template in a combined reverse transcription/amplification reaction (RT-PCR). In this step, the nucleic acid sequences in the COVID-19 virus RNA are transcribed using a reverse transcriptase enzyme to generate COVID-19 complementary DNA (cDNA) that is amplified in the same reaction using COVID-19 virus selective fluorescent labeled primer pairs to generate fluorescent labeled COVID-19 virus amplicons. Each fluorescent labeled primer pair comprises an unlabeled primer and a fluorescently labeled primer in about 4-fold to about 8-fold excess of the unlabeled primer whereby, upon completion of the reaction, the fluorescently labelled amplicon is primarily single stranded (that is, the reaction is a type of “asymmetric PCR”).


Hybridization of the fluorescent labeled COVID-19 virus amplicons to the plurality of nucleic acid probes attached at specific positions on a microarray support is performed as described supra. The nucleic acid probes may have a sequence corresponding to a sequence determinant that discriminates among the Clade variants of the COVID-19 virus and are specific to the target region of the gene in the COVID-19 virus, as discussed supra. This enables hybridization of the one fluorescent labeled COVID-19 virus amplicon generated to the Spike gene-specific nucleic acid probe thereby discriminating the Clade variants of the COVID-19 virus in the sample. In a non-limiting example, the target region is in a Spike gene in the COVID-19 virus and the nucleic acid probes have a sequence shown in SEQ ID NO: 31 to SEQ ID: 63 (Table 3). Controls are as described supra and shown in (Table 3).


Further to this embodiment, prior to the harvesting step, the method comprises mixing the sample with an RNA stabilizer. A representative RNA stabilizer is a chemical stabilizer, as described supra.


In both embodiments one or more of the at least one fluorescent labeled second primer pair is selective for a panel of target nucleotide sequences within a target region of a gene in the COVID-19 virus; and the nucleic acid probes are specific to the target region of the gene, whereby the at least one fluorescent labeled COVID-19 virus amplicon generated is hybridized to the nucleic acid probe thereby discriminating the clade variants of the COVID-19 virus in the sample. Further to this embodiment the method comprises detecting the at least one fluorescent signal from the hybridized at least one fluorescent labeled COVID-19 virus amplicons associated with the panel of target nucleotide sequences within the target region of the gene; and generating an intensity distribution profile unique to each of the clade variants, whereby each of the clade variants is distinguishable from others. Particularly, the gene may be the Spike gene.


In a non-limiting example, the target region may be in the Spike gene in the COVID-19 virus and the fluorescent labeled second primer pairs may have forward (odd numbers) and reverse (even number) sequences shown in SEQ ID NO: 9 to SEQ ID NO: 18 (Table 2). Controls including, but not limited to a RNAse P control having a primer pair with forward primer SEQ ID NO: 66 and reverse primer SEQ ID NO: 67 are also used herein (Table 2).


In all embodiments the COVID-19 gene, the clade variants of the COVID-19 virus, the at least one fluorescent labeled primer pair, the fluorescent label, the nucleic acid probes, and the samples are as described supra. Also in all embodiments the fluorescently labeled primer may be in an excess of about 4-fold to about 8-fold over the unlabeled primer in the fluorescent labeled primer pair. Exemplary nucleotide sequences for the fluorescent labeled primer pairs including, for example, RNAse P controls, are shown in Table 2. Exemplary nucleotide sequences for the nucleic acid probes, including, for example, RNAse P controls and negative controls, are shown in Table 3.


Provided herein are methods of nucleic acid analysis to detect stable genetic variation such as a clade variation in a viral pathogen which is based on simultaneous analysis of multiple sequence domains in a gene, such as for example the Spike gene in the RNA genome of CoV-2, to measure clade variation in SARS-CoV-2. In one method for detecting the stable genetic variation, total RNA from the harvested viruses is isolated and used in a combined reverse transcription and first amplification reaction (RT-PCR) to generate COVID-19 virus cDNA amplicons. These amplicons are used as template in a second amplification reaction that uses fluorescent labeled second primer pair selective for a panel of target nucleotide sequences within a target region of a gene in the COVID-19 virus such as for example, the gene for the Spike protein, to generate fluorescent labeled COVID-19 virus amplicons. In a second method, a combined reverse transcription and asymmetric PCR amplification reaction is performed using at least one fluorescent labeled primer pair selective for the panel of target nucleotide sequences within a target region of a gene in the COVID-19 virus to generate fluorescent labeled COVID-19 virus amplicons. In either method, the fluorescent labeled COVID-19 virus amplicons are hybridized to nucleic acid probes attached at specific positions on a microarray.


This method allows positive hybridization signals to be validated on each sample tested based on internal “mismatched” and “sequence specific” controls. Additionally, at least one fluorescent signal from the hybridized amplicons associated with the panel of target nucleotide sequences within the target region of the gene is detected and an intensity distribution profile unique to each of the Clade variants generated, whereby each of the Clade variants is distinguishable from others.


In the embodiments described supra, the solid microarray support is made of any suitable material known in the art including but not limited to borosilicate glass, a thermoplastic acrylic resin (e.g., poly(methyl methacrylate-VSUVT) a cycloolefin polymer (e.g. ZEONOR 1060R), a metal including, but not limited to gold and platinum, a plastic including, but not limited to polyethylene terephthalate, polycarbonate, nylon, a ceramic including, but not limited to TiO2, and Indium tin oxide (ITO) and engineered carbon surfaces including, but not limited to graphene. A combination of these materials may also be used. The solid support has a front surface and a back surface and is activated on the front surface by chemically activatable groups for attachment of the nucleic acid probes. In this embodiment, the chemically activatable groups include but are not limited to epoxysilane, isocyanate, succinimide, carbodiimide, aldehyde and maleimide. These materials are well known in the art and one of ordinary skill in this art would be able to readily functionalize any of these supports as desired. In a preferred embodiment, the solid support is epoxysilane functionalized borosilicate glass support.


The nucleic acid probes are attached either directly to the solid microarray support, or indirectly attached to the support using bifunctional polymer linkers. In this embodiment, the bifunctional polymer linker has a top domain and a bottom end. On the bottom end is attached a first reactive moiety that allows covalent attachment to the chemically activatable groups in the solid support. Examples of first reactive moieties include but are not limited to an amine group, a thiol group and an aldehyde group. In one aspect the first reactive moiety is an amine group. On the top domain of the bifunctional polymer linker is provided a second reactive moiety that allows covalent attachment to the oligonucleotide probe. Examples of second reactive moieties include but are not limited to nucleotide bases like thymidine, adenine, guanine, cytidine, uracil and bromodeoxyuridine and amino acid like cysteine, phenylalanine, tyrosine glycine, serine, tryptophan, cystine, methionine, histidine, arginine and lysine. The bifunctional polymer linker may be an oligonucleotide such as OLIGOdT, an amino polysaccharide such as chitosan, a polyamine such as spermine, spermidine, cadaverine and putrescine, a polyamino acid, with a lysine or histidine, or any other polymeric compounds with dual functional groups which can be attached to the chemically activatable solid support on the bottom end, and the nucleic acid probes on the top domain. Preferably, the bifunctional polymer linker is OLIGOdT having an amine group at the 5′ end.


The bifunctional polymer linker may be unmodified with a fluorescent label. Alternatively, the bifunctional polymer linker has a fluorescent label attached covalently to the top domain, the bottom end, or internally. The second fluorescent label is different from the fluorescent label in the fluorescent labeled primers. Having a fluorescent label (fluorescent tag) attached to the bifunctional polymer linker is beneficial since it allows the user to image and detect the position of the individual nucleic acid probes (“spot”) printed on the microarray. By using two different fluorescent labels, one for the bifunctional polymer linker and the second for the amplicons generated from the viral RNA being queried, the user can obtain a superimposed image that allows parallel detection of those nucleic acid probes which have been hybridized with amplicons. This is advantageous since it helps in identifying the virus comprised in the sample using suitable computer and software, assisted by a database correlating nucleic acid probe sequence and microarray location of this sequence with a known RNA signature in viruses. Examples of fluorescent labels include, but are not limited to CYS, DYLIGHT™ DY647, ALEXA FLUOR 647, CY3, DYLIGHT™ DY547, or ALEXA FLUOR 550. The fluorescent labels may be attached to any reactive group including but not limited to, amine, thiol, aldehyde, sugar amido and carboxy on the bifunctional polymer linker. In one aspect, the bifunctional polymer linker is CY5-labeled OLIGOdT having an amino group attached at its 3′terminus for covalent attachment to an activated surface on the solid support.


Moreover, when the bifunctional polymer linker also is fluorescently labeled a second fluorescent signal image is detected in the imaging step. Superimposing the first fluorescent signal image and second fluorescent signal image allows identification of the virus by comparing the sequence of the nucleic acid probe at one or more superimposed signal positions on the microarray with a database of signature sequence determinants for a plurality of viral RNA. This embodiment is particularly beneficial since it allows identification of more than one type of virus in a single assay.


The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.


EXAMPLE 1
Microarray Assay for Clade Variant Detection

Provided herein is a method of nucleic acid analysis to detect stable genetic variation in a pathogen which is based on simultaneous analysis of multiple sequence domains in a gene, such as the Spike gene in the RNA genome of CoV-2, to measure clade variation in SARS-CoV-2. For CoV-2, the sequence domains are processed for nucleic acid analysis by converting them into a set of amplicons via a multiplex RT-PCR reaction. In a present preferred implementation, the sequence of those multiplex RT-PCR products is identified relative to that of the underlying CoV-2 Spike gene, by the Horizontal Black Bars in the bottom of Tables 4-8.


The product of that multiplex RT-PCR reaction is analyzed by hybridization to a matrix of synthetic oligonucleotide probes positioned as a microarray test (see the boxes in Tables 4-8). As seen in Tables 4-8, in a preferred implementation of the present invention for CoV-2, there are (15) such Spike Gene Target Regions containing meaningful local sequence variation. (See top Row of Tables 4-8 for their identification).


In terms of detailed test design, the oligonucleotide probes resident at each Target Region of the Spike surface protein are each produced as 3 closely related probe variants, which may be referred to as “Wild Type”, “Mutant” and “Universal”.


Wild Type Probes

In the present invention, a “Wild Type” probe refers to an oligonucleotide probe sequence, generally 14-25 bases long that is specific to the Wuhan progenitor Clade sequence. The pattern of Multiplex RT-PCR amplicon binding to such Wild Type Probes in the present invention are displayed as superscript “2” in Table 4 and as superscript “1” in Table 6.


Mutant Probes

“Mutant” probes correspond to an oligonucleotide probe sequence, also 15-25 bases long specific to the Sequence Change relative to the Wuhan progenitor manifest at the Spike gene location of interest are displayed as superscript “1” in Table 4 and as superscript “1” in Table 5.


Universal Probes

A “Universal” probe refers to an oligonucleotide probe sequence (15-30 bases long) which has been designed to bind to both “Wild Type” and “Mutant” sequences at each site with similar affinity. The patterns of Multiplex RT-PCR amplicon binding to such “Universal” Probes in the present invention are displayed are displayed as superscript “1” in Table 7.









TABLE 4





Combinatorial Analysis of CoV-2 Variants

























Spike Gene Target Region












(Codon) Amino Acid Change
S13I
L18F
T20N
P26S
Δ69-70
D80A
D138Y
Y144DEL
W152C
R190S





Mutation specific Probe coverage
1

1

1
1
1

1


Wuhan reference specific probe/s coverage
2

2

2
2
2

2


Locus specific Probe coverage




N/A






















REGION
LINEAGE DESIGNATION var
































Denmark
Mink V
B.1.1.298
S2
L3
T2
P3
Δ1
D2
D2
Y3
W2
R3


UK
GR/501Y.V1
B.1.1.7
S2
L3
T2
P3
Δ1
D2
D2
Δ3
W2
R3


SA
GH/501Y.V2
B.1.351
S2
L3
T2
P3
HV2
A1
D2
Y3
W2
R3


Brazil/Japan
P.1

S2
F3
N1
S3
HV2
D2
Y1
Y3
W2
S3


Brazil
P.2

S2
L3
T2
P3
HV2
D2
D2
Y3
W2
R3


California
CAL.20C-GH/452R.V1
B.1.429
I1
L3
T2
P3
HV2
D2
D2
Y3
C1
R3


India
(Andhra Pradesh)
N440K
S2
L3
T2
P3
HV2
D2
D2
Y3
W2
R3


WUHAN

WUHAN
S2
L3
T2
P3
HV2
D2
D2
Y3
W2
R3











PCR Amplimer length (bases)
(1) 101
(2) 104
(3) 129

















Spike Gene Target Region









(Codon) Amino Acid Change
D215G
A243del
R246I
K417N/T
N440K
L452R
Y453F





Mutation specific Probe coverage
1


1
1
1


Wuhan reference specific probe/s coverage
2


2
2
2


Locus specific Probe coverage





















REGION
LINEAGE DESIGNATION var


























Denmark
Mink V
B.1.1.298
D2
A3
R3
K2
N2
L2
F3


UK
GR/501Y.V1
B.1.1.7
D2
A3
R3
K2
N2
L2
Y3


SA
GH/501Y.V2
B.1.351
G1
Δ3
I3
N1
N2
L2
Y3


Brazil/Japan
P.1

D2
A3
R3
T1
N2
L2
Y3


Brazil
P.2

D2
A3
R3
K2
N2
L2
Y3


California
CAL.20C-GH/452R.V1
B.1.429
D2
A3
R3
K2
N2
R1
Y3


India
(Andhra Pradesh)
N440K
D2
A3
R3
K2
K1
L2
Y3


WUHAN

WUHAN
D2
A3
R3
K2
N2
L2
Y3









PCR Amplimer length (bases)
(4) 160
(5) 199


















Spike Gene Target Region










(Codon) Amino Acid Change
E484K
N501Y
A570D
D614G
H655Y
P681H
I692V
A701V





Mutation specific Probe coverage
1
1

1

1

1


Wuhan reference specific probe/s coverage
2
2

2

2

2


Locus specific Probe coverage
























REGION
LINEAGE DESIGNATION var




























Denmark
Mink V
B.1.1.29
E2
N2
A3
G1
H3
P2
V
A2


UK
GR/501Y.V1
B.1.1.7
E2
Y1
D3
G1
H3
H1
I
A2


SA
GH/501Y.V2
B.1.351
K1
Y1
A3
G1
H3
P2
I
V1


Brazil/Japan
P.1

K1
Y1
A3
G1
Y3
P2
I
A2


Brazil
P.2

K1
N2
A3
G1
H3
P2
I
A2


California
CAL.20C-GH/452R.V1
B.1.429
E2
N2
A3
G1
H3
P2
I
A2


India
(Andhra Pradesh)
N440K
E2
N2
A3
G1
H3
P2
I
A2


WUHAN

WUHAN
E2
N2
A3
D2
H3
P2
I
A2












PCR Amplimer length (bases)
(6) 151

(7) 88

(8) 135


















Spike Gene Target Region









(Codon) Amino Acid Change
T716I
S982A
T1027I
D1118H
V1176F
M1229I







Mutation specific Probe coverage



Wuhan reference specific probe/s coverage



Locus specific Probe coverage
















REGION
LINEAGE DESIGNATION var



























Denmark
Mink V
B.1.1.29
T3
S3
T3
D3
V3
I3



UK
GR/501Y.V1
B.1.1.7
I3
A3
T3
H3
V3
M3



SA
GH/501Y.V2
B.1.351
T3
S3
T3
D3
V3
M3



Brazil/Japan
P.1

T3
S3
I3
D3
F3
M3



Brazil
P.2

T3
S3
T3
D3
F3
M3



California
CAL.20C-GH/452R.V1
B.1.429
T3
S3
T3
D3
V3
M3



India
(Andhra Pradesh)
N440K
T3
S3
T3
D3
V3
M3



WUHAN

WUHAN
T3
S3
T3
D3
V3
M3















PCR Amplimer length (bases)








1AA mutation - hybridizes to mutation specific probe





2AA identical to hCoV-19/Wuhan/WIV04/2019 (WIV04) - official reference sequence employed by GISAID (EPI_ISL_402124) Hybridizes to reference specific probe





3Potential probe target














TABLE 5





Combinatorial Analysis of CoV-2 Variants - Mutant Probes

























Spike Gene Target Region












(Codon) Amino Acid Change
S13I
L18F
T20N
P26S
Δ69-70
D80A
D138Y
Y144DEL
W152C
R190S





Mutation specific Probe coverage
1

1

1
1
1

1

















REGION
LINEAGE DESIGNATION var































Denmark
Mink V
B.1.1.298
S
L
T
P
Δ1
D
D
Y
W
R


UK
GR/501Y.V1
B.1.1.7
S
L
T
P
Δ1
D
D
Δ
W
R


SA
GH/501Y.V2
B.1.351
S
L
T
P
HV
A
D
Y
W
R


Brazil/Japan
P.1

S
F
N1
S
HV
D
Y1
Y
W
S


Brazil
P.2

S
L
T
P
HV
D
D
Y
W
R


California
CAL.20C-GH/452R.V1
B.1.429
I1
L
T
P
HV
D
D
Y
C1
R


India
(Andhra Pradesh)
N440K
S
L
T
P
HV
D
D
Y
W
R


WUHAN

WUHAN
S
L
T
P
HV
D
D
Y
W
R











PCR Amplimer length (bases)
(1) 101
(2) 104
(3) 129

















Spike Gene Target Region









(Codon) Amino Acid Change
D215G
A243del
R246I
K417N/T
N440K
L452R
Y453F





Mutation specific Probe coverage
1


1
1
1















REGION
LINEAGE DESIGNATION var


























Denmark
Mink V
B.1.1.298
D
A
R
K
N
L
F


UK
GR/501Y.V1
B.1.1.7
D
A
R
K
N
L
Y


SA
GH/501Y.V2
B.1.351
G1
Δ
I
N1
N
L
Y


Brazil/Japan
P.1

D
A
R
T1
N
L
Y


Brazil
P.2

D
A
R
K
N
L
Y


California
CAL.20C-GH/452R.V1
B.1.429
D
A
R
K
N
R1
Y


India
(Andhra Pradesh)
N440K
D
A
R
K
K1
L
Y


WUHAN

WUHAN
D
A
R
K
N
L
Y









PCR Amplimer length (bases)
(4) 160
(5) 199


















Spike Gene Target Region










(Codon) Amino Acid Change
E484K
N501Y
A570D
D614G
H655Y
P681H
I692V
A701V





Mutation specific Probe coverage
1
1

1

1

1
















REGION
LINEAGE DESIGNATION var




























Denmark
Mink V
B.1.1.298
E
N
A
G1
H
P
V
A


UK
GR/501Y.V1
B.1.1.7
E
Y1
D
G1
H
H1
I
A


SA
GH/501Y.V2
B.1.351
K1
Y1
A
G1
H
P
I
V1


Brazil/Japan
P.1

K1
Y1
A
G1
Y
P
I
A


Brazil
P.2

K1
N
A
G1
H
P
I
A


California
CAL.20C-GH/452R.V1
B.1.429
E
N
A
G1
H
P
I
A


India
(Andhra Pradesh)
N440K
E
N
A
G1
H
P
I
A


WUHAN

WUHAN
E
N
A
D
H
P
I
A












PCR Amplimer length (bases)
(6) 151

(7) 88

(8) 135


















Spike Gene Target Region









(Codon) Amino Acid Change
T716I
S982A
T1027I
D1118H
V1176F
M1229I







Mutation specific Probe coverage
















REGION
LINEAGE DESIGNATION var



























Denmark
Mink V
B.1.1.298
T
S
T
D
V
I



UK
GR/501Y.V1
B.1.1.7
I
A
T
H
V
M



SA
GH/501Y.V2
B.1.351
T
S
T
D
V
M



Brazil/Japan
P.1

T
S
I
D
F
M



Brazil
P.2

T
S
T
D
F
M



California
CAL.20C-GH/452R.V1
B.1.429
T
S
T
D
V
M



India
(Andhra Pradesh)
N440K
T
S
T
D
V
M



WUHAN

WUHAN
T
S
T
D
V
M










PCR Amplimer length (bases)








1AA mutation - hybridizes to mutation specific probe














TABLE 6





Combinatorial Analysis of CoV-2 Variants - Wild Type Probes

























Spike Gene Target Region












(Codon) Amino Acid Change
S13I
L18F
T20N
P26S
Δ69-70
D80A
D138Y
Y144DEL
W152C
R190S





Wuhan reference specific probe/S coverage
1

1

1
1
1

1












REGION
LINEAGE DESIGNATION var


























Denmark
Mink V
B. 1.1.298
S1
L
T1
P
Δ
D1
D1
Y
W1
R


UK
GR/501Y.V1
B.1.1.7
S1
L
T1
P
Δ
D1
D1
Δ
W1
R


SA
GH/501Y.V2
B.1.351
S1
L
T1
P
HV
A
D1
Y
W1
R


Brazil/Japan
P.1

S1
F
N
S
HV
D1
Y
Y
W1
S


Brazil
P.2

S1
L
T1
P
HV
D1
D1
Y
W1
R


California
CAL.20C-GH/452R.V1
B.1.429
I
L
T1
P
HV
D1
D1
Y
C
R


India
(Andhra Pradesh)
N440K
S1
L
T1
P
HV
D1
D1
Y
W1
R


WUHAN

WUHAN
S1
L
T1
P
HV
D1
D1
Y
W1
R











PCR Amplimer length (bases)
(1) 101
(2) 104
(3) 129

















Spike Gene Target Region









(Codon) Amino Acid Change
D215G
A243del
R246I
K417N/T
N440K
L452R
Y453F





Wuhan reference specific probe/S coverage
1


1
1
1













REGION
LINEAGE DESIGNATION var
























Denmark
Mink V
B. 1.1.298
D1
A
R
K1
N1
L1
F


UK
GR/501Y.V1
B.1.1.7
D1
A
R
K1
N1
L1
Y


SA
GH/501Y.V2
B.1.351
G
Δ
I
N
N1
L1
Y


Brazil/Japan
P.1

D1
A
R
T
N1
L1
Y


Brazil
P.2

D1
A
R
K1
N1
L1
Y


California
CAL.20C-GH/452R.V1
B.1.429
D1
A
R
K1
N1
R
Y


India
(Andhra Pradesh)
N440K
D1
A
R
K1
K
L1
Y


WUHAN

WUHAN
D1
A
R
K1
N1
L1
Y









PCR Amplimer length (bases)
(4) 160
(5) 199


















Spike Gene Target Region










(Codon) Amino Acid Change
E484K
N501Y
A570D
D614G
H655Y
P681H
1692V
A701V





Wuhan reference specific probe/S coverage
1
1

1

1

1
















REGION
LINEAGE DESIGNATION var




























Denmark
Mink V
B.1.1.298
E1
N1
A
G
H
P1
V
A1


UK
GR/501Y.V1
B.1.1.7
E1
Y
D
G
H
H
I
A1


SA
GH/501Y.V2
B.1.351
K
Y
A
G
H
P1
I
V


Brazil/Japan
P.1

K
Y
A
G
Y
P1
I
A1


Brazil
P.2

K
N1
A
G
H
P1
I
A1


California
CAL.20C-GH/452R.V1
B.1.429
E1
N1
A
G
H
P1
I
A1


India
(Andhra Pradesh)
N440K
E1
N1
A
G
H
P1
I
A1


WUHAN

WUHAN
E1
N1
A
D1
H
P1
I
A1












PCR Amplimer length (bases)
(6) 151

(7) 88

(8) 135


















Spike Gene Target Region









(Codon) Amino Acid Change
T716I
S982A
T1027I
D1118H
V1176F
M1229I







Wuhan reference specific probe/S coverage
















REGION
LINEAGE DESIGNATION var



























Denmark
Mink V
B.1.1.298
T
S
T
D
V
I



UK
GR/501Y.V1
B.1.1.7
I
A
T
H
V
M



SA
GH/501Y.V2
B.1.351
T
S
T
D
V
M



Brazil/Japan
P.1

T
S
I
D
F
M



Brazil
P.2

T
S
T
D
F
M



California
CAL.20C-GH/452R.V1
B.1.429
T
S
T
D
V
M



India
(Andhra Pradesh)
N440K
T
S
T
D
V
M



WUHAN

WUHAN
T
S
T
D
V
M















PCR Amplimer length (bases)








1AA identical to hCoV-19/Wuhan/WIV04/2019 (WIV04) - official reference sequence employed by GISAID (EPI_ISL_402124) Hybridizes to reference specific probe














TABLE 7





Combinatorial Analysis of CoV-2 Variants - Universal Probes

























Spike Gene Target Region












(Codon) Amino Acid Change
S13I
L18F
T20N
P26S
Δ69-70
D80A
D138Y
Y144DEL
W152C
R190S





Locus specific Probe coverage
1



N/A
1
1

1


















REGION
LINEAGE DESIGNATION var
































Denmark
Mink V
B.1.1.298
S1
L
T1
P
Δ
D1
D1
Y
W1
R


UK
GR/501Y.V1
B.1.1.7
S1
L
T1
P
Δ
D1
D1
Δ
W1
R


SA
GH/501Y.V2
B.1.351
S1
L
T1
P
HV
A1
D1
Y
W1
R


Brazil/Japan
P.1

S1
F
N1
S
HV
D1
Y1
Y
W1
S


Brazil
P.2

S1
L
T1
P
HV
D1
D1
Y
W1
R


California
CAL.20C-GH/452R.V1
B.1.429
I1
L
T1
P
HV
D1
D1
Y
C1
R


India
(Andhra Pradesh)
N440K
S1
L
T1
P
HV
D1
D1
Y
W1
R


WUHAN

WUHAN
S1
L
T1
P
HV
D1
D1
Y
W1
R











PCR Amplimer length (bases)
(1) 101
(2) 104
(3) 129

















Spike Gene Target Region









(Codon) Amino Acid Change
D215G
A243del
R246I
K417N/T
N440K
L452R
Y453F





Locus specific Probe coverage
1


1
1
1















REGION
LINEAGE DESIGNATION var


























Denmark
Mink V
B.1.1.298
D1
A
R
K1
N1
L1
F


UK
GR/501Y.V1
B.1.1.7
D1
A
R
K1
N1
L1
Y


SA
GH/501Y.V2
B.1.351
G1
Δ
I
N1
N1
L1
Y


Brazil/Japan
P.1

D1
A
R
T1
N1
L1
Y


Brazil
P.2

D1
A
R
K1
N1
L1
Y


California
CAL.20C-GH/452R.V1
B.1.429
D1
A
R
K1
N1
R1
Y


India
(Andhra Pradesh)
N440K
D1
A
R
K1
K1
L1
Y


WUHAN

WUHAN
D1
A
R
K1
N1
L1
Y









PCR Amplimer length (bases)
(4) 160
(5) 199


















Spike Gene Target Region










(Codon) Amino Acid Change
E484K
N501Y
A570D
D614G
H655Y
P681H
I692V
A701V





Locus specific Probe coverage
























REGION
LINEAGE DESIGNATION var




























Denmark
Mink V
B.1.1.298
E1
N1
A
G1
H
P1
V
A1


UK
GR/501Y.V1
B.1.1.7
E1
Y1
D
G1
H
H1
I
A1


SA
GH/501Y.V2
B.1.351
K1
Y1
A
G1
H
P1
I
V1


Brazil/Japan
P.1

K1
Y1
A
G1
Y
P1
I
A1


Brazil
P.2

K1
N1
A
G1
H
P1
I
A1


California
CAL.20C-GH/452R.V1
B.1.429
E1
N1
A
G1
H
P1
I
A1


India
(Andhra Pradesh)
N440K
E1
N1
A
G1
H
P1
I
A1


WUHAN

WUHAN
E1
N1
A
D1
H
P1
I
A1












PCR Amplimer length (bases)
(6) 151

(7) 88

(8) 135


















Spike Gene Target Region









(Codon) Amino Acid Change
T716I
S982A
T1027I
D1118H
V1176F
M1229I







Locus specific Probe coverage
















REGION
LINEAGE DESIGNATION var



























Denmark
Mink V
B.1.1.298
T
S
T
D
V
I



UK
GR/501Y.V1
B.1.1.7
I
A
T
H
V
M



SA
GH/501Y.V2
B.1.351
T
S
T
D
V
M



Brazil/Japan
P.1

T
S
I
D
F
M



Brazil
P.2

T
S
T
D
F
M



California
CAL.20C-GH/452R.V1
B.1.429
T
S
T
D
V
M



India
(Andhra Pradesh)
N440K
T
S
T
D
V
M



WUHAN

WUHAN
T
S
T
D
V
M















PCR Amplimer length (bases)








1Both Mutant and Wuhan reference sequence virus hybridize to Locus specific probe














TABLE 8





Combinatorial Analysis of CoV-2 Variants





















Spike_S13I
Spike_L18F
Spike_T20N
Spike_P26S
Spike_Δ69-70
















Mutation specific Probe coverage
3

3

3


Wuhan reference specific probe/S coverage
1

1

1


Locus specific Probe coverage
4

4

N/A













REGION
LINEAGE DESIGNATION var






















Denmark
Mink V
B.1.1.298
S2
L4
T2
P4
Δ3


UK
GR/501Y.V1
B.1.1.7
S2
L4
T2
P4
Δ3


SA
GH/501Y.V2
B.1.351
S2
L4
T2
P4
HV


Brazil/Japan
P.1

S2
F5
N3
S5
HV


Brazil
P.2

S
L4
T2
P4
HV


California
CAL.20C-GH/452R.V1
B.1.429
I3
L4
T2
P4
HV


India
(Andhra Pradesh)
N440K
S2
L4
T2
P4
HV


WUHAN

WUHAN
S1
L4
T1
P4

HV1











AMP 1
AMP 2



101 BASE
104 BASE
















Spike_D80A
Spike_D138Y
Spike_Y144DE
Spike_W152C















Mutation specific Probe coverage
3
3

3


Wuhan reference specific probe/S coverage
1
1

1


Locus specific Probe coverage
4
4

4












REGION
LINEAGE DESIGNATION var




















Denmark
Mink V
B.1.1.298
D2
D2
Y4
W2


UK
GR/501Y.V1
B.1.1.7
D2
D2
Δ5
W2


SA
GH/501Y.V2
B.1.351
A3
D2
Y4
W2


Brazil/Japan
P.1

D2
Y3
Y4
W2


Brazil
P.2

D2
D2
Y4
W2


California
CAL.20C-GH/452R.V1
B.1.429
D2
D2
Y4
C3


India
(Andhra Pradesh)
N440K
D2
D2
Y4
W2


WUHAN

WUHAN
D1
D1
Y4
W1










AMP 2
AMP 3



104 BASE
129 BASE
















Spike_R190S
Spike_D215G
Spike_A243del
Spike_R246I















Mutation specific Probe coverage

3




Wuhan reference specific probe/S coverage

1


Locus specific Probe coverage

4












REGION
LINEAGE DESIGNATION var




















Denmark
Mink V
B.1.1.298
R4
D2
A4
R4


UK
GR/501Y.V1
B.1.1.7
R4
D2
A4
R4


SA
GH/501Y.V2
B.1.351
R4
G3
Δ5
I5


Brazil/Japan
P.1

S5
D2
A4
R4


Brazil
P.2

R4
D2
A4
R4


California
CAL.20C-GH/452R.V1
B.1.429
R4
D2
A4
R4


India
(Andhra Pradesh)
N440K
R4
D2
A4
R4


WUHAN

WUHAN
R4
D1
A4
R4









AMP 4



160 BASE
















Spike_K417N
Spike_N440K
Spike_L452R
Spike_Y453F















Mutation specific Probe coverage
3
3
3



Wuhan reference specific probe/S coverage
1
1
1


Locus specific Probe coverage
4
4
4












REGION
LINEAGE DESIGNATION var




















Denmark
Mink V
B.1.1.298
K2
N2
L2
F5


UK
GR/501Y.V1
B.1.1.7
K2
N2
L2
Y4


SA
GH/501Y.V2
B.1.351
N3
N2
L2
Y4


Brazil/Japan
P.1

T3
N2
L2
Y4


Brazil
P.2

K2
N2
L2
Y4


California
CAL.20C-GH/452R.V1
B.1.429
K2
N2
R3
Y4


India
(Andhra Pradesh)
N440K
K2
K3
L2
Y4


WUHAN

WUHAN
K1
N1
L1
Y4









AMP 5



199 BASE

















Spike_E484K
Spike_N501Y
Spike_A570D
Spike_D614G
Spike_H655Y
















Mutation specific Probe coverage
3
3

3



Wuhan reference specific probe/S coverage
1
1

1


Locus specific Probe coverage
4
4

4













REGION
LINEAGE DESIGNATION var






















Denmark
Mink V
B.1.1.298
E2
N2
A4
G3
H4


UK
GR/501Y.V1
B.1.1.7
E2
Y3
D5
G3
H4


SA
GH/501Y.V2
B.1.351
K3
Y3
A4
G3
H4


Brazil/Japan
P.1

K3
Y3
A4
G3
Y5


Brazil
P.2

K3
N2
A4
G3
H4


California
CAL.20C-GH/452R.V1
B.1.429
E2
N2
A4
G3
H4


India
(Andhra Pradesh)
N440K
E2
N2
A4
G3
H4


WUHAN

WUHAN
E1
N1
A4
D1
H4












AMP 6

AMP 7




151 BASE

88 BASE

















Spike_P681H
Spike_I692V
Spike_A701V
Spike_T716I
Spike_S982A
















Mutation specific Probe coverage
3

3




Wuhan reference specific probe/S coverage
1

1


Locus specific Probe coverage
4

4













REGION
LINEAGE DESIGNATION var






















Denmark
Mink V
B.1.1.298
P2
V5
A2
T4
S4


UK
GR/501Y.V1
B.1.1.7
H3
I4
A2
I5
A5


SA
GH/501Y.V2
B.1.351
P2
I4
V3
T4
S4


Brazil/Japan
P.1

P2
I4
A2
T4
S4


Brazil
P.2

P2
I4
A2
T4
S4


California
CAL.20C-GH/452R.V1
B.1.429
P2
I4
A2
T4
S4


India
(Andhra Pradesh)
N440K
P2
I4
A2
T4
S4


WUHAN

WUHAN
P1
I4
A1
T4
S4











AMP 8





135 BASE
















Spike_T1027I
Spike_D1118H
Spike_V1176F
Spike_M1229I

















Mutation specific Probe coverage







Wuhan reference specific probe/S coverage



Locus specific Probe coverage














REGION
LINEAGE DESIGNATION var























Denmark
Mink V
B.1.1.298
T4
D4
V4
I5



UK
GR/501Y.V1
B.1.1.7
T4
H5
V4
M4



SA
GH/501Y.V2
B.1.351
T4
D4
V4
M4



Brazil/Japan
P.1

I5
D4
F5
M4



Brazil
P.2

T4
D4
F5
M4



California
CAL.20C-GH/452R.V1
B.1.429
T4
D4
V4
M4



India
(Andhra Pradesh)
N440K
T4
D4
V4
M4



WUHAN

WUHAN
T4
D4
V4
M4








1AA of hCoV-19/Wuhan/WIV04/2019 (WIV04) - official reference sequence employed by GISAID (EPI_ISL_402124)





2AA Identical to (WIV04) - hybridizes to Wuhan reference probe





3AA mutation - hybridizes to mutation specific probe





4Potential probe target identical to (WIV04)





5Potential AA mutation probe target







EXAMPLE 2
Biological Rationale for the Design of the Present Invention

The oligonucleotide probes of the microarray and the PCR primers to generate RT-PCR amplicons were developed to accommodate a specific CoV-2 Clade Variant set of international interest in 2021, as specified in the Left-most Column in Tables 4-8. But, as can already be seen among the Clade Variant strain these tables, the pattern of local sequence change manifest in each Clade Variant comprises a unique combination derived from a larger set of specific local sequence variation chosen at discrete sites in the Spike gene.


For the Spike protein of the CoV-2 virus and for pathogens more universally, spontaneous local mutation in a surface protein such as Spike is likely to inactivate the protein, thus disabling the pathogen. As such, most spontaneous mutations in surface proteins do not propagate and hence go undetected.


On occasion, however, such random mutation produces a surface protein change that confers a selective advantage to a pathogen, such as enhanced infectivity, better resistance to vaccination or drug therapy and thus the mutation propagates in an infection and ultimately be detected at population scale. Such positively selected local mutational changes are generally rare and thus localized to a relatively small number of discrete segments within a pathogen surface protein such as Spike, often localized to specific sites where the protein contacts host cells, or sites which present peptides for interaction with a protective host antibody or sites where a drug might bind. In many cases such altered surface protein features may function in an additive way (enhanced cell binding+diminished neutralizing antibody binding may be selected for, concurrently) to produce a Clade Variant presenting a combination derived from the set of available local sequence changes that confer functional superiority to the pathogen.


The present invention takes advantage of the fundamental matrix-like character of such selectable (discrete, local) surface protein changes and the ability of a matrix of hybridization probes (as in a microarray) to query many sites of local surface protein sequence change simultaneously (Tables 4-7). As such, this oligonucleotide probe set can interrogate (at the nucleic acid level) many possible combinations of such surface protein change as a single combinatorial test.


Based on the core design test design embodied in Tables 4-7, new, as-yet unknown, functionally relevant local sequence change can be added, once known, as new probes to the microarray (cells with superscript “3” in Table 4). It is expected that many other CoV-2 Clades could be detected and discriminated, in a similar combinatorial fashion, via such relatively minor expansion of the core invention depicted in Table 4.


EXAMPLE 3
Test Manufacturing Considerations

The present implementation of such an oligonucleotide probe panel for analysis of the CoV-2 Spike gene is based upon detection of 15 positively selected local mutational changes in the Spike gene, i.e. Tables 4-7, each with 3 probe sequence variants at each site, “Mutant”, “Wild Type”, “Universal”, thus generating a set of 15×3=45 oligonucleotide probes to be used for the purpose of combinatorial Clade Variant Analysis.


In the present implementation, if that set of 45 probes is manufactured in triplicate, a 3×45=135 probe microarray is thus generated, which when printed along with positive and negative controls appropriate for CoV-2 testing (such as RNAse P) the present Clade Chip Assay consumes the full microarray content capacity presented by the standard 150 probe, 96-Well format described in applications U.S. Ser. No. 16/950,171 and U.S. Ser. No. 16/950,210, both hereby incorporated by reference in their entireties.


It is useful to note that the information content of such a 150-probe microarray becomes resident in a single well of the 96-well microarray format and thus generates information content similar to that of re-sequencing of the entire gene and content that is equivalent to that obtained from 150 q-RT-PCR assays performed in parallel. As seen below, a first preferred implementation of such a Clade Chip prototype has been fabricated via standard mass production methods described in the above-referenced patent applications.


Performance

In a first preferred implementation, the sample preparation methods of the Clade Chip are optimized for both NP-Swab and Saliva collection and designed to detect CoV-2 at 5 virus/RT-PCR reaction sensitivity (500 cp/ml) and resolve multiple CoV-2 Clade variants of present international concern (Denmark, UK, S Africa, Brazil/Japan, India, CA L452R, Wuhan), as depicted in Tables 4-7.


So long as any new CoV-2 Clade may be detected and discriminated via its pattern of Spike gene gRNA sequence change, that additional Clade sequence content (cells with superscript “3” in Table 4) can be designed and added to the manufacture of the present invention in less than 2-weeks, as the need for new or broader-range CoV-2 Clade Variant detection emerges.


The Clade-Chip Assay in the present preferred implementation is based on a standard 96-well plate microarray processing workflow already described in applications having U.S. Ser. No. 16/950,171 and U.S. Ser. No. 16/950,210 both hereby incorporated by reference in their entireties and is deployed in that standard 96-well format as a manual or automated test. However, the matrix of oligonucleotide probes of the present invention to detect CoV-2 Clade Variants via Combinatorial Analysis could, in principle, be implemented by other methods of microarray manufacture or via alternative methods of bead-based solution phase nucleic acid hybridization. Additionally, the same principles of Combinatorial Analysis could be used to develop analogous tests for clade variation in other viruses, bacterial and fungi in the microarray or other hybridization formats.


EXAMPLE 4
Clade Array Manufacturing Quality and Functional Characterization

The Clade Chip Test Design summary is shown in Table 9 and is suited for combinatorial analysis among multiple Spike Targets. The following are its features;

    • 1. Core Content, Completed (11) Target Sites, >3 Probes Each (Universal, Mutant, Wild Type)=11×3×3=99 probe spots
    • 2. Additional (Future Clade) Content Array Real-estate,
      • a. Up to 8 additional Target sites can be added to current Clade Array
      • b. 9×3×3=81 additional probe spots.


Clade Variant Content as Printed

A Clade Chip Probe layout was set up in duplicate. The probe content included three (3) probes for each Spike target site (Universal, Mutant, Wild Type). Validation testing was used to pick the best” of the two closely related “redundant” lead designs for each of the three probes. In addition to the core set of 11 spike targets, new probe designs were included to expand the content of the assay. The full set of redundant probe content was printed in duplicate as a 12 x 16 probe array in a 96-well format (Table 10). The forward (odd numbers) and reverse (even numbers) primer sequences for each amplimer employed in this assay are shown in Table 11.









TABLE 9





Validation of test design for the five prevalent Clade variants























Spike Gene Target Region










(Codon) Amino Acid Change
S13I
L18F
T20N
P26S
Δ69-70
D80A
D138Y
Y144D





Mutation specific Probe coverage




1
1
1


Wuhan reference specific probe/S coverage




2
2
2


Locus specific Probe coverage




N/A


















Region
Lineage Designation Var




























Denmark
Mink V
B.1.1.298
S3
L3
T3
P3
Δ1
D2
D2
Y3


UK
GR/501Y.V1
B.1.1.7
S3
L3
T3
P3
Δ1
D2
D2
Δ3


SA
GH/501Y.V2
B.1.351
S3
L3
T3
P3
HV2
A1
D2
Y3


Brazil/Japan
P.1

S3
F3
N3
S3
HV2
D2
Y1
Y3


Brazil
P.2

S3
L3
T3
P3
HV2
D2
D2
Y3


California
CAL.20C-GH/452R.V1
B.1.429
I3
L3
T3
P3
HV2
D2
D2
Y3


India
(Andhra Pradesh)
N440K
S3
L3
T3
P3
HV2
D2
D2
Y3


S. US
20G/B.1.2
Q677P/H
S3
L3
T3
P3
HV2
D2
D2
Y3


WUHAN

WUHAN
S3
L3
T3
P3
HV2
D2
D2
Y3









4
5
4











(1) 101
(2)
(3)129



















Spike Gene Target Region










(Codon) Amino Acid Change
W152C
R190S
D215G
A243del
R246I
K417N
N440K
L452R





Mutation specific Probe coverage
1





1
1


Wuhan reference specific probe/S coverage
2





2
2


Locus specific Probe coverage
























Region
Lineage Designation Var




























Denmark
Mink V
B.1.1.298
W2
R3
D3
A3
R3
K3
N2
L2


UK
GR/501Y.V1
B.1.1.7
W2
R3
D3
A3
R3
K3
N2
L2


SA
GH/501Y.V2
B.1.351
W2
R3
G3
Δ3
I3
N3
N2
L2


Brazil/Japan
P.1

W2
S3
D3
A3
R3
T3
N2
L2


Brazil
P.2

W2
R3
D3
A3
R3
K3
N2
L2


California
CAL.20C-GH/452R.V1
B.1.429
C1
R3
D3
A3
R3
K3
N2
R1


India
(Andhra Pradesh)
N440K
W2
R3
D3
A3
R3
K3
K1
L2


S. US
20G/B.1.2
Q677P/H
W2
R3
D3
A3
R3
K3
N2
L2


WUHAN

WUHAN
W2
R3
D3
A3
R3
K3
N2
L2





4





4
4












(3)129

(4) 160
(5) 199



















Spike Gene Target Region










(Codon) Amino Acid Change
Y453F
E484K
N501Y
A570D
D614G
H655Y
Q677P/H
P681H





Mutation specific Probe coverage

1
1

1


1


Wuhan reference specific probe/S coverage

2
2

2


2


Locus specific Probe coverage
























Region
Lineage Designation Var




























Denmark
Mink V
B.1.1.298
F3
E2
N2
A3
G1
H3
Q3
P2


UK
GR/501Y.V1
B.1.1.7
Y3
E2
Y1
D3
G1
H3
Q3
H1


SA
GH/501Y.V2
B.1.351
Y3
K1
Y1
A3
G1
H3
Q3
P2


Brazil/Japan
P.1

Y3
K1
Y1
A3
G1
Y3
Q3
P2


Brazil
P.2

Y3
K1
N2
A3
G1
H3
Q3
P2


California
CAL.20C-GH/452R.V1
B.1.429
Y3
E2
N2
A3
G1
H3
Q3
P2


India
(Andhra Pradesh)
N440K
Y3
E2
N2
A3
G1
H3
Q3
P2


S. US
20G/B.1.2
Q677P/H
Y3
E2
N2
A3
G1
H3
P/H
P2


WUHAN

WUHAN
Y3
E2
N2
A3
D2
H3
Q3
P2






5
4

4


4














(5) 199
(6) 151

(7) 88

(8) 135



















Spike Gene Target Region










(Codon) Amino Acid Change
I692V
A701V
T716I
S982A
T1027I
D1118
V1176
M1229I





Mutation specific Probe coverage

1


Wuhan reference specific probe/S coverage

2


Locus specific Probe coverage


















Region
Lineage Designation Var




























Denmark
Mink V
B.1.1.298
V3
A2
T3
S3
T3
D3
V3
I3


UK
GR/501Y.V1
B.1.1.7
I3
A2
I3
A3
T3
H3
V3
M3


SA
GH/501Y.V2
B.1.351
I3
V1
T3
S3
T3
D3
V3
M3


Brazil/Japan
P.1

I3
A2
T3
S3
I3
D3
F3
M3


Brazil
P.2

I3
A2
T3
S3
T3
D3
F3
M3


California
CAL.20C-GH/452R.V1
B.1.429
I3
A2
T3
S3
T3
D3
V3
M3


India
(Andhra Pradesh)
N440K
I3
A2
T3
S3
T3
D3
V3
M3


S. US
20G/B.1.2
Q677P/H
I3
A2
T3
S3
T3
D3
V3
M3


WUHAN

WUHAN
I3
A2
T3
S3
T3
D3
V3
M3






4















(8) 135








1AA mutation - hybridizes to mutation specific probe





2AA identical to hCoV-19/Wuhan/WIV04/2019 (WIV04) - official reference sequence employed by GISAID (EPI_ISL_402124). Hybridizes to reference specific probe





3Potential probe target





4No Probe Adjustment Necessary





5Minor Probe Adjustment Necessary














TABLE 10







Clade chip probe layout















Amplimer




Row
Col.
SEQ ID NOS.
#
Target
Probe Sequence (5′ to 3′)





1
 1
SEQ ID NO: 30
1
AA S13I
TTTTTCTAGTCTCTAKTCAGTGTGTTTTTT





1
 2
SEQ ID NO: 64
1
AA S13_ (1)
TTTTTTTGTCTCTAGTCAGTGTTTTTTTTT





1
 3
SEQ ID NO: 65
1
AA S13_ (2)
TTTTTTTAGTCTCTAGTCAGTGTTTTTTTT





1
 4
SEQ ID NO: 66
1
AA_13I (1)
TTTTTTAGTCTCTATTCAGTGTTTTTTTTT





1
 5
SEQ ID NO: 67
1
AA_13I (2)
TTTTTTTAGTCTCTATTCAGTGTTTTTTTT





1
 6
SEQ ID NO: 68
1
AA T20N
TTTTTTAATYTTACAAMCAGAACTCTTTTT





1
 7
SEQ ID NO: 69
1
AA T20_ (1)
TTTTTTTATCTTACAACCAGAACCTTTTTT





1
 8
SEQ ID NO: 70
1
AA T20_ (2)
TTTTTTTATCTTACAACCAGAACTTTTTTT





1
 9
SEQ ID NO: 71
1
AA_20N (1)
TTTTTTATTTTACAAACAGAACTTTTTTTT





1
10
SEQ ID NO: 72
1
AA_20N (2)
TTTTTCAATTTTACAAACAGAACTTTTTTT





1
11
SEQ ID NO: 68

RNAse P control
TTTTTTTTCTGACCTGAAGGCTCTGCGCGTTTTT





1
12
SEQ ID NO: 73
2
AA69-70 HV (1)
TTTTTTATGCTATACATGTCTCTGTTTTTT





2
 1
SEQ ID NO: 31
2
AA69-70 HV (2)
TTTTTCCCATGCTATACATGTCTCTGTTTTTT





2
 2
SEQ ID NO: 74
2
AA69-70 DEL (1)
TTTTTTACCATGCTATCTCTGGGATTTTTT





2
 3
SEQ ID NO: 32
2
AA69-70 DEL (2)
TTTTTTTTTCCATGCTATCTCTGGGATTTTTT





2
 4
SEQ ID NO: 33
2
AA D80A
TTTTTCAGAGGTTTGMTAACCCTGTCTTTTTT





2
 5
SEQ ID NO: 34
2
AA D80_ (1)
TTTTTTTGGTTTGATAACCCTGCTTTTTTT





2
 6
SEQ ID NO: 75
2
AA D80_ (2)
TTTTTCTAGGTTTGATAACCCTGCTTTTTT





2
 7
SEQ ID NO: 76
2
AA_80A (1)
TTTTTTTAGGTTTGCTAACCCTCTTTTTTT





2
 8
SEQ ID NO: 35
2
AA_80A (2)
TTTTTTTGGTTTGCTAACCCTGCTTTTTTT





2
 9
SEQ ID NO: 36
3
AA D138Y
TTTTATTTTGTAATKATCCATTTTTGTTTT





2
10
SEQ ID NO: 37
3
AA D138_ (1)
TTTTTCTTTGTAATGATCCATTTTCTTTTT





2
11
SEQ ID NO: 77
3
AA D138_ (2)
TTTTTTCTTTGTAATGATCCATTTCTTTTT





2
12
SEQ ID NO: 38
3
AA_138Y (1)
TTTTTTTTTGTAATTATCCATTTTCTTTTT





3
 1
SEQ ID NO: 78
3
AA_138Y (2)
TTTTTCTTTGTAATTATCCATTTTCTTTTT





3
 2
SEQ ID NO: 39
3
AA W152C
TTTTTAGTTGKATGGAAAGTGAGTTCTTTT





3
 3
SEQ ID NO: 40
3
AA W152_ (1)
TTTCTCTAAAAGTTGGATGGAAACTCTTCT





3
 4
SEQ ID NO: 79
3
AA W152_ (2)
TTTTCTTCAAAGTTGGATGGAAACTCTTTT





3
 5
SEQ ID NO: 41
3
AA_152C (1)
TTTCTTCAAAGTTGTATGGAAAGCCTTCTT





3
 6
SEQ ID NO: 80
3
AA_152C (2)
TTTCTCTAAAAGTTGTATGGAAACTCTTCT





3
 7
SEQ ID NO: 81
4
AA D215G
TTTTTTAGTGCGTGRTCTCCCTCATTTTTT





3
 8
SEQ ID NO: 82
4
AA D215_ (1)
TTTTTTCTGCGTGATCTCCCTCATTTTTTT





3
 9
SEQ ID NO: 83
4
AA D215_ (2)
TTTTTTTCTGCGTGATCTCCCTCTTTTTTT





3
10
SEQ ID NO: 84
4
AA_215G (1)
TTTTTTTTGCGTGGTCTCCCTCTTTTTTTT





3
11
SEQ ID NO: 85
4
AA_215G (2)
TTTTTTTTTGCGTGGTCTCCCTTTTTTTTT





3
12
SEQ ID NO: 86
5
AA K417N
TTTTAACTGGAAAKATTGCTGATTATTTTT





4
 1
SEQ ID NO: 87
5
AA K417_ (1)
TTTCTTCTCTGGAAAGATTGCTGCTTTTTT





4
 2
SEQ ID NO: 88
5
AA K417_ (2)
TTCTTCTCTGGAAAGATTGCTGACTTTTTT





4
 3
SEQ ID NO: 89
5
AA_417N (1)
TTTTTCTCTGGAAATATTGCTGACTTTTTT





4
 4
SEQ ID NO: 90
5
AA_417N (2)
TTTTCTCTGGAAATATTGCTGATCTTTTTT





4
 5
SEQ ID NO: 91
5
AA_417T (1)
TTTTTTTACTGGAACGATTGCTTTTTTTTT





4
 6
SEQ ID NO: 92
5
AA_417T (2)
TTTTTTCCTGGAACGATTGCTGTTTTTTTT





4
 7
SEQ ID NO: 42
5
AA N439K + N440K
TTTTTAATTCTAAMAAKCTTGATTCTAATTTT





4
 8
SEQ ID NO: 93
5
AA N439_ + N440_ (1)
TTTTTTATTCTAACAATCTTGATTTCTTTT





4
 9
SEQ ID NO: 43
5
AA N439_ + N440_ (2)
TTTTTAATTCTAACAATCTTGATTTCTTTT





4
10
SEQ ID NO: 94
5
AA N439_ + _440K (1)
TTTTTTTTTCTAACAAGCTTGATTTTTTTT





4
11
SEQ ID NO: 44
5
AA N439_ + _440K (2)
TTTTTTATTCTAACAAGCTTGATTTTTTTT





4
12
SEQ ID NO: 45
5
AA_439K + N440_ (1)
TTTTCTATTCTAAAAATCTTGATTTCTTTT





5
 1
SEQ ID NO: 95
5
AA_439K + N440_ (2)
TTCTTAATTCTAAAAATCTTGATTTCTTTT





5
 2
SEQ ID NO: 46
5
AA L452R
TTTCTATAATTACCTGTATAGATTGTCTTT





5
 3
SEQ ID NO: 96
5
AA L452_ (1)
TTTTTCATAATTACCTGTATAGACTTTCTT





5
 4
SEQ ID NO: 47
5
AA L452_ (2)
TTTTTTTAATTACCTGTATAGATTTCTTTT





5
 5
SEQ ID NO: 48
5
AA_452R (1)
TTTTTCATAATTACTGGTATAGATCTTTTT





5
 6
SEQ ID NO: 97
5
AA_452R (2)
TTTTTTCAATTACCGGTATAGATCTTTTTT





5
 7
SEQ ID NO: 49
6
AA S477_
TTTTTTCGCCGGTAGCACACCTCTTTTTTT





5
 8
SEQ ID NO: 98
6
AA_478I (1)
TTTTTTTTGGTAGCATACCTTGTTTTTTTT





5
 9
SEQ ID NO: 99
6
AA_478I (2)
TTTTTTTCGGTAGCATACCTTGTTTTTTT





5
10
SEQ ID NO: 50
6
AA_477N (1)
TTTTCTTCCGGTAACACACCTTTTTTTTTT





5
11
SEQ ID NO: 100
6
AA_477N (2)
TTTTTTCGCCGGTAACACACCTCTTTTTTT





5
12
SEQ ID NO: 101
6
AA_476S (1)
TTTTTTTTCAGGCCAGTAGCACTTTTTTTT





6
1
SEQ ID NO: 51
6
AA V483A + E484K
TTTTTTAATGGTGTTRAAGGTTTTAATTTTTT





6
2
SEQ ID NO: 52
6
AA V483_ + E484_ (1)
TTTTTTCTGGTGTTGAAGGTTTTACTTTTT





6
3
SEQ ID NO: 102
6
AA V483_ + E484_ (2)
TTTTTCTGGTGTTGAAGGTTTTATCTTTTT





6
4
SEQ ID NO: 103
6
AA V483_ + _484K (1)
TTTTTTCTGGTGTTAAAGGTTTTACTTTTT





6
5
SEQ ID NO: 53
6
AA V483_ + _484K (2)
TTTTTTTATGGTGTTAAAGGTTTTCTTTTT





6
6
SEQ ID NO: 54
6
AA_483A + E484_ (1)
TTTTTTTATGGTGCTGAAGGTTCTTTTTTT





6
7
SEQ ID NO: 104
6
AA_483A + E484_ (2)
TTTTTTCAATGGTGCTGAAGGTTCTTTTTT





6
8
SEQ ID NO: 55
6
AA N501Y
TTTTTTTTCCAACCCACTWATGGTGTTTTTTTT





6
9
SEQ ID NO: 56
6
AA N501_ (1)
TTTTTTTTACCCACTAATGGTGTCTTTTTT





6
10
SEQ ID NO: 105
6
AA N501_ (2)
TTTTTTTAACCCACTAATGGTGTCTTTTTT





6
11
SEQ ID NO: 57
6
AA_501Y (1)
TTTTTTTTACCCACTTATGGTGTCTTTTTT





6
12
SEQ ID NO: 106
6
AA_501Y (2)
TTTTTTTAACCCACTTATGGTGTCTTTTTT





7
1
SEQ ID NO: 107
7
AA D614G
TTTTTCTCTTTATCARGRTGTTAACTGCTTTTTT





7
2
SEQ ID NO: 108
7
AA D614_
TTTTTCTTATCAGGATGTTAACTTTTTTTT





7
3
SEQ ID NO: 109
7
AA_614G + 613 (CAG)
TTTTTTCCTATCAGGGTGTTAACTTTTTTT





7
4
SEQ ID NO: 110
7
AA_614G + 613 (CAA)
TTTTTTCCTATCAAGGTGTTAACTTTTTTT





7
5
SEQ ID NO: 111
7
AA_614G
TTTTTTCCTATCARGGTGTTAACTTTTTTT





7
6
SEQ ID NO: 58
8
AA P681H
TTTTTTCAGACTAATTCTCMTCGGCTTTTT





7
7
SEQ ID NO: 112
8
AA P681_ (1)
TTTTTTTTAATTCTCCTCGGCGTTTTTTTT





7
8
SEQ ID NO: 59
8
AA P681_ (2)
TTTTTTTCTAATTCTCCTCGGCGTTTTTTT





7
9
SEQ ID NO: 60
8
AA_681H (1)
TTTTTTTTTAATTCTCATCGGCGTTTTTTT





7
10
SEQ ID NO: 113
8
AA_681H (2)
TTTTTTTCTAATTCTCATCGGCGTTTTTTT





7
11
SEQ ID NO: 61
8
AA A701V
TTTTCACTTGGTGYAGAAAATTCAGTTTTT





7
12
SEQ ID NO: 62
8
AA A701_ (1)
TCTTCTTCTTGGTGCAGAAAATTATTCTTT





8
1
SEQ ID NO: 114
8
AA A701_ (2)
TTCTTCTACTTGGTGCAGAAAATTATTCTT





8
2
SEQ ID NO: 63
8
AA_701V (1)
TCTTCTTCTTGGTGTAGAAAATTATTCTTT





8
3
SEQ ID NO: 115
8
AA_701V (2)
TTTCTTTCTTGGTGTAGAAAATTCTTTTTT





8
4
SEQ ID NO: 116

N2
TTTTTTACAATTTGCCCCCAGCGTCTTTTT





8
5
SEQ ID NO: 117

SARS-2003 N2
TTTTTTTTTGCTCCRAGTGCCTCTTTTTTT





8
6
SEQ ID NO: 70

Negative Control
TTTTTTCTACTACCTATGCTGATTCACTCTTTTT





8
7
EMPTY








8
8
EMPTY








8
9
EMPTY








8
10
EMPTY








8
11
EMPTY








8
12
EMPTY
















TABLE 11







Amplimer primer sequences












Amplimer


Primer Sequence 


SEQ ID NOS.
#
Target
Gene
(5′ to 3′)





SEQ ID NO: 25
2
AA66-85
Spike
TTCTTTTCCAATGTTACTTGGTT






CCATG





SEQ ID NO: 26
2
AA66-85
Spike
Cy3-TTTCAAAATAAACACCATC






ATTAAATGG





SEQ ID NO: 11
3
AA126-157
Spike
TTTCTTATTGTTAATAACGCTAC






TAATG





SEQ ID NO: 12
3
AA126-157
Spike
Cy3-TTTCATTCGCACTAGAATA






AACTCTGAA





SEQ ID NO: 27
5
AA413-458
Spike
TTTGATGAAGTCAGACAAATCG






CTCCAG





SEQ ID NO: 28
5
AA413-458
Spike
Cy3-TTTCTCTCAAAAGGTTTGA






GATTAGACT





SEQ ID NO: 15
6
AA475-506
Spike
TTTTATTTCAACTGAAATYTATCA






GGCC





SEQ ID NO: 16
6
AA475-506
Spike
Cy3-TTTAAAGTACTACTACTCT






GTATGGTTG





SEQ ID NO: 29
7
AA610-618
Spike
TTTCAAATACTTCTAACCAGGTT






GCTGT





SEQ ID NO: 24
7
AA610-618
Spike
Cy3-TTTTGCATGAATAGCAACA






GGGACTTCT





SEQ ID NO: 17
8
AA677-707
Spike
TTTTATATGCGCTAGTTATCAGA






CTCAG





SEQ ID NO: 18
8
AA677-707
Spike
Cy3-TTTTGGTATGGCAATAGAG






TTATTAGAG









EXAMPLE 5
Clade Array Functional Characterization
Experiment 1

Samples Used for Testing. Analysis was performed with a highly characterized, purified Wuhan gRNA standard (Quantitative Standard obtained from ATCC-BEI) or with synthetic “mutant” targets designed by PDx, obtained by SGI fabrication (IDT).


RT-PCR Conditions. RT-PCR was performed using the [UNG+One-Step RT-PCR] protocol. As is customary in optimization of multiplex RT-PCR, the data presented comprise the use of Single PCR primer pairs as a single reaction. Based on these data multiplex RT-PCR conditions are optimized.


Clade Array Hybridization & Imaging. Conditions of Hybridization, Washing and Imaging were exactly as described. Following the completion of the multiplex RT-PCR, the DNA microarray was prepared for hybridization with brief water washes, and an incubation in prehybridization buffer (0.6M NaCl, 0.06M sodium citrate solution, 0.1% Ficoll, 0.1% Polyvinylpyrrolidone 0.1% Bovine Serum Albumin). Following aspiration of the prehybridization buffer, a mixture of amplicon and hybridization buffer (0.6M NaCl, 0.06M sodium citrate solution, 0.1% Ficoll, 0.1% Polyvinylpyrrolidone 0.1% Bovine Serum Albumin) was added to the DNA microarray and allowed to incubate for 2 hours. The microarray was prepared for imaging with one quick wash of wash buffer (22.5 mM NaCl, 2.25mM sodium citrate solution) and a 10-minute incubation (22.5 mM NaCl, 2.25 mM sodium citrate solution). The microarray plate was then spun dry for 5 minutes at 2200 rpm. The underside of the plate was wiped clean with 70% ethanol and lens tissue until all dust particles were removed. The plate was scanned on the Sensospot utilizing Sensovation software. Cy5 exposure time was set at 312 ms, and the Cy3 exposure times at 115 ms and 578 ms. Upon image scanning completion, the folder containing all of the scanned data was saved to a thumb drive and uploaded to Dropbox for Augury Analysis.


Data Analysis. Data for all (11) Core Spike Target Sites are presented in FIGS. 1-11. For all Spike Target sites, data is presented as a bar graph, where the ratio of hybridization signal strength for Wild Type vs Mutant Probes define the specificity of analysis.

    • a) Left Side of each bar graph shows hybridization data derived from analysis of the “Mutant” synthetic CoV-2 target sequence appropriate for that site.
    • b) Right Side of each bar graph displays the corresponding data obtained for the “Wild Type” Wuhan gRNA reference sequence.
    • c) In the analysis of each bar graph at each target site, the parameter of analytical importance is the hybridization signal strength (RFU) ratio obtained by comparison of RT-PCR amplimer hybridization to the Mutant-Specific vs Wild-Type specific probe which can be presented as [RFUwt/RFUmt].
    • i. Criterion #1. Primary Data. The [RFUwt/RFUmt] ratio should differ significantly (>5×) upon comparison of “Wild Type” CoV2 genome hybridization at that site, vs “Mutant” Template hybridization measured at that same site.
    • ii. Criterion #2. Optimal but not necessarily. The [RFUwt/RFUmt] ratio should change qualitatively upon CoV2 hybridization analysis of a “Wild Type” CoV2 genome at that site, vs a “Mutant” Template measured at that site. i.e.
      • Wild-Type Template 4→[RFUwt/RFUmt]>1
      • Mutant Template 4→[RFUwt/RFUmt]<1


Clade Array Results



  • a) The Data, Presented in FIGS. 1-11 demonstrate that all (11) Spike Target sites show generally excellent hybridization-based discrimination between Wild Type vs Mutant Sequence Variants by the primary Criterion #1 listed above.

  • b) For (9) of the 11, Both Criterion #1 and Criterion #2 have been met. All such sites are thus marked among FIGS. 1-11 as “No Additional Probe Optimization Required” and are marked by a “*” below their location in the probe matrix of Table 9.

  • c) For two of the 11 Spike Target sites (D80A and E484K, FIGS. 2 and 7), Criterion #2 was not met adequately.

  • d) Only two Probes need Optimization. In two cases (Target Sites D80A and E484K) although the Wild Type (Wuhan gRNA) is easily distinguished from the Mutant based on significant differences in relative hybridization (Criterion #1, above), the Mutant/Wild Type distinction does not change sign (Criterion #2). Thus, although the data obtained at those (2) sites (D80A, E484K) is adequate at present to make accurate “Clade Calls”, modest Optimization (1 Probe at each site) can be deployed quickly to enhance the quality of the data obtained there.

  • e) Experience and general understanding of nucleic acid biophysics suggest that the needed optimization (Target Sites D80A and E484K) was obtainable by simply reducing the length of one probe at each site by 2 bases. As soon as the need for such.



SUMMARY

The Clade Array Probe Content was found to be fully functional. An optimized shorter probe was seen to improve Cov-2 mutant analysis at target sites D80A and E484K.


Experiment 2

A second 15 Plate Manufacturing Run (#2) of DETECTX-Cv, similar to the one described in Experiment 1 above was implemented to complete validation of the Multiplex assay. In this assessment, print quality passed the test for all 96 (160 probe) arrays among all 15 plates.


The second set of validation tests sought to evaluate the preferred method of multiplexing of the RT-PCR reaction, using the UNG combined with One Step RT-PCR condition. The primary goal was to deliver a first RT-PCR Multiplex capable of distinguishing the five prevalent Clade Variants—UK (B.1.1.7), S Africa (B.1.351) Brazil (P.1) Brazil (P.2) US California (B.1.429) shown in Table 12. The validation materials comprised a purified Wuhan gRNA reference (ATCC-BEI). The data obtained subsequent to RT-PCR, hybridization and washing revealed that an initial deployment of a specific 4-plex RT-PCR reaction, comprising amplimers [2, 3, 6, 8] was sufficient to distinguish, as a single multiplex assay, these five prevalent Clade variants (FIGS. 12A-12B).



FIG. 12A shows the raw microarray hybridization data for the eight (8) target sites covered by amplimer sets 2, 3, 6 and 8 with data normalized to the Universal Probe at “100%” to emphasize the sensitivity of Universal vs Wild-Type/Mutant Target Detection. FIG. 12B shows the same raw hybridization data for the eight (8) target sites covered by the amplimer sets 2, 3, 6 and 8, but normalized to the Wild-Type probe signal, to emphasize the specificity of discrimination between Wild-Type vs Mutant target sequence.


Experiment 3
Fully Multiplexed DETECTX-Cv

A third round of validation was performed to evaluate the preferred method of multiplexing of the RT-PCR reaction, using the UNG combined with One Step RT-PCR condition. The primary goal was to deliver a second (N=5) RT-PCR Multiplex capable of distinguishing the six (6) prevalent US Clade Variants—UK (B.1.1.7), S Africa (B.1.351) Brazil (P.1) Brazil (P.2) a second redundant target in US California (B.1.429) and India N440K. shown in Table 12. The validation materials comprised a purified Wuhan gRNA reference (ATCC-BEI). The data obtained subsequent to RT-PCR, hybridization and washing revealed that a second deployment of a specific 5-plex RT-PCR reaction, comprising a N=5 multiplex of amplimers [2, 3, 5, 6, 8] was sufficient to distinguish, as a single multiplex assay, these six prevalent Clade Variants (FIGS. 13A-13B).



FIG. 13A shows the raw microarray hybridization data for the eleven (11) target sites covered by amplimer sets 2, 3, 5, 6 and 8 with data normalized to the Universal Probe at “100%” to emphasize the sensitivity of Universal vs Wild-Type/Mutant Target Detection. FIG. 13B shows the same raw hybridization data for the eleven (11) target sites covered by the amplimer sets 2, 3, 5, 6 and 8, but normalized to the Wild-Type probe signal, to emphasize the specificity of discrimination between Wild-Type vs Mutant target sequence.









TABLE 12





Validation of test design for the five prevalent Clade variants
























Spike Gene Target Region











(Codon) Amino Acid Change
S13I
L18F
T20N
P26S
Δ69-70
D80A
T95I
D138Y
Y144D





Mutation specific Probe coverage




1
1

1


Wuhan reference specific probe/S coverage




2
2

2


Locus specific Probe coverage




N/A




















Region
Lineage Designation Var






























Denmark
Mink V
B.1.1.298
S3
L3
T3
P3
Δ1
D2
T3
D2
Y3


UK
GR/501Y.V1
B.1.1.7
S3
L3
T3
P3
Δ1
D2
T3
D2
Δ3


SA
GH/501Y.V2
B.1.351
S3
L3
T3
P3
HV2
A1
T3
D2
Y3


Brazil
P.1

S3
F3
N3
S3
HV2
D2
T3
Y1
Y3


Brazil
P.2

S3
L3
T3
P3
HV2
D2
T3
D2
Y3


California
CAL.20C-
B.1.429
I3
L3
T3
P3
HV2
D2
T3
D2
Y3



GH/452R.V1


India
(Andhra
N440K
S3
L3
T3
P3
HV2
D2
T3
D2
Y3



Pradesh)


S. US
B.1.596/
Q677P/H
S3
L3
T3
P3
HV2
D2
T3
D2
Y3



B.1.2


NY
Ho et al.
B.1.526a
S3
L3
T3
P3
HV2
D2
I3
D2
Y3




B.1.526b
S3
L3
T3
P3
HV2
D2
I3
D2
Y3


WUHAN

WUHAN
S3
L3
T3
P3
HV2
D2
T3
D2
Y3









4
5

4










PCR Amplimer length (bases)
(1) 101
(2B)150
(3)129



















Spike Gene Target Region











(Codon) Amino Acid Change
W152C
R190S
D215G
A243del
R246I
D253G
K417N
N440K
L452R





Mutation specific Probe coverage
1


1
1
1
1
1
1


Wuhan reference specific probe/S coverage
2


2
2
2
2
2
2


Locus specific Probe coverage


























Region
Lineage Designation Var






























Denmark
Mink V
B.1.1.298
W2
R3
D3
A2
R2
D2
K2
N2
L2


UK
GR/501Y.V1
B.1.1.7
W2
R3
D3
A2
R2
D2
K2
N2
L2


SA
GH/501Y.V2
B.1.351
W2
R3
G3
Δ1
I1
D2
N1
N2
L2


Brazil
P.1

W2
S3
D3
A2
R2
D2
T1
N2
L2


Brazil
P.2

W2
R3
D3
A2
R2
D2
K2
N2
L2


California
CAL.20C-
B.1.429
C1
R3
D3
A2
R2
D2
K2
N2
R1



GH/452R.V1


India
(Andhra
N440K
W2
R3
D3
A2
R2
D2
K2
K1
L2



Pradesh)


S. US
B.1.596/
Q677P/H
W2
R3
D3
A2
R2
D2
K2
N2
L2



B.1.2


NY
Ho et al.
B.1.526a
W2
R3
D3
A2
R2
G1
K2
N2
L2




B.1.526b
W2
R3
D3
A2
R2
G1
K2
N2
L2


WUHAN

WUHAN
W2
R3
D3
A2
R2
D2
K2
N2
L2





4






4
4












PCR Amplimer length (bases)
(3)129


(4B) 160
(5) 199



















Spike Gene Target Region











(Codon) Amino Acid Change
Y453F
S477N
E484K
N501Y
A570D
D614G
H655Y
Q677P/H
P681H





Mutation specific Probe coverage

1
1
1

1

1
1


Wuhan reference specific probe/S coverage

2
2
2

2

2
2


Locus specific Probe coverage


























Region
Lineage Designation Var






























Denmark
Mink V
B.1.1.298
F3
S2
E2
N2
A3
G1
H3
Q2
P2


UK
GR/501Y.V1
B.1.1.7
Y3
S2
E2
Y1
D3
G1
H3
Q2
H1


SA
GH/501Y.V2
B.1.351
Y3
S2
K1
Y1
A3
G1
H3
Q2
P2


Brazil
P.1

Y3
S2
K1
Y1
A3
G1
Y3
Q2
P2


Brazil
P.2

Y3
S2
K1
N2
A3
G1
H3
Q2
P2


California
CAL.20C-
B.1.429
Y3
S2
E2
N2
A3
G1
H3
Q2
P2



GH/452R.V1


India
(Andhra
N440K
Y3
S2
E2
N2
A3
G1
H3
Q2
P2



Pradesh)


S. US
B.1.596/
Q677P/H
Y3
S2
E2
N2
A3
G1
H3
P/ H1
P2



B.1.2


NY
Ho et al.
B.1.526a
Y3
S2
K1
N2
A3
G1
H3
Q2
P2




B.1.526b
Y3
N1
E2
N2
A3
G1
H3
Q2
P2


WUHAN

WUHAN
Y3
S2
E2
N2
A3
D2
H3
Q2
P2







5
4

4


4













PCR Amplimer length (bases)
(5) 199
(6) 151

(7)88

(8) 135




















Spike Gene Target Region











(Codon) Amino Acid Change
1692V
A701V
T716I
S982A
T1027I
D1118
V1176
M1229I







Mutation specific Probe coverage

1



Wuhan reference specific probe/S coverage

2



Locus specific Probe coverage




















Region
Lineage Designation Var































Denmark
Mink V
B.1.1.298
V3
A2
T3
S3
T3
D3
V3
I3



UK
GR/501Y.V1
B.1.1.7
I3
A2
I3
A3
T3
H3
V3
M3



SA
GH/501Y.V2
B.1.351
I3
V1
T3
S3
T3
D3
V3
M3



Brazil
P.1

I3
A2
T3
S3
I3
D3
F3
M3



Brazil
P.2

I3
A2
T3
S3
T3
D3
F3
M3



California
CAL.20C-
B.1.429
I3
A2
T3
S3
T3
D3
V3
M3




GH/452R.V1



India
(Andhra
N440K
I3
A2
T3
S3
T3
D3
V3
M3




Pradesh)



S. US
B.1.596/
Q677P/H
I3
A2
T3
S3
T3
D3
V3
M3




B.1.2



NY
Ho et al.
B.1.526a
I3
V1
T3
S3
T3
D3
V3
M3





B.1.526b
I3
A/V
T3
S3
T3
D3
V3
M3



WUHAN

WUHAN
I3
A2
T3
S3
T3
D3
V3
M3







4
















PCR Amplimer length (bases)
(8) 135








1AA mutation - hybridizes to mutation specific probe





2AA identical to hCoV-19/Wuhan/WIV04/2019 (WIV04) - official reference sequence employed by GISAID (EPI_ISL_402124)





3Potential probe target





4No Probe Adjustment Necessary





5Minor Probe Adjustment Necessary







Table 13 shows the information content for the fully multiplexed (2, 3, 5, 6, 8) data obtained via the multiplex RT-PCR reaction in this DETECTX-Cv assay, which is sufficient to discriminate the five clade variants (superscript “1”). It was determined that including Amplimer 5 to the multiplex adds redundancy (superscript “2”) thereby allowing unambiguous discrimination of the India Mutant (B.1.36.29). Similarly, addition of amplimer 4 (for NY B.1.526) and Q677P/H probes (for Southern US B.1.596/13.1.2) to the multiplex enabled discrimination of Southern US and NY Clade variants (superscript “3”). Importantly, the emerging Southern US Clade variants (B.1.596/1.1.2) does not require modification of the present multiplex reaction since inclusion of probes at Q677P/H would be sufficient. Analytical specificity was established as described earlier via analysis of both wild type (Wuhan) gRNA and synthetic, Clade specific fragments.












Information content obtained by addition of amplimers















Information






obtained by





Information
adding




Information
obtained
Amplimer 4 +




obtained with
by adding
New Clade




Amplimers 2, 3,
Amplimer
variant


Region
Lineage Designation Var
6, 8
5
probes















Denmark
Mink V
B.1.1.298
1




UK
GR/501Y.V1
B.1.1.7
1


SA
GH/501Y.V2
B.1.351
1
2
3


Brazil/Japan
P.1

1
2


Brazil
P.2

1


California
CAL.20C-
B.1.429
1
2



GH/452R.V1


India
(Andhra Pradesh)
N440K

2


S. US
B.1.596/B.1.2
Q677P/H


3


NY
Ho et al.
B.1.526


3




B.1.526.2


3


WUHAN


1
2
3









Augury Software Modification

Current deployment of Augury software was modified to include automated capacity for determining “Wild-Type” vs “Mutant” at each of the (11) Spike target sites of the present DETECTX-Cv assay described above (the columns in Table 12. As modified, Augury is capable of calling the identity of the clade variant, based on the pattern of mutant presentation among the sites (that is, a “look-up” table comprising the pattern of each row of Table 12). Coding to enable such autonomous calling is based on allelotyping methods previously developed for HLA allelotyping. In the present case, the clade variant test is also an exercise in spike gene allelotyping. Such spike gene allelotypes (the rows in Table 12) have already been determined as being the preferred marker for CoV-2 Clade Variation.









TABLE 14







New Clade variant probe″ sequences











Amplimer

Probe Sequence


SEQ ID NOS.
#
Target
(5′ to 3′)





SEQ ID NO: 118
4
AA A243_
TTTTTTTTCAAACTTTACTTGCTTTACTC





TTT





SEQ ID NO: 119
4
AA_243DEL
TTTTTTTTCAAACTTTACATAGAAGCCTT





TTT





SEQ ID NO: 120
4
AA R246_
TTTTCTACATAGAAGTTATTTGACTCCCT





TTT





SEQ ID NO: 121
4
AA_246I
TTTTCTGCTTTACATATGACTCCTGGTTT





TTT





SEQ ID NO: 122
4
AA D253G
TTTCTACTCCTGGTGRTTCTTCTTCATTT





T





SEQ ID NO: 123
4
AA D253_
TTTTTTCCCTGGTGATTCTTCTTTCTTTT





T





SEQ ID NO: 124
4
AA_253G
TTTTTTCCCTGGTGGTTCTTCTTTTTTTT





T





SEQ ID NO: 125
8
AA_Q677P/H
TTTTTTATCAGACTCMGACTAATTCTCTT





TTT





SEQ ID NO: 126
8
AA Q677_
TTTTTTCCAGACTCAGACTAATTTCTTTT





T





SEQ ID NO: 127
8
AA_677P
TTTTTCTTCAGACTCCGACTAATCTTTTT





T





SEQ ID NO: 128
8
AA_677H1
TTTTTTCCAGACTCATACTAATTTCTTTT





T





SEQ ID NO: 129
8
AA_677H2
TTTTTTCCAGACTCACACTAATTTCTTTT





T









EXAMPLE 6
Augury Modification with Clade ID Module

The current deployment of the Augury software for wild type COV-2 was modified to include automated capacity for determining “Wild-Type” vs “Mutant” at each of the Spike target sites of the DETECTX-Cv assay (the columns in Table 15) and to identify the Clade variant based on the pattern of mutant presentation among the sites (the rows in Tables 15 and 16). Coding for the software is based on allelotyping formalism previously developed for HLA allelotyping.


Augury Software for DETECTX-Cv

All DETECTX-Cv probe sequences and their information content were added to a database (“Dot Score” file) within Augury. This database defined the DETECTX-Cv probe content (Mutant, Wild Type, Universal) at each of the eleven (11) Spike target regions (the columns in Table 15).


Establishment of DETECTX-Cv Version Control

The Augury Software is configured to read the bar code associated with each 96-well plate of microarrays for DETECTX-Cv and use the information in the bar code to create a “Dot Score” file for the probe content introduced into DETECTX-Cv. Further, Augury is configured to incorporate a new “Dot Score” file as appropriate for any new Clade Variant content with additional probes in the array (Table 15). Additionally, Augury is intrinsically cloud enabled and configured to deploy software modification downloaded from the cloud. When useful for analysis of DETECTX-Cv, data such as those from the RADx Rosalind initiative can also be introduced directly into Augury autonomously, to update the list of prevalent clade variants.


Manual Deployment Version of Augury for DETECTX-Cv

The core functionality of Augury has been used as a manual product for deployment at TriCore. This version of Augury automatically is enabled to read DETECTX-Cv plate bar codes, perform microarray image analysis, create “Dot Score” files and present the resulting averaged, background subtracted DETECTX-Cv data as a spread sheet matrix, which can be compared to the Clade Variant Hybridization patterns such as described in Table 15. This manual deployment version has been tested on DETECTX-Cv synthetic Clade variant standards.


Clade Variant “Look up Table”

All prevalent Cov-2 Clades have been programmed into Augury to generate a “Look-up Table” (equivalent in content to the pattern of boxes having superscript 1 and 2 in Table 15). The Augury internal “Lookup Table” is formatted to function as part of a Boolean pattern search as developed previously for allelotype analysis of all genes.









TABLE 15





Validation of Clade variants


























Spike Gene Target Region





A67V/







(Codon) Amino Acid Change
S13I
L18F
T20N
P26S
Q52R
Δ69-70
D80A
T95I
D138Y
Y144DEL
W152C





Mutation specific Probe coverage





1
1

1
1
1


Wuhan reference specific probe/S coverage





2
2

2
2
2


Locus specific Probe coverage





N/A
























Street name
Pango lineage - (Clade Nexstrain)
















UK
B.1.1.7 - (20I/501Y.V1)
S3
L3
T3
P3
Q3
Δ1
D2
T3
D2
Δ1
W2


SA
B.1.351 - (20H/501Y.V2)
S3
L3
T3
P3
Q3
HV2
A1
T3
D2
Y2
W2


US
B.1.375
S3
L3
T3
P3
Q3
Δ1
D2
T3
D2
Y2
W2


Brazil
P.1 - (20J/501Y.V3)
S3
F
N
S
Q3
HV2
D2
T3
Y1
Y2
W2


Cal L452R
B.1.429/427 - (20C/S:452R)
I
L3
T3
P3
Q3
HV2
D2
T3
D2
Y2
C1


Rio de Jan.
B.1.1.28
S3
L3
T3
P3
Q3
HV2
D2
T3
D2
Y2
W2


Andrah Pradesh
B.1.36.29
S3
L3
T3
P3
Q3
HV2
D2
T3
D2
Y2
W2


S. US/Q677P/H
(S:677P.B.1.596)
S3
L3
T3
P3
Q3
HV2
D2
T3
D2
Y2
W2



(S:677H.B.1.2)


NYC (Ho etal)
B.1.526a - (20C/S:484K)
S3
L3
T3
P3
Q3
HV2
D2
I
D2
Y2
W2



B.1.526b
S3
L3
T3
P3
Q3
HV2
D2
I
D2
Y2
W2


NYC
B.1.525 - (20A/S:484K)
S3
L3
T3
P3
R
V/Δ1
D
T3
D
Δ1
W



A.23.1
S3
L3
T3
P3
Q3
HV2
D2
T3
D2
Y2
W2



B.1.258 - (20A/S:439K)
S3
L3
T3
P3
Q3
Δ1
D2
T3
D2
Y2
W2



B.1.1.33
S3
L3
T3
P3
Q3
HV2
D2
T3
D2
Y2
W2



B.1.177 - (20E (EU1)(S:A222V))
S3
L3
T3
P3
Q3
HV2
D2
T3
D2
Y2
W2



B.1.1.207
S3
L3
T3
P3
Q3
HV2
D2
T3
D2
Y2
W2


Mink/Cluster V
B.1.1.298 (S:Y453F)
S3
L3
T3
P3
Q3
Δ1
D2
T3
D2
Y2
W2


WUHAN
WUHAN
S3
L3
T3
P3
Q3
HV2
D2
T3
D2
Y2
W2









4
5

4

4











PCR Amplimer length (bases)
(1) 101

(2B) 150
(3) 129


















Spike Gene Target Region










(Codon) Amino Acid Change
F157L
R190S
D215G
A222V
A243del
R246I
D253G
V367F





Mutation specific Probe coverage




1
1
1


Wuhan reference specific probe/S coverage




2
2
2


Locus specific Probe coverage























Street name
Pango lineage - (Clade Nexstrain)













UK
B.1.1.7 - (20I/501Y.V1)
F3
R3
D3
A3
A2
R2
D2
V3


SA
B.1.351 - (20H/501Y.V2)
F3
R3
G
A3
Δ1
I1
D2
V3


US
B.1.375
F3
R3
D3
A3
A2
R2
D2
V3


Brazil
P.1 - (20J/501Y.V3)
F3
S
D3
A3
A2
R2
D2
V3


Cal L452R
B.1.429/427 - (20C/S:452R)
F3
R3
D3
A3
A2
R2
D2
V3


Rio de Jan.
B.1.1.28
F3
R3
D3
A3
A2
R2
D2
V3


Andrah Pradesh
B.1.36.29
F3
R3
D3
A3
A2
R2
D2
V3


S. US/Q677P/H
(S:677P.B.1.596)
F3
R3
D3
A3
A2
R2
D2
V3



(S:677H.B.1.2)


NYC (Ho etal)
B.1.526a - (20C/S:484K)
F3
R3
D3
A3
A2
R2
G1
V3



B.1.526b
F3
R3
D3
A3
A2
R2
G1
V3


NYC
B.1.525 - (20A/S:484K)
F3
R3
D3
A3
A
R
D
V3



A.23.1
L
R3
D3
A3
A2
R2
D2
F



B.1.258 - (20A/S:439K)
F3
R3
D3
A3
A2
R2
D2
V3



B.1.1.33
F3
R3
D3
A3
A2
R2
D2
V3



B.1.177 - (20E (EU1)(S:A222V))
F3
R3
D3
V
A2
R2
D2
V3



B.1.1.207
F3
R3
D3
A3
A2
R2
D2
V3


Mink/Cluster V
B.1.1.298 (S:Y453F)
F3
R3
D3
A3
A2
R2
D2
V3


WUHAN
WUHAN
F3
R3
D3
A3
A2
R2
D2
V3













PCR Amplimer length (bases)




(4B) 160


















Spike Gene Target Region










(Codon) Amino Acid Change
K417N/T
N439K
N440K
L452R
Y453F
S477N
E484K
N501Y





Mutation specific Probe coverage
1
1
1
1
1
1
1
1


Wuhan reference specific probe/S coverage
2
2
2
2
2
2
2
2


Locus specific Probe coverage





N/A


















Street name
Pango lineage - (Clade Nexstrain)













UK
B.1.1.7 - (20I/501Y.V1)
K2
N2
N2
L2
Y2
S2
E2
Y1


SA
B.1.351 - (20H/501Y.V2)
N1
N2
N2
L2
Y2
S2
K1
Y1


US
B.1.375
K2
N2
N2
L2
Y2
S2
E2
N2


Brazil
P.1 - (20J/501Y.V3)
T1
N2
N2
L2
Y2
S2
K1
Y1


Cal L452R
B.1.429/427 - (20C/S:452R)
K2
N2
N2
R1
Y2
S2
E2
N2


Rio de Jan.
B.1.1.28
K2
N2
N2
L2
Y2
S2
K1
N2


Andrah Pradesh

K2
N2
K1
L2
Y2
S2
E2
N2


S. US/Q677P/H
(S:677P.P.B.1.596)
K2
N2
N2
L2
Y2
S2
E2
N2



(S:677H.B.1.2)


NYC (Ho etal)
B.1.526a - (20C/S:484K)
K2
N2
N2
L2
Y2
S2
K1
N2



B.1.526b
K2
N2
N2
L2
Y2
N1
E2
N2


NYC
B.1.525 - (20A/S:484K)
K
N
N
L
Y
S
K1
N



A.23.1
K2
N2
N2
L2
Y2
S2
E2
N2



B.1.258 - (20A/S:439K)
K2
K1
N2
L2
Y2
S2
E2
N2



B.1.1.33
K2
N2
N2
L2
Y2
S2
K
N2



B.1.177 - (20E (EU1)(S:A222V))
K2
N2
N2
L2
Y2
S2
E2
N2



(22.5 mM NaCl, 2.25 mM
K2
N2
N2
L2
Y2
S2
E2
N2



sodium citrate)


Mink/Cluster V
B.1.1.298 (S:Y453F)
K2
N2
N2
L2
F
S2
E2
N2


WUHAN
WUHAN
K2
N2
N2
L2
Y2
S2
E2
N2






4
4


5
4









PCR Amplimer length (bases)
(5) 199
(6) 151


















Spike Gene Target Region










(Codon) Amino Acid Change
A570D
Q613H
D614G
H655Y
Q677P/H
P681H
1692V
A701V





Mutation specific Probe coverage

1
1

1
1
1
1


Wuhan reference specific probe/S coverage

2
2

2
2
2
2


Locus specific Probe coverage
























Street name
Pango lineage - (Clade Nexstrain)













UK
B.1.1.7 - (20I/501Y.V1)
D
Q2
G1
H3
Q2
H1
I2
A2


SA
B.1.351 - (20H/501Y.V2)
A3
Q2
G1
H3

3Q2

P2
I2
V1


US
B.1.375
A3
Q2
G1
H3
Q2
P2
I2
A2


Brazil
P.1 - (20J/501Y.V3)
A3
Q2
G1
Y
Q2
P2
I2
A2


Cal L452R
B.1.429/427 - (20C/S:452R)
A3
Q2
G1
H3
Q2
P2
I2
A2


Rio de Jan.
B.1.1.28
A3
Q2
G1
H3
Q2
P2
I2
A2


Andrah Pradesh

A3
Q2
G1
H3
Q2
P2
I2
A2


S. US/Q677P/H
(S:677P.P.B.1.596)
A3
Q2
G1
H3
P/H1
P2
I2
A2



(S:677H.B.1.2)


NYC (Ho etal)
B.1.526a - (20C/S:484K)
A3
Q2
G1
H3
Q2
P2
I2
V1



B.1.526b
A3
Q2
G1
H3
Q2
P2
I2
A/V1


NYC
B.1.525 - (20A/S:484K)
A3
Q
G1
H3
H1
P
I
A



A.23.1
A3
H1
D2
H3
Q2
R1
I2
A2



B.1.258 - (20A/S:439K)
A3
Q2
G1
H3
Q2
P2
I2
A2



B.1.1.33
A3
Q2
G
H3
Q2
P2
I2
A2



B.1.177 - (20E (EU1)(S:A222V))
A3
Q2
G
H3
Q2
P2
I2
A2



(22.5 mM NaCl, 2.25 mM
A3
Q2
G
H3
Q2
H
I2
A2



sodium citrate)


Mink/Cluster V
B.1.1.298 (S:Y453F)
A3
Q2
G
H3
Q2
P2
V
A2


WUHAN
WUHAN
A3
Q2
D2
H3
Q2
P2
I2
A2






4


4

4











PCR Amplimer length (bases)

(7) 88

(8) 135



















Spike Gene Target Region










(Codon) Amino Acid Change
T716I
F888L
S982A
T1027I
D1118H
V1176F
M1229I







Mutation specific Probe coverage



Wuhan reference specific probe/S coverage



Locus specific Probe coverage

















Street name
Pango lineage - (Clade Nexstrain)














UK
B.1.1.7 - (20I/501Y.V1)
I
F3
A
T
H
V3
M3



SA
B.1.351 - (20H/501Y.V2)
T3
F3
S3
T3
D3
V3
M3



US
B.1.375
T3
F3
S3
T3
D3
V3
M3



Brazil
P.1 - (20J/501Y.V3)
T3
F3
S3
I
D3
F
M3



Cal L452R
B.1.429/427 - (20C/S:452R)
T3
F3
S3
T3
D3
V3
M3



Rio de Jan.
B.1.1.28
T3
F3
S3
T3
D3
F
M3



Andrah Pradesh

T3
F3
S3
T3
D3
V3
M3



S. US/Q677P/H
(S:677P.P.B.1.596)
T3
F3
S3
T3
D3
V3
M3




(S:677H.B.1.2)



NYC (Ho etal)
B.1.526a - (20C/S:484K)
T3
F3
S3
T3
D3
V3
M3




B.1.526b
T3
F3
S3
T3
D3
V3
M3



NYC
B.1.525 - (20A/S:484K)
T3
L
S3
T3
D3
V3
M3




A.23.1
T3
F3
S3
T3
D3
V3
M3




B.1.258 - (20A/S:439K)
T3
F3
S3
T3
D3
V3
M3




B.1.1.33
T3
F3
S3
T3
D3
V3
M3




B.1.177 - (20E (EU1)(S:A222V))
T3
F3
S3
T3
D3
V3
M3




(22.5 mM NaCl, 2.25 mM
T3
F3
S3
T3
D3
V3
M3




sodium citrate)



Mink/Cluster V
B.1.1.298 (S:Y453F)
T3
F3
S3
T3
D3
V3
I



WUHAN
WUHAN
T3
F3
S3
T3
D3
V3
M3
















PCR Amplimer length (bases)








1AA mutation - hybridizes to mutation specific probe





2AA identical to hCoV-19/Wuhan/WIV04/2019 (WIV04) - official reference sequence employed by GISAID (EPI_ISL_402124)





3Potential probe target





4No Probe Adjustment Necessary





5Minor Probe Adjustment Necessary














TABLE 16







Information content obtained by addition of amplimers
















Potential Probe
Amplimer 4 +



Pango lineage
Amplimers
Amplimer
content as described
New Clade


Street Name
(Clade Nextstrain open-source toolkit)
2, 3, 6, 8
5
in Table 14
variant probes





UK
B.1.1.7 - (20I/501Y.V1)
1

3



SA
B.1.351 - (20H/501Y.V2)
1
2

4


US
B.1.375
1


Brazil
P.1 - (20J/501Y.V3)
1
2


California L452R
B.1.429/427 - (20C/S: 452R)
1
2


Rio de Janeiro
B.1.1.28
1


Andhra Pradesh


2


S. US/Q677P/H
(S: 677P. Pelican) (S: 677H. Robin1)


3


NYC (Ho et al.)
B.1,526a - (20C/S: 484K)



4



B.1.526a - (20C/S: 484K)



4


NYC
B.1.525 - (20A/S: 484K)


3


Mink/Cluster V
(S: Y453F)
1

3


WUHAN

1
2
3
4






1Information obtained by adding Amplimers 2, 3, 6 and 8




2Information obtained by adding Amplimer 5




3Information obtained by adding Potential probe content




4Information obtained by adding Amplimer 4 + New Clade variant probes







Analytical Threshold Values

Multiplex RT-PCR [2, 3, 5, 6, 8] were performed in the absence of template (0 copies/reaction) to obtain the mean and STD from the mean for LoB signals. This “blank” data collection data is used by Augury to obtain the analytical threshold for each probe (3.2×STD+Mean) to yield Mutant threshold (Tm), Wild Type threshold (Tw) and Universal threshold (Tu) values for all thirty-three (33) probes comprising the content of DETECTX-Cv.


Deployment of Automatic Mutant vs Wild Type detection (“Delta”)

Threshold values were introduced as constants into Augury for autonomous Mutant vs Wild Type determination at all eleven (11) sites. This was performed using the following relationship analytical approach;





Delta=([RFUm−Tm]/Tm)−([RFUw−Tw]/Tw)  (Equation 1)


where,

    • RFUm=mutant probe RFU signal in a sample
    • RFUw=wild type probe RFU signal in a sample
    • Tm=mutant probe RFU Threshold−a constant obtained from CLSI (LoB) analysis
    • Tw=wild type probe RFU Threshold−a constant obtained from CLSI (LoB) analysis
    • [RFUm−Tm]=Mutant Probe Signal strength above Threshold. By definition, this is a non-zero value.
    • [RFUw−Tw]=Wild Type Signal strength above Threshold. By definition, this is a non-zero value.
    • Delta=Difference in Signal Strength above Threshold normalized to Threshold


If Delta>0, within experimental accuracy, then “Mutant” (i.e. boxes having superscript 1 in Table 15). If Delta<0, within experimental accuracy, then “Wild Type” (i.e. boxes having superscript 2 in Table 15).


EXAMPLE 7
Clade Variant Array Deployment-1

1. Analytical LoD Determination. A first determination of analytical LoD was performed for DETECTX-Cv, among all eleven (11) Spike target sites deployed using the [UNG+One Step RT-PCR] conditions. For this analysis, validation materials comprised a purified Wuhan gRNA reference (ATCC-BEI) and a cocktail of five (5) synthetic fragments designed by PathogenDx and fabricated by Integrated DNA Technologies, Inc. (IDT, Coralville, Iowa), comprising each region targeted for amplification via the [2, 3, 5, 6, 8] multiplex RT-PCR reaction (deployed as N=5 multiplex).


To support the multiplex reaction, all 5 synthetic CoV-2 fragments were mixed [1:1:1:1:1] in strand equivalents. Copy number values listed in Table 15 refer to the copy number of each fragment (in the equimolar mix) applied to the RT-PCR reaction. The primary goal here is to deploy the (N=5) RT-PCR multiplex to obtain a preliminary analytical LoD in units of copies/reaction for each of the probes comprising the set associated with each of the (n) target sites—LoDn (Universal), LoDn (Wild Type), LoDn (Mutant). The analytical LoD associated with the Universal probes (LoDn) were lower than that of either LoDn or LoDn, due to the intentionally longer probe sequence for the universal probe, which is associated with a higher affinity for its complementary amplicon sequence.


Results

Subsequent to RT-PCR the DNA microarray was prepared for hybridization with brief water washes, and an incubation in prehybridization buffer (0.6M NaCl, 0.06M sodium citrate solution, 0.1% Ficoll, 0.1% Polyvinylpyrrolidone 0.1% Bovine Serum Albumin). Following aspiration of the prehybridization buffer, a mixture of amplicon and hybridization buffer (0.6M NaCl, 0.06M sodium citrate, 0.1% Ficoll, 0.1% Polyvinylpyrrolidone 0.1% Bovine Serum Albumin) was added to the DNA microarray and allowed to incubate for 2 hours. The microarray is then washed with wash buffer (22.5 mM NaCl, 2.25 mM sodium citrate) and dried via centrifugation. The glass portion of the microarray was cleaned with lens tissue and 70% ethanol and images were acquired on the Sensospot. Images were then uploaded for Augury analysis. Following image acquisition and upload to Augury, it was found that the 5-plex RT-PCR reaction, comprising a N=5 multiplex of amplimers [2, 3, 5, 6, 8] was sufficient to obtain a first determination of analytical LODs [LoDn (Universal), LoDn (Wild Type), and LoDn (Mutant)].



FIGS. 14A-14Y shows analytical LoD data for a series of synthetic G-block fragments corresponding to domains 2-8. The synthetic copy number was determined by IDT and used as such, in subsequent dilutions. Fragments fabricated to display “signature” mutations as defined in boxes showing superscript 1 in Table 15 were mixed into a series of “cocktails” to emulate different clade variant types.



FIGS. 14A, 14C, 14E, 14G, 14I, 14K, 14M, 14O, 14Q, 14S, 14U, 14V and 14X
FIGS. 4(a-q) show a comparison of signals for Mutant (Synthetic) Cov-2, followed by hybridization to DETECTX-Cv to yield Wild-Type Probe (open circle) vs Mutant Probe (closed triangle) to emphasize the specificity of discrimination between Wild-Type vs Mutant target sequence. The signals were derived from microarray hybridization data (N=2 Repeats) for the N=5 multiplex RT-PCR amplification of Mutant (Synthetic) Cov-2, followed by hybridization to DETECTX-Cv. Table 17 summarizes the analytical LoDn (Wild Type) and LoDn (Mutant) values. FIGS. 14B, 14D, 14F, 14H, 14J, 14L, 14N, 14P, 14R, 14T, 14W and 14YFIGS. 4(a-q) show microarray hybridization data (N=2 Repeats) for the N=5 multiplex RT-PCR amplification of Mutant (Synthetic) Cov-2, followed by hybridization to DETECTX-Cv, to yield Universal Probe Hybridization probe signals (open square). These data emphasize the high sensitivity of analysis obtained via hybridization to the (longer) Universal Probe, generally manifested as a lower LoDn (Universal).









TABLE 17







Summary of analytical LoD values measured for each of the eleven Spike


gene target sites, for Universal, Wild-Type and Mutant probes.












Target Site

LoDn*
LoDn*
LoDn§
LoDn


(n)
Amplicon
(Universal) WT
(Universal) MT
(Wild Type)
(Mutant)















69-70(del)
2
NA
NA
50
  <10 **


D80A
2
50
<10
50
<10


D138Y
3
100
<10
100
<10


W152C
3
100
<10
500
<10


N440K
5
50
<10
50
<10


L452R
5
50
<10
50
<10


S477N
6
10
<10
50
<10


E484K
6
10
<10
10
<10


N501Y
6
100
<10
100
<10


P681H
8
10
<10
10
<10


A701V
8
10
<10
50
<10





*LoDn (Universal). Analytical LoD Values for Universal Probes as defined from the input target density (in copies per RT-PCR reaction) at which the signal obtained from the Universal probe becomes indistinguishable from the present estimate of background. There are two related values obtained for LoDn (Universal). One value is obtained upon titration with Wild Type (Wuhan) genomic gRNA (LoDn (Universal) and the other obtained upon titration with Mutant Synthetic Fragments (LoDn (Universal) MT)



§LoDn (Wild Type). Analytical LoD Values for Analysis of Wild Type (Wuhan)as defined from the input target density (measured in copies per RT-PCR reaction) at which the signal obtained from the Wild Type probe becomes indistinguishable from background.




LoDn (Mutant). Analytical LoD Values for Analysis of Mutant (Synthetic Fragment)as defined from the input target density (measured in copies per RT-PCR reaction) at which the signal obtained from the Mutant probe becomes indistinguishable from background.







EXAMPLE 8
Analysis of “Synthetic Clade Variant” Standards for Deployment to TriCore and Other Labs
1. Synthetic Clade Variant Analysis.

The (N=5) RT-PCR Multiplex (2, 3, 5, 6, 8) described in Example 7 was deployed to obtain a full eleven (11) site Clade variant profile using standard hybridization and wash procedures described above.


2. Synthetic Clade Variant Cocktails.

A set of five (5) different “Synthetic Clade Variant Standards” corresponding to UK (B.1.1.7), SA (B.1.351), CA452 (B.1.429), Brazil (P.1) and India N440K (B.1.36.29) were prepared each containing a synthetic gene fragment (IDT, Coralville, Iowa) identical to each of the Spike domains amplified by the present RT-PCR multiplex.


3. Synthetic Clade Variant Data Analysis.

Data were obtained at 100 copies/reaction for each of the five (5) synthetic cocktails. Hybridization analysis was performed, and the hybridization data thus obtained was plotted as described above.

    • 4. Results.


Raw data from this analysis presented in FIGS. 15A-15E shows that the ratio of Mutant (open bars) to Wild Type signal (black bars) readily identify the state of each of the eleven (11) target domains. Spike target sites expected to display a “Mutant” Signal (i.e. open bars>black bars) are marked with brackets.


EXAMPLE 9
CoV-2 Detection and Pooling Via (Oasis) Pure-SAL Saliva Collection

Clinical LoD Range Finding and Clinical LoD analysis were performed on contrived samples, comprising clinical negatives from healthy volunteers, collected in PURE-SAL™ collection device (OASIS DIAGNOSTICS° Corporation, WA). The samples were contrived with heat attenuated CoV-2 (Wuhan, BEI).


Contrived samples were subjected to viral gRNA capture and purification on Zymo silica magnetic beads or Ceres magnetic beads. Five microliters of purified RNA was added to the RT-PCR mix in a PCR plate. The plate was sealed and placed in a thermocycler to undergo 20 minutes of reverse transcription and 45 cycles of asymmetric PCR. Upon PCR completion, the DNA microarray was prepared for hybridization with brief water washes, and an incubation in prehybridization buffer (0.6M NaCl, 0.06M sodium citrate solution, 0.1% Ficoll, 0.1% Polyvinylpyrrolidone 0.1% Bovine Serum Albumin). Following aspiration of the prehybridization buffer, a mixture of amplicon and hybridization buffer (0.6M NaCl, 0.06M sodium citrate, 0.1% Ficoll, 0.1% Polyvinylpyrrolidone 0.1% Bovine Serum Albumin) was added to the DNA microarray and allowed to incubate for 2 hours. The microarray is then washed with wash buffer (22.5 mM NaCl, 2.25mM sodium citrate) and dried via centrifugation. The glass portion of the microarray was cleaned with lens tissue and 70% ethanol and images were acquired on the Sensospot. Images were then uploaded for Augury analysis.


Clinical LoD Results: Clinical LoD range finding was performed as described above (N=6 repeats) using clinically negative saliva samples (PURE-SAL™) to which were added heat inactivated CoV-2 that were processed using Zymo bead capture. FIG. 16 shows that the clinical LoD is close to 1000 copies/ml. A follow-up experiment was performed at N=20, where the resulting clinical LoD is defined as the point at which nineteen of the twenty (19/20) repeated samples produced positive detection (Table 18), which corresponds to a clinical LoD of 1000 copies/ml, a value that is identical within experimental accuracy to that obtained via the same DETECTX-Cv assay of contrived NP-VTM samples with Ceres bead collection as follows. Twenty microliters of beads were added to 400 μL of clinical sample and 800 μL of viral DNA/RNA buffer and mixed on a shaker at 1200 rpm for 10 minutes. The samples were placed on the magnet and supernatant was removed before the addition and pipette-mixing of Zymo Wash Buffer 1. This was repeated for Zymo Wash Buffer 2 and two washes with 100% ethanol. All washes were performed at a volume of 500 μL. The beads were dried at 55° C. Once completely dried, 50 μL of water was added to the beads and mixed well. After placing the samples on the magnet, the supernatant was transfer to another plate for RNA storage. Five microliters of purified RNA were added to the RT-PCR mix in a PCR plate. The plate was sealed and placed in a thermocycler to undergo 20 minutes of reverse transcription and 45 cycles of asymmetric PCR. Upon PCR completion, the DNA microarray was prepared for hybridization with brief water washes, and an incubation in prehybridization buffer (0.6M NaCl, 0.06M sodium citrate solution, 0.1% Ficoll, 0.1% Polyvinylpyrrolidone 0.1% Bovine Serum Albumin). Following aspiration of the prehybridization buffer, a mixture of amplicon and hybridization buffer (0.6M NaCl, 0.06M sodium citrate solution, 0.1% Ficoll, 0.1% Polyvinylpyrrolidone 0.1% Bovine Serum Albumin) was added to the DNA microarray and allowed to incubate for 2 hours. The microarray is then washed with wash buffer (22.5mM NaCl, 2.25 mM sodium citrate) and dried via centrifugation. The glass portion of the microarray was cleaned with lens tissue and 70% ethanol and images were acquired on the Sensospot. Images were then uploaded for Augury analysis.









TABLE 18







Summary of LoD Experiment Results











Input
SARS-
SARS-
Positive
% Positive


Concentration
CoV-2 N1
CoV-2 N2
Final Call
Final Call





1500 cp/mL
20/20
19/20
20/20
100% 


1000 cp/mL
19/20
13/20
19/20
95%







Final LoD











1000 cp/mL
19/20
13/20
19/20
95%









Pure-SAL Saliva. Pooling Range Finding

The ability to pool CoV-2 contrived clinical negative PURE-SAL™ saliva samples was tested. Contrived clinical negative samples were pooled at (1) Positive Clinical Sample (100 L)+(4) Clinical samples (100 (L each), to yield a final pooled sample where the viral complement of the original contrived clinical positive is diluted 5×. The entire pooled specimen was then subjected to Zymo magnetic bead purification, RT-PCR and Hybridization to DETECTX-Cv as described above. The results shown in FIG. 17 suggest that subsequent to 5× pooling, the LoD is reduced less than the full 5× expected from a simple 5× dilution, thus demonstrating feasibility of the N=5 PURE-SAL™ pooling.


EXAMPLE 10
Autonomous Analysis

DETECTX-Cv analysis was performed by hands-free, autonomous analysis of raw DETECTX-microarray data obtained from Sensovation Scans to generate “Mutant” vs “Wild Type” calls among the ten (10) Spike target sites Table 19. These calls were subsequently used for Clade identification. The autonomous analysis is presented here along with manual Augury analysis.


The following multiple functional modules were added to Augury to enable autonomous analysis of DETECTX-Cv data as follows;

  • (1) Look-Up Table. A database (a “Look-Up Table”) directly related to a Clade Variant vs Mutation data matrix (Table 19) was programmed into Augury. The database is flexible, resident within Augury and can be increased in size as needed to include a larger number of Spike Gene Targets (i.e. more columns as in Table 19) or Clade Variant Targets (i.e. more Rows as in Table 19). Augury is intrinsically linked to the cloud. Further, the Clade Variant Look-Up Table in Augury can be updated in real time via secure inputs such as those which could be provided by Rosalind (San Diego, Calif.).
  • (2) Comparison among probe data sets. Augury was modified to compare probe information to be used for data quality (QA/QC) and for interpretation of the RFU data (Clade ID):
    • a) QA/QC based on signal strength (signal intensity) . The universal probes described earlier were used to measure data quality. If universal probe signals were <10,000 (resulting from sample degradation or low concentration), the data associated with the corresponding Mutant and Wild type data at a Spike Target Site are not used by Augury for Clade variant identification.
    • b) Data Interpretation: Primary. “Wild Type” and “Mutant” Probe data (RFU) were compared automatically, along with clinical threshold data stored in Augury to generate a “Delta” value (see Example 6). A Delta value greater than 0 returns a “Mutant” call, whereas a Delta value less than 0 returns a “Wild Type” call at each Spike Target Site.
    • c) Data Interpretation: Secondary. The pattern of Wild Type vs Mutant calls (i.e. the rows in Table 19) obtained from the Primary Data Interpretation were automatically compared to patterns associated with known Clade variants. The most likely Clade variant pattern is automatically reported. A statistical probability is also assignable to the Clade Variant call and alternative calls based on DETECTX-Cv analysis of multiple Clade Variant samples.
    • d) Data Reports. A Standard Report Format was chosen.


DETECTX-Cv Analysis of Synthetic Clade Variant Standards at TriCore

Five (5) synthetic Clade variant standards described earlier (UK, SA, CA452, Brazil P.1, India, Examples 8 and 9) were used for on-site validation. Each standard contained a synthetic gene fragment (IDT) identical to each of the Spike domains amplified by the RT-PCR multiplex. DETECTX-Cv data were obtained at TriCore at 100 copies/reaction for each of the five (5) synthetic cocktails. Analysis of the hybridization data were plotted as described previously. Table 20 shows a plate map, PCR recipe and cycling conditions for this analysis. DNA fragment cocktails were utilized as reference.









TABLE 19





Spike Gene Target Region (Codon) Amino Acid Change























L5F
S13I
L18F
T20N
P26S
Q52R
A67V




















CDC %
Incidence %





















Pango
Mar. 14-27,
Gisaid March
Signal
S1 subunit (14-685)



Street name
lineage
2021 (US)
2021
(1-13)
N-terminal domain (14-305)






















VOC
UK
B.1.1.7
44.10% 
49.81%
L3
S2
L3
T2
P3
Q3
A3


VOC
California L452R
B.1.427
6.90%
2.08%
L3
I1
L3
T2
P3
Q3
A3


VOC

B.1.427
2.90%
0.90%
L3
S/I1
L3
T2
P3
Q3
A3


VOC
Brazil
P.1
1.40%
0.39%
L3
S2
F
N1
S
Q3
A3


VOC
SA
B.1.351
0.70%
1.13%
L3
S2
L3
T2
P3
Q3
A3


VOC
NYC (Ho et al.)
B.1.526
9.20%
0.82%
L/F
S2
L3
T2
P3
Q3
A3


VOC
NYC
B.1.525
0.50%
0.10%
L3
S2
L3
T2
P3
R
V


VOC
Rio de Janeiro
P.2
0.30%
0.36%
L3
S2
L3
T2
P3
Q3
A3




B.1.2
10.00% 
7.83%
L3
S2
L3
T2
P3
Q3
A3




B.1, B.1.1,
2.4%/
2.6%/
L3
S2
L3
T2
P3
Q3
A3




B.1.234
0.9%/
1.5%/





0.5% 
0.5%




B.1.1.519
4.10%
1.50%
L3
S2
L3
T2
P3
Q3
A3




B.1.526.1
3.90%
0.35%
F
S2
L3
T2
P3
Q3
A3




B.1.526.2
2.90%
0.18%
F
S2
L3
T2
P3
Q3
A3




B.1.596
1.70%
1.04%
L3
S2
L3
T2
P3
Q3
A3




R.1
1.20%
0.20%
L3
S2
L3
T2
P3
Q3
A3




B.1.575
1.10%
0.19%
L3
S2
L3
T2
P3
Q3
A3




B.1.243,
0.60%
0.84%
L3
S2
L3
T2
P3
Q3
A3




B.1.1.207



US
B.1.375
<1%
0.03%
L3
S2
L3
T2
P3
Q3
A3




B.1.1.1,
<1%
0.50%
L3
S2
L3
T2
P3
Q3
A3




B.1.416,




B.1.1.33,




B.1.311,




B.1.1.122



Brazil (Original)
B.1.1.28
<1%
0.10%
L3
S2
L3
T2
P3
Q3
A3



Andhra Pradesh
B.1.36.29
<1%
0.08%
L3
S2
F
T2
P3
Q3
A3




A.23.1
<1%
0.05%
L3
S2
L3
T2
P3
Q3
A3




A.27
<1%
0.05%
L3
S2
F
T2
P3
Q3
A3




A.28
<1%
0.02%
L3
S2
L3
T2
P3
Q3
A3



Mink/Cluster V
B.1.1.298
<1%
0.00%
L3
S2
L3
T2
P3
Q3
A3




B.1.1.318
<1%
0.01%
L3
S2
L3
T2
P3
Q3
A3




B.1.160
<1%
1.76%
L3
S2
L3
T2
P3
Q3
A3




B.1.177
<1%
3.19%
L3
S2
F
T2
P3
Q3
A3




B.1.177.80
<1%
0.04%
L3
S2
F
T2
P3
Q3
A3




B.1.258
<1%
1.15%
L3
S2
L3
T2
P3
Q3
A3




B.1.258.14
<1%
0.06%
L3
S2
L3
T2
P3
Q3
A3




B.1.258.17
<1%
1.02%
L3
S2
L3
T2
P3
Q3
A3




B.1.517
<1%
0.25%
L3
S2
L3
T2
P3
Q3
A3



WUHAN
WUHAN


L3
S2
L3
T2
P3
Q3
A3









PCR Amplimer length (bases)
(1) 101
(2B) 150






















Δ69-
D80A/




F157L/





70
G
T95I
D138Y
Y144DEL
W152C
S







CDC %
Incidence %














Pango
Mar. 14-27,
Gisaid March
S1 subunit (14-685)



Street name
lineage
2021 (US)
2021
N-terminal domain (14-305)






















VOC
UK
B.1.1.7
44.10% 
49.81%
Δ1
D2
T3
D2
Δ1
W2
F3


VOC
California L452R
B.1.427
6.90%
2.08%
HV2
D2
T3
D2
Y2
C1
F3


VOC

B.1.427
2.90%
0.90%
HV2
D2
T3
D2
Y2
W/C2
F3


VOC
Brazil
P.1
1.40%
0.39%
HV2
D2
T3
Y1
Y2
W2
F3


VOC
SA
B.1.351
0.70%
1.13%
HV2
A1
T3
D2
Y2
W2
F3


VOC
NYC (Ho et al.)
B.1.526
9.20%
0.82%
HV2
D2
I
D2
Y2
W2
F3


VOC
NYC
B.1.525
0.50%
0.10%
Δ1
D2
T3
D2
Δ1
W2
F3


VOC
Rio de Janeiro
P.2
0.30%
0.36%
HV2
D2
T3
D2
Y2
W2
F3




B.1.2
10.00% 
7.83%
HV2
D2
T3
D2
Y2
W2
F3




B.1, B.1.1,
2.4%/
2.6%/
HV2
D2
T3
D2
Y2
W2
F3




B.1.234
0.9%/
1.5%/





0.5% 
0.5%




B.1.1.519
4.10%
1.50%
HV2
D2
T3
D2
Y2
W2
F3




B.1.526.1
3.90%
0.35%
HV2
G
I
D2
Δ1
W2
S




B.1.526.2
2.90%
0.18%
HV2
D2
T3
D2
Y2
W2
F3




B.1.596
1.70%
1.04%
HV2
D2
T3
D2
Y2
W2
F3




R.1
1.20%
0.20%
HV2
D2
T3
D2
Y2
L
F3




B.1.575
1.10%
0.19%
HV2
D2
T3
D2
Y2
W2
F3




B.1.243,
0.60%
0.84%
HV2
D2
T3
D2
Y2
W2
F3




B.1.1.207



US
B.1.375
<1%
0.03%
Δ1
D2
T3
D2
Y2
W2
F3




B.1.1.1,
<1%
0.50%
HV2
D2
T3
D2
Y2
W2
F3




B.1.416,




B.1.1.33,




B.1.311,




B.1.1.122



Brazil (Original)
B.1.1.28
<1%
0.10%
HV2
D2
T3
D2
Y2
W2
F3



Andhra Pradesh
B.1.36.29
<1%
0.08%
HV2
D2
T3
D2
Y2
W2
F3




A.23.1
<1%
0.05%
HV2
D2
T3
D2
Y2
W2
L




A.27
<1%
0.05%
HV2
D2
T3
D2
Y2
W2
F3




A.28
<1%
0.02%
Δ1
D2
T3
D2
Y2
W2
F3



Mink/Cluster V
B.1.1.298
<1%
0.00%
Δ1
D2
T3
D2
Y2
W2
F3




B.1.1.318
<1%
0.01%
HV2
D2
T3
D2
Δ1
W2
F3




B.1.160
<1%
1.76%
HV2
D2
T3
D2
Y2
W2
F3




B.1.177
<1%
3.19%
HV2
D2
T3
D2
Y2
W2
F3




B.1.177.80
<1%
0.04%
HV2
D2
T3
D2
Δ/Y1
W2
F3




B.1.258
<1%
1.15%
HV/Δ1
D2
T3
D2
Y2
W2
F3




B.1.258.14
<1%
0.06%
HV2
D2
T3
D2
Y2
W2
F3




B.1.258.17
<1%
1.02%
Δ1
D2
T3
D2
Y2
W2
F3




B.1.517
<1%
0.25%
HV2
D2
T3
D2
Y2
W2
F3



WUHAN
WUHAN


HV2
D2
T3
D2
Y2
W2
F3











PCR Amplimer length (bases)
(2B) 150

(3) 129






















L189F
R190S
D215G
A222V
A243del
G252V
D253G







CDC %
Incidence %














Pango
Mar. 14-27,
Gisaid March
S1 subunit (14-685)



Street name
lineage
2021 (US)
2021
N-terminal domain (14-305)






















VOC
UK
B.1.1.7
44.10% 
49.81%
L3
R3
D3
A3
A2
G3
D2


VOC
California L452R
B.1.427
6.90%
2.08%
L3
R3
D3
A3
A2
G3
D2


VOC

B.1.427
2.90%
0.90%
L3
R3
D3
A3
A2
G3
D2


VOC
Brazil
P.1
1.40%
0.39%
L3
S
D3
A3
A2
G3
D2


VOC
SA
B.1.351
0.70%
1.13%
L3
R3
G
A3
Δ1
G3
D2


VOC
NYC (Ho et al.)
B.1.526
9.20%
0.82%
L3
R3
D3
A3
A2
G3
G1


VOC
NYC
B.1.525
0.50%
0.10%
L3
R3
D3
A3
A2
G3
D2


VOC
Rio de Janeiro
P.2
0.30%
0.36%
L3
R3
D3
A3
A2
G3
D2




B.1.2
10.00% 
7.83%
L3
R3
D3
A3
A2
G3
D2




B.1, B.1.1,
2.4%/
2.6%/
L3
R3
D3
A3
A2
G3
D2




B.1.234
0.9%/
1.5%/





0.5% 
0.5%




B.1.1.519
4.10%
1.50%
L3
R3
D3
A3
A2
G3
D2




B.1.526.1
3.90%
0.35%
L3
R3
D3
A3
A2
G3
D/G1




B.1.526.2
2.90%
0.18%
L3
R3
D3
A3
A2
G3
G1




B.1.596
1.70%
1.04%
L3
R3
D3
A3
A2
G3
D2




R.1
1.20%
0.20%
L3
R3
D3
A3
A2
G3
D2




B.1.575
1.10%
0.19%
L3
R3
D3
A3
A2
G3
D2




B.1.243,
0.60%
0.84%
L3
R3
D3
A3
A2
G3
D2




B.1.1.207



US
B.1.375
<1%
0.03%
L3
R3
D3
A3
A2
G3
D2




B.1.1.1,
<1%
0.50%
L3
R3
D3
A3
A2
G3
D2




B.1.416,




B.1.1.33,




B.1.311,




B.1.1.122



Brazil (Original)
B.1.1.28
<1%
0.10%
L3
R3
D3
A3
A2
G3
D2



Andhra Pradesh
B.1.36.29
<1%
0.08%
L3
R3
D3
A3
A2
G3
D2




A.23.1
<1%
0.05%
L3
R3
D3
A3
A2
G3
D2




A.27
<1%
0.05%
L3
R3
D3
A3
A2
G3
D2




A.28
<1%
0.02%
L3
R3
D3
A3
A2
G3
D2



Mink/Cluster V
B.1.1.298
<1%
0.00%
L3
R3
D3
A3
A2
G3
D2




B.1.1.318
<1%
0.01%
L3
R3
D3
A3
A2
G3
D2




B.1.160
<1%
1.76%
L3
R3
D3
A3
A2
G3
D2




B.1.177
<1%
3.19%
L3
R3
D3
V
A2
G3
D2




B.1.177.80
<1%
0.04%
L3
R3
D3
V
A2
G3
D2




B.1.258
<1%
1.15%
L3
R3
D3
A3
A2
G3
D2




B.1.258.14
<1%
0.06%
L3
R3
D3
A3
A2
G3
D2




B.1.258.17
<1%
1.02%
F
R3
D3
A3
A2
G3
D2




B.1.517
<1%
0.25%
L3
R3
D3
A3
A2
G/V
D2



WUHAN
WUHAN


L3
R3
D3
A3
A2
G3
D2












PCR Amplimer length (bases)




(4B) 160


















V367F
K417N/T
N439K
N440K
L452R
Y453F
S477N



















CDC %
Incidence %




Street
Pango
Mar. 14-27,
Gisaid March
S1 subunit (14-685)



name
lineage
2021 (US)
2021
RBD (319-541)






















VOC
UK
B.1.1.7
44.10% 
49.81%
V3
K2
N2
N2
L2
Y2
S2


VOC
California
B.1.427
6.90%
2.08%
V3
K2
N2
N2
R1
Y2
S2



L452R


VOC

B.1.429
2.90%
0.90%
V3
K2
N2
N2
R1
Y2
S2


VOC
Brazil
P.1
1.40%
0.39%
V3
K2
N2
N2
L2
Y2
S2


VOC
SA
B.1.351
0.70%
1.13%
V3
K2
N2
N2
L2
Y2
S2


VOC
NYC
B.1.526
9.20%
0.82%
V3
K2
N2
N2
L2
Y2
S/N1



(Ho et al.)


VOC
NYC
B.1.525
0.50%
0.10%
V3
K2
N2
N2
L2
Y2
S2


VOC
Rio de
P.2
0.30%
0.36%
V3
K2
N2
N2
L2
Y2
S2



Janeiro




B.1.2
10.00% 
7.83%
V3
K2
N2
N2
L2
Y2
S2




B.1,
2.4%/
2.6%/
V3
K2
N2
N2
L2
Y2
S2




B.1.1,
0.9%/
1.5%/




B.1.234
0.5% 
0.5%




B.1.1.519
4.10%
1.50%
V3
K2
N2
N2
L2
Y2
S2




B.1.526.1
3.90%
0.35%
V3
K2
N2
N2
R1
Y2
S2




B.1.526.2
2.90%
0.18%
V3
N/T1
N2
N2
L2
Y2
N1




B.1.596
1.70%
1.04%
V3
N1
N2
N2
L2
Y2
S2




R.1
1.20%
0.20%
V3
K2
N2
N2
L2
Y2
S2




B.1.575
1.10%
0.19%
V3
K2
N2
N2
L2
Y2
S2




B.1.243,
0.60%
0.84%
V3
K2
N2
N2
L2
Y2
S2




B.1.1.207



US
B.1.375
<1%
0.03%
V3
K2
N2
N2
L2
Y2
S2




B.1.1.1,
<1%
0.50%
V3
K2
N2
N2
L2
Y2
S2




B.1.416,




B.1.1.33,




B.1.311,




B.1.1.122



Brazil
B.1.1.28
<1%
0.10%
V3
K2
N2
N2
L2
Y2
S2



Original



Andhra
B.1.36.29
<1%
0.08%
V3
K2
N2
K1
L2
Y2
S2



Pradesh




A.23.1
<1%
0.05%
F
K2
N2
N2
L2
Y2
S2




A.27
<1%
0.05%
V3
K2
N2
N2
R1
Y2
S2




A.28
<1%
0.02%
V3
K2
N2
N2
L2
Y2
S2



Mink/
B.1.1.298
<1%
0.00%
V3
K2
N2
N2
L2
F1
S2



Cluster V




B.1.1.318
<1%
0.01%
V3
K2
N2
N2
L2
Y2
S2




B.1.160
<1%
1.76%
V3
K2
N2
N2
L2
Y2
N1




B.1.177
<1%
3.19%
V3
K2
N2
N2
L2
Y2
S2




B.1.177.80
<1%
0.04%
V3
K2
N2
N2
L2
Y2
S2




B.1.258
<1%
1.15%
V3
K2
K1
N2
L2
Y2
S2




B.1.258.14
<1%
0.06%
V3
K2
K1
N2
L2
Y2
S2




B.1.258.17
<1%
1.02%
V3
K2
K1
N2
L2
Y2
S2




B.1.517
<1%
0.25%
V3
K2
N2
N2
L2
Y2
S2



WUHAN
WUHAN


V3
K2
N2
N2
L2
Y2
S2










PCR Amplimer length (bases)

(5) 199
(6) 151






















V483A
E484K
S494P
N501Y/T
A570D
Q613H
D614G







CDC %
Incidence %













Street
Pango
Mar. 14-27,
Gisaid March
S1 subunit (14-685)
















name
lineage
2021 (US)
2021
RBD (319-541)

























VOC
UK
B.1.1.7
44.10% 
49.81%
V2
E/K1
S/P
Y1
D
Q2
G1


VOC
California
B.1.427
6.90%
2.08%
V2
E2
S3
N2
A3
Q2
G1



L452R


VOC

B.1.429
2.90%
0.90%
V2
E2
S3
N2
A3
Q2
G1


VOC
Brazil
P.1
1.40%
0.39%
V2
K1
S3
Y1
A3
Q2
G1


VOC
SA
B.1.351
0.70%
1.13%
V2
K1
S3
Y1
A3
Q2
G1


VOC
NYC
B.1.526
9.20%
0.82%
V2
E/K1
S3
N2
A3
Q2
G1



(Ho et al.)


VOC
NYC
B.1.525
0.50%
0.10%
V2
K1
S3
N2
A3
Q2
G1


VOC
Rio de
P.2
0.30%
0.36%
V2
K1
S3
N2
A3
Q2
G1



Janeiro




B.1.2
10.00% 
7.83%
V2
E2
S3
Y1
A3
Q2
G1




B.1,
2.4%/
2.6%/
V2
E2
S3
N2
A3
Q2
G1




B.1.1,
0.9%/
1.5%/




B.1.234
0.5% 
0.5%




B.1.1.519
4.10%
1.50%
V2
E2
S3
N2
A3
Q2
G1




B.1.526.1
3.90%
0.35%
V2
K1
S3
N2
A3
Q2
G1




B.1.526.2
2.90%
0.18%
V2
E2
S3
N2
A3
Q2
G1




B.1.596
1.70%
1.04%
V2
E2
S3
N2
A3
Q2
G1




R.1
1.20%
0.20%
V2
K1
S3
N2
A3
Q2
G1




B.1.575
1.10%
0.19%
V2
E2
P
N2
A3
Q2
G1




B.1.243,
0.60%
0.84%
V2
E2
S3
N2
A3
Q2
G1




B.1.1.207



US
B.1.375
<1%
0.03%
V2
E2
S3
N2
A3
Q2
G1




B.1.1.1,
<1%
0.50%
V2
E2
S3
N2
A3
Q2
G1




B.1.416,




B.1.1.33,




B.1.311,




B.1.1.122



Brazil
B.1.1.28
<1%
0.10%
V2
E2
S3
N2
A3
Q2
G1



Original



Andhra
B.1.36.29
<1%
0.08%
V2
E2
S3
N2
A3
Q2
G1



Pradesh




A.23.1
<1%
0.05%
V2
E/K1
S3
N2
A3
H1
D2




A.27
<1%
0.05%
V2
E2
S3
Y1
A3
Q2
D2




A.28
<1%
0.02%
V2
E2
S3
T
A3
Q2
D2



Mink/
B.1.1.298
<1%
0.00%
V2
E2
S3
N2
A3
Q2
G1



Cluster V




B.1.1.318
<1%
0.01%
V2
K1
S3
N2
A3
Q2
G1




B.1.160
<1%
1.76%
V2
E2
S3
N2
A3
Q2
G1




B.1.177
<1%
3.19%
V2
E2
S3
N2
A3
Q2
G1




B.1.177.80
<1%
0.04%
V2
E2
S3
N2
A3
Q2
G1




B.1.258
<1%
1.15%
V2
E2
S3
N2
A3
Q2
G1




B.1.258.14
<1%
0.06%
V2
E2
S3
N2
A3
Q2
G1




B.1.258.17
<1%
1.02%
V2
E2
S3
N2
A3
Q2
G1




B.1.517
<1%
0.25%
V2
E2
S3
T
A3
Q/H1
G1



WUHAN
WUHAN


V2
E2
S3
N2
A3
Q2
D2










PCR Amplimer length (bases)
(6) 151

(7) 88


























H655Y
Q677P/H
P681H
I692V
A701V
T716I
G769V









CDC %
Incidence %



Street
Pango
Mar. 14-27,
Gisaid March













name
lineage
2021 (US)
2021
S2 subunit (686-1273)






















VOC
UK
B.1.1.7
44.10% 
49.81%
H3
Q2
H1
I2
A2
I
G3


VOC
California
B.1.427
6.90%
2.08%
H3
Q2
P2
I2
A2
T3
G3



L452R


VOC

B.1.429
2.90%
0.90%
H3
Q2
P2
I2
A2
T3
G3


VOC
Brazil
P.1
1.40%
0.39%
Y
Q2
P2
I2
A2
T3
G3


VOC
SA
B.1.351
0.70%
1.13%
H3
Q2
P2
I2
V1
T3
G3


VOC
NYC
B.1.526
9.20%
0.82%
H3
Q2
P2
I2
A/V1
T3
G3



(Ho et al.)


VOC
NYC
B.1.525
0.50%
0.10%
H3
H1
P2
I2
A2
T3
G3


VOC
Rio de
P.2
0.30%
0.36%
H3
Q2
P2
I2
A2
T3
G3



Janeiro




B.1.2
10.00% 
7.83%
H3
Q2
P2
I2
A2
T3
G3




B.1,
2.4%/
2.6%/
H3
Q2
P2
I2
A2
T3
G3




B.1.1,
0.9%/
1.5%/




B.1.234
0.5% 
0.5%




B.1.1.519
4.10%
1.50%
H3
Q2
H1
I2
A2
T3
G3




B.1.526.1
3.90%
0.35%
H3
Q2
P2
I2
A/V1
T3
G3




B.1.526.2
2.90%
0.18%
H3
Q2
P2
I2
A2
T3
G3




B.1.596
1.70%
1.04%
H3
Q/P1
P2
I2
A2
T3
G3




R.1
1.20%
0.20%
H3
Q2
P2
I2
A2
T3
V




B.1.575
1.10%
0.19%
H3
Q2
H1
I2
A2
I
G3




B.1.243,
0.60%
0.84%
H3
Q2
H1
I2
A2
T3
G3




B.1.1.207



US
B.1.375
<1%
0.03%
H3
Q2
P2
I2
A2
T3
G3




B.1.1.1,
<1%
0.50%
H3
Q2
P2
I2
A2
T3
G3




B.1.416,




B.1.1.33,




B.1.311,




B.1.1.122



Brazil
B.1.1.28
<1%
0.10%
H3
Q2
P2
I2
A2
T3
G3



Original



Andhra
B.1.36.29
<1%
0.08%
H3
Q2
P2
I2
A2
T3
G3



Pradesh




A.23.1
<1%
0.05%
Y
Q2
R1
I2
A2
T3
G3




A.27
<1%
0.05%
Y
Q2
P2
I2
A2
T3
G3




A.28
<1%
0.02%
H3
Q2
P2
I2
A2
T3
G3



Mink/
B.1.1.298
<1%
0.00%
H3
Q2
P2
I2
A2
T3
G3



Cluster V




B.1.1.318
<1%
0.01%
H3
Q2
H1
I2
A2
T3
G3




B.1.160
<1%
1.76%
H3
Q2
P2
I2
A2
T3
G3




B.1.177
<1%
3.19%
H3
Q2
P2
I2
A2
T3
G3




B.1.177.80
<1%
0.04%
H3
Q2
P2
I2
A2
T3
G3




B.1.258
<1%
1.15%
H3
Q2
P2
I2
A2
T3
G3




B.1.258.14
<1%
0.06%
H3
Q2
P2
I2
A2
T3
G3




B.1.258.17
<1%
1.02%
H3
Q2
P2
I2
A2
T3
G3




B.1.517
<1%
0.25%
H3
Q2
P2
I2
A2
T3
G3



WUHAN
WUHAN


H3
Q2
P2
I2
A2
T3
G3











PCR Amplimer length (bases)

(8) 135

















D796V
F888L
S982A
T1027I
D1118H
V1176F



















CDC %
Incidence %
S2 subunit (686-1273)



Street
Pango
Mar. 14-27,
Gisaid March
Fusion peptide



name
lineage
2021 (US)
2021
(788-806)





















VOC
UK
B.1.1.7
44.10% 
49.81%
D3
F3
A
T3
H
V3


VOC
California
B.1.427
6.90%
2.08%
D3
F3
S3
T3
D3
V3



L452R


VOC

B.1.429
2.90%
0.90%
D3
F3
S3
T3
D3
V3


VOC
Brazil
P.1
1.40%
0.39%
D3
F3
S3
I
D3
F


VOC
SA
B.1.351
0.70%
1.13%
D3
F3
S3
T3
D3
V3


VOC
NYC
B.1.526
9.20%
0.82%
D3
F3
S3
T3
D3
V3



(Ho et al.)


VOC
NYC
B.1.525
0.50%
0.10%
D3
L
S3
T3
D3
V3


VOC
Rio de
P.2
0.30%
0.36%
D3
F3
S3
T3
D3
F



Janeiro




B.1.2
10.00% 
7.83%
D3
F3
S3
T3
D3
V3




B.1,
2.4%/
2.6%/
D3
F3
S3
T3
D3
V3




B.1.1,
0.9%/
1.5%/




B.1.234
0.5% 
0.5%




B.1.1.519
4.10%
1.50%
D3
F3
S3
T3
D3
V3




B.1.526.1
3.90%
0.35%
D3
F3
S3
T3
D3
V3




B.1.526.2
2.90%
0.18%
D3
F3
S3
T3
D3
V3




B.1.596
1.70%
1.04%
D3
F3
S3
T3
D3
V3




R.1
1.20%
0.20%
D3
F3
S3
T3
D3
V3




B.1.575
1.10%
0.19%
D3
F3
S3
T3
D3
V3




B.1.243,
0.60%
0.84%
D3
F3
S3
T3
D3
V3




B.1.1.207



US
B.1.375
<1%
0.03%
D3
F3
S3
T3
D3
V3




B.1.1.1,
<1%
0.50%
D3
F3
S3
T3
D3
V3




B.1.416,




B.1.1.33,




B.1.311,




B.1.1.122



Brazil
B.1.1.28
<1%
0.10%
D3
F3
S3
T3
D3
F



Original



Andhra
B.1.36.29
<1%
0.08%
D3
F3
S3
T3
D3
V3



Pradesh




A.23.1
<1%
0.05%
D3
F3
S3
T3
D3
V3




A.27
<1%
0.05%
Y
F3
S3
T3
D3
V3




A.28
<1%
0.02%
D3
F3
S3
T3
D3
V3



Mink/
B.1.1.298
<1%
0.00%
D3
F3
S3
T3
D3
V3



Cluster V




B.1.1.318
<1%
0.01%
D3
F3
S3
T3
D3
V3




B.1.160
<1%
1.76%
D3
F3
S3
T3
D3
V3




B.1.177
<1%
3.19%
D3
F3
S3
T3
D3
V3




B.1.177.80
<1%
0.04%
D3
F3
S3
T3
D3
V3




B.1.258
<1%
1.15%
D3
F3
S3
T3
D3
V3




B.1.258.14
<1%
0.06%
D3
F3
S3
T3
D3
V3




B.1.258.17
<1%
1.02%
D3
F3
S3
T3
D3
V3




B.1.517
<1%
0.25%
D3
F3
S3
T3
D3
V3



WUHAN
WUHAN


D3
F3
S3
T3
D3
V3













PCR Amplimer length (bases)






1AA mutation - hybridizes to mutation specific probe




2AA identical to hCoV-19/Wuhan/WIV04/2019 (WIV04) - official reference sequence employed by GISAID (EPI_ISL_402124)




3Potential probe target














TABLE 20







Plate map, PCR recipe and Cycling conditions used in the analysis










RT-PCR Mix











Per
RT-PCR conditions












Plate Map

reaction

Temp

















1
2
Components
(μL)
Steps
(° C.)
Time
Cycles




















A
300 copies
300
ACCESSQUICK ™
25
1
55
20
min
1



S. Africa
copies
Mastermix




UK


B
100 copies
100
Primer
2
2
94
2
min
1



S. Africa
copies




UK


C
300 copies
300
Avian
1
3
94
30
s
45



California
copies
Myeloblastosis




Wuhan
Virus (AMV)




gRNA
Enzyme mix


D
100 copies
100
Water
17
4
55
30
s



California
copies




Wuhan




gRNA




E
300 copies
NTC
Total
45
5
68
30
s



India















F
100 copies
NTC

6
68
7
min
1



India














G
300 copies
NTC

7
4





Brazil











H
100 copies
NTC





Brazil









Results


FIGS. 18A-18E and Table 21 show the results from analysis of synthetic clade variant standards at TriCore. The data shows that the raw data, i.e. the ratio of Mutant (open bars) to Wild Type signal (black bars) readily identifies the state of each of the ten (10) target domains. Spike target sites, which were expected to display a “Mutant” signal (i.e. open bars>black bars), are marked in square brackets. As shown, the Mutant vs Wild type signals obtained by TriCore on synthetic Clade variant standards were as expected. The data established that the DETECTX-Cv workflow is easily deployable in any high throughput COVID-19 clinical testing lab.









TABLE 21





DETECTX-Cv analysis of synthetic Clade


variant standards at TriCore







Reference FIG. 18A












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












H/V69
ON

100.00%



_80A

ON
100.00%



D138
ON

100.00%



W152
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484

ON
100.00%



_501Y

ON
100.00%



P681
ON

100.00%



_701V

ON
100.00%







UNHYBRIDIZED PROBES












_152C

OFF
98.80%



_681H

OFF
100.00%



A701
OFF

97.40%













Pattern consistent with S Africa (B.1.351)



Reference FIG. 18B












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












H/V69
ON

100.00%



D80
ON

100.00%



D138
ON

100.00%



_152C

ON
100.00%



N439
ON

100.00%



N440
ON

100.00%



_452R

ON
100.00%



E484
ON

100.00%



N501
ON

100.00%



P681
ON

100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_484K

OFF
100.00%



_501Y

OFF
99.80%













Pattern consistent with California (B.1.429/427)



Reference FIG. 18C












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












H/V69
ON

100.00%



D80
ON

100.00%



D138
ON

100.00%



W152
ON

100.00%



N439
ON

100.00%



_440K

ON
100.00%



L452
ON

100.00%



E484
ON

100.00%



N501
ON

100.00%



P681
ON

100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_152C

OFF
98.80%



_484K

OFF
100.00%



_501Y

OFF
98.20%













Pattern consistent with India (N440K)



Reference FIG. 18D












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












H/V69
ON

100.00%



D80
ON

100.00%



_138Y

ON
100.00%



W152
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484
ON

100.00%



_501Y

ON
100.00%



P681
ON

100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












D138
OFF

100.00%



_152C

OFF
98.80%













Pattern consistent with Brazil (P1)



Reference FIG. 18E












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












_69(del)

ON
100.00%



D80
ON

100.00%



D138
ON

100.00%



W152
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484
ON

100.00%



_501Y

ON
100.00%



_681H

ON
100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












H/V69
OFF

100.00%



_484K

OFF
100.00%













Pattern consistent with UK (B.1.1.7)













TABLE 22







Hybridization plate map for 28 SARS-


CoV-2 positive clinical samples












3
4
5
6

















A
Sample 1
Sample 9
Sample 17
Sample 25



B
Sample 2
Sample 10
Sample 18
Sample 26



C
Sample 3
Sample 11
Sample 19
Sample 27



D
Sample 4
Sample 12
Sample 20
Sample 28



E
Sample 5
Sample 13
Sample 21



F
Sample 6
Sample 14
Sample 22



G
Sample 7
Sample 15
Sample 23



H
Sample 8
Sample 16
Sample 24










DETECTX-Cv Analysis of Clinical Positive Samples Performed at Tricore

The Biomerieux EASYMAG® Magnetic Bead platform (bioMérieux, St. Louis, Mo.) was used to extract Covid-19 RNA from 28 clinical positive (NP-VTM) samples (positivity previously determined by Cobas 6800 analysis). The extracted RNA (5 L) was processed using the DETECTX-Cv method. Table 22 shows a plate map for 28 SARS-CoV-2 positive clinical samples. The PCR recipe and cycling conditions were as described in Table 20.


Results FIGS. 19A-19K and Table 23 show the results of the DETECTX-Cv analysis for the clinical samples. It was determined that 68% (19/28) of samples generated data that passed QA/QC in terms of Universal Probe signal strength and were therefore fit for manual or autonomous Augury Clade calls. These data thus demonstrate that high quality DETECTX-Cv data is obtainable with minimal training on clinical positive samples.









TABLE 23





DETECTX-Cv analysis of clinical positive


samples performed at TriCore







Reference FIG. 19A TriCore Sample 2-B












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












H/V69
ON

100.00%



D80
ON

100.00%



D138
ON

100.00%



W152
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484
ON

100.00%



N501
ON

100.00%



P681
ON

100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_152C

OFF
98.80%



_484K

OFF
100.00%



_501Y

OFF
93.30%













Pattern consistent with: Wuhan Progenitor



Reference FIG. 19B TriCore Sample 7-B












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












H/V69
ON

100.00%



D80
ON

100.00%



D138
ON

100.00%



_152C

ON
100.00%



N439
ON

100.00%



N440
ON

100.00%



_452R

ON
100.00%



E484
ON

100.00%



N501
ON

100.00%



P681
ON

100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_80A

OFF
100.00%



_484K

OFF
100.00%



_501Y

OFF
100.00%













Pattern consistent with: California (B.1.429/427)







Reference FIG. 19C TriCore Sample 9-B












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












H/V69
ON

100.00%



D80
ON

100.00%



D138
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



_452R

ON
100.00%



E484
ON

100.00%



N501
ON

69.70%



P681
ON

100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_80A

OFF
100.00%



_138Y

OFF
100.00%



W152
OFF

100.00%



N439_/_440K

OFF
100.00%



L452
OFF

97.40%



_484K

OFF
100.00%



_501Y

OFF
100.00%



_701V

OFF
95.40%













Pattern consistent with: California (B.1.429/427)







Reference FIG. 19D TriCore Sample 17-B












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












_69(del)

ON
100.00%



D80
ON

100.00%



D138
ON

100.00%



W152
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484
ON

100.00%



_501Y

ON
100.00%



_681H

ON
100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












H/V69
OFF

100.00%



_152C

OFF
98.80%



_484K

OFF
100.00%













Pattern consistent with: UK (B.1.1.7)







Reference FIG. 19E TriCore Sample 18-B












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












_69(del)

ON
100.00%



D80
ON

100.00%



D138
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484
ON

100.00%



_501Y

ON
100.00%



_681H

ON
100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












H/V69
OFF

100.00%



_80A

OFF
100.00%



_138Y

OFF
100.00%



_152C

OFF
98.80%



W152
OFF

98.30%



N439_/_440K

OFF
100.00%



_484K

OFF
100.00%



N501
OFF

100.00%













Pattern consistent with: UK (B.1.1.7)







Reference FIG. 19F TriCore Sample 21-B












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












_69(del)

ON
100.00%



D80
ON

100.00%



D138
ON

100.00%



W152
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484
ON

100.00%



_501Y

ON
100.00%



_681H

ON
100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












H/V69
OFF

100.00%



_138Y

OFF
100.00%



_152C

OFF
98.80%



_484K

OFF
100.00%













Pattern consistent with: UK (B.1.1.7)



Reference FIG. 19G TriCore Sample 22-B












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












H/V69
ON

100.00%



D80
ON

100.00%



D138
ON

100.00%



W152
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484
ON

100.00%



N501
ON

100.00%



_681H

ON
100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_152C

OFF
98.80%



_484K

OFF
100.00%



_501Y

OFF
99.80%













Pattern consistent with: B.1.1.207



Reference FIG. 19H TriCore Sample 24-B












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












H/V69
ON

100.00%



D80
ON

100.00%



D138
ON

100.00%



W152
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484
ON

100.00%



N501
ON

100.00%



_681H

ON
100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_152C

OFF
97.60%



_501Y

OFF
98.60%













Pattern consistent with: B.1.1.207







Reference FIG. 191 TriCore Sample 27-B












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












H/V69
ON

100.00%



D80
ON

100.00%



D138
ON

100.00%



_152C

ON
85.40%



N439
ON

100.00%



N440
ON

100.00%



_452R

ON
100.00%



E484
ON

100.00%



N501
ON

100.00%



P681
ON

100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_80A

OFF
100.00%



_138Y

OFF
100.00%



_152C

OFF
69.40%



W152
OFF

100.00%



N439_/_440K

OFF
98.30%



_484K

OFF
100.00%



_501Y

OFF
100.00%













Pattern consistent with: California (B.1.429/427)







Reference FIG. 19J TriCore Sample 1-B










Probe
Wild Type
Mutant Type
Confidence










HYBRIDIZED PROBES










_69(del)

ON
87.50%







UNHYBRIDIZED PROBES










H/V69
OFF

99.70%


Pattern consistent with:


1
UK (B.1.1.7)


2
B.1.525


3
B.1.375


4
Denmark


5
B.1.258










Reference FIG. 19K TriCore Sample 4-B


START_WELL_20


SPECIMEN_ID Sample #20












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












_69(del)

ON
100.00%







UNHYBRIDIZED PROBES









Pattern consistent with: NA



END_WELL_20










DETECTX-Cv analysis of TriCore Clinical Positive Samples at Pathogen Dx

Sixty (60) clinical positive NP-VTM samples collected by TriCore were sent to PathogenDx for DETECTX-Cv analysis. RNA was extracted from these samples using the Zymo Magnetic Bead platform. The extracted RNA (5 L) was processed using the DETECTX-Cv method. The PCR recipe and cycling conditions were as described in Table 20.


Results


FIGS. 20A-20J and Table 24 show the results of this analysis. It was determined that 65% (39/60) of these samples generated data which passed QC/QA in terms of signal strength and were thus fit for manual or autonomous Augury Clade calls. The data shows all NP-VTM specimens which passed QA/QC and which displayed nonstandard clade variants (other than Wuhan) and representative data (2) for which QA/QC were inadequate either due to low RNA concentration or degraded RNA in the sample.









TABLE 24





DETECTX-Cv analysis of clinical positive


samples performed at PathogenDx







Reference FIG. 20A TriCore 238480 -d Sample 1












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












D80
ON

100.00%



D138
ON

100.00%



W152
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484
ON

100.00%



N501
ON

100.00%



P681
ON

100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_69(del)

OFF
100.00%



_80A

OFF
100.00%



_138Y

OFF
100.00%



_152C

OFF
98.80%



_452R

OFF
98.00%



_484K

OFF
100.00%



_501Y

OFF
100.00%



_681H

OFF
87.00%



_701V

OFF
100.00%













Pattern consistent with: Wuhan Progenitor







Reference FIG. 20B TriCore 238484-d Sample 4












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












D80
ON

100.00%



D138
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



_452R

ON
100.00%



E484
ON

100.00%



N501
ON

100.00%



P681
ON

100.00%



A701
ON

99.90%







UNHYBRIDIZED PROBES












_69(del)

OFF
100.00%



_80A

OFF
89.00%



_152C

OFF
98.80%



W152
OFF

100.00%



L452
OFF

100.00%



_484K

OFF
100.00%



_701V

OFF
100.00%













Pattern consistent with: No Clade Call, likely California







Reference FIG. 20C TriCore 238485-d Sample 5












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












D80
ON

100.00%



D138
ON

100.00%



_152C

ON
100.00%



N439
ON

100.00%



N440
ON

100.00%



_452R

ON
100.00%



E484
ON

100.00%



N501
ON

100.00%



P681
ON

100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_69(del)

OFF
100.00%



_80A

OFF
100.00%



W152
OFF

100.00%



L452
OFF

90.50%



_484K

OFF
100.00%



_501Y

OFF
100.00%



_681H

OFF
83.60%



_701V

OFF
99.90%













Pattern consistent with: California (B.1.429/427)







Reference FIG. 20D TriCore 238487-d Sample 6












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES


UNHYBRIDIZED PROBES












_69(del)

OFF
100.00%













Pattern consistent with: NA







Reference FIG. 20E TriCore 238488-d Sample 7












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












D80
ON

100.00%



D138
ON

100.00%



_152C

ON
100.00%



N439
ON

100.00%



N440
ON

100.00%



_452R

ON
100.00%



E484
ON

100.00%



N501
ON

100.00%



P681
ON

100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_69(del)

OFF
100.00%



_80A

OFF
100.00%



W152
OFF

100.00%



L452
OFF

98.50%



_484K

OFF
100.00%



_501Y

OFF
100.00%













Pattern consistent with: California (B.1.429/427)



Reference FIG. 20F TriCore 238498-d Sample 12












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












D80
ON

100.00%



D138
ON

100.00%



_152C

ON
97.60%



N439
ON

100.00%



N440
ON

100.00%



_452R

ON
100.00%



E484
ON

100.00%



P681
ON

100.00%



A701
ON

78.60%







UNHYBRIDIZED PROBES












_69(del)

OFF
100.00%



_80A

OFF
100.00%



_138Y

OFF
100.00%



W152
OFF

100.00%



L452
OFF

100.00%



_484K

OFF
100.00%



_501Y

OFF
100.00%



_681H

OFF
100.00%



_701V

OFF
100.00%













Pattern consistent with: California (B.1.429/427)



Reference FIG. 20G TriCore 238499-d Sample 13












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












D80
ON

100.00%



D138
ON

100.00%



_152C

ON
100.00%



N439
ON

100.00%



N440
ON

100.00%



_452R

ON
100.00%



E484
ON

100.00%



N501
ON

100.00%



P681
ON

100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_69(del)

OFF
100.00%



_80A

OFF
100.00%



_138Y

OFF
100.00%



W152
OFF

100.00%



L452
OFF

94.40%



_484K

OFF
100.00%



_501Y

OFF
100.00%



_681H

OFF
98.40%



_701V

OFF
100.00%













Pattern consistent with: California (B.1.429/427)



Reference FIG. 20H TriCore 238504-d Sample 16












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












D80
ON

100.00%



D138
ON

100.00%



W152
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484
ON

100.00%



N501
ON

100.00%



_681H

ON
100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_69(del)

OFF
99.70%



_80A

OFF
100.00%



_138Y

OFF
100.00%



_152C

OFF
97.40%



_452R

OFF
94.60%



_484K

OFF
100.00%



_501Y

OFF
100.00%



_701V

OFF
99.80%













Pattern consistent with: B.1.1.207



Reference FIG. 20I TriCore 236310-P Sample 39












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












D80
ON

100.00%



D138
ON

100.00%



W152
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484
ON

100.00%



N501
ON

100.00%



_681H

ON
100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_69(del)

OFF
100.00%



_80A

OFF
100.00%



_138Y

OFF
100.00%



_152C

OFF
98.80%



_452R

OFF
98.90%



_484K

OFF
100.00%



_501Y

OFF
100.00%



_701V

OFF
100.00%













Pattern consistent with: B.1.1.207



Reference FIG. 20J TriCore 236315-P Sample 42












Probe
Wild Type
Mutant Type
Confidence











HYBRIDIZED PROBES












D80
ON

100.00%



D138
ON

100.00%



W152
ON

100.00%



N439
ON

100.00%



N440
ON

100.00%



L452
ON

100.00%



E484
ON

100.00%



N501
ON

100.00%



P681
ON

100.00%



A701
ON

100.00%







UNHYBRIDIZED PROBES












_69(del)

OFF
100.00%



_80A

OFF
100.00%



_138Y

OFF
100.00%



_152C

OFF
98.80%



_452R

OFF
98.00%



_484K

OFF
100.00%



_501Y

OFF
100.00%



_681H

OFF
87.00%



_701V

OFF
100.00%













Pattern consistent with: Wuhan Progenitor






Conclusion

Described here is a “DETECTX-Cv” technology designed to combine the practicality of field deployable Q-RT-PCR testing with the high-level information content of targeted NGS. Population scale deployment of DETECTX-Cv is enabled in a way that is simple enough that it can be “drop-shipped” with minimal set up cost and training into any laboratory performing conventional Q-RT-PCR based COVID-19 screening. Initial field deployment demonstrated the ability of DETECTX-Cv to identify clinical positives per shift per Q-RT-PCR screening and analysis without additional sample prep for a large panel of CoV-2 clade variants (UK, Denmark, South Africa, Brazil, US (CA, NY, Southern US) and Wuhan) incorporated into the content of the assay.


In conclusion, the technology encompassed in this invention enables DETECTX-Cv to perform very low-cost microarray analyses in a field-deployable format. DETECTX-Cv is based on proprietary technology of PathogenDx for designing DNA microarray probes and so, the resulting microarrays can be mass produced to deliver >24,000 tests/day. DETECTX-Cv also enables sequence-based testing on these microarrays via open-format room temperature hybridization and washing, much like the processing of ELISA assays. Like an ELISA plate, DETECTX-Cv is mass produced in a 96-well format, ready for manual or automated fluid handling and has the capability to handle up to 576 probe spots per well, at full production scale.


DETECTX-Cv is a combinatorial assay with several targets in the CoV-2 Spike gene comprising an exceptionally large set of gain-of-function Spike mutants, which are believed to be selected for enhanced infectivity or resistance to natural or vaccine induced host immunity. Based on analysis of the rapidly growing CoV-2 resequencing effort (600,000 genomes in GISAID, April 2021) “terminal differentiation” of the Spike gene marker “basis set” into a set of about thirty-five (35) informative Spike gene target sites is expected, which can be built into and mass produced into the same 96-well format described above. DETECTX-Cv is therefore expected to be beneficial as a true discovery tool that is capable of unbiased identification of new CoV-2 clade variants based on detection of novel combinations of the underlying Spike Variant “basis set”. Thus, DETECTX-Cv is expected to become the basis for field deployed seasonal COVID testing with military and civilian applications


The DETECTX-Cv test content comprises Spike Gene Target sequence analysis among eleven (11) discrete information-rich domains, produced as triplicate tests per array, with positive and negative controls, to produce core content that is deployed as about 140 independent hybridization tests (per well) on each sample. The DETECTX-Cv technology described here is based on multiplex (n=5) asymmetric, endpoint RT-PCR amplification of viral RNA purified as for Q-RT-PCR screening. The RT-PCR product resulting from amplification is fluorescently tagged and used as-is without cleanup for the subsequent steps of hybridization and washing, which are performed at room temperature (RT). Subsequent to hybridization and washing, the DETECTX-Cv plate is subjected to fluorescent plate reading, data processing and analysis that occurs automatically with no intervention by a human user to result in an output of detected CoV-2 clades which can be used locally for diagnosis and/or simultaneously uploaded to a secure, cloud-based portal for use by medical officials for military or public health tracking and epidemiology analysis.

Claims
  • 1. A method for detecting clade variants in a Coronavirus disease 2019 virus (COVID-19) in a sample, comprising: obtaining the sample;harvesting viruses from the sample;isolating a total RNA from the harvested viruses;performing a combined reverse transcription and first amplification reaction on the total RNA using at least one first primer pair selective for all COVID-19 viruses to generate COVID-19 virus cDNA amplicons;performing a second amplification using the COVID-19 virus cDNA amplicons as template and at least one fluorescent labeled second primer pair selective for a target nucleotide sequence in the COVID-19 virus cDNA to generate at least one fluorescent labeled COVID-19 virus amplicon;hybridizing the fluorescent labeled COVID-19 virus amplicons to a plurality of nucleic acid probes, each having a sequence corresponding to a sequence determinant that discriminates among the clade variants of the COVID-19 virus, said nucleic acid probes attached to a solid microarray support;washing the microarray at least once; andimaging the microarray to detect at least one fluorescent signal from the hybridized fluorescent labeled COVID-19 virus amplicons, thereby detecting the clade variants of the COVID-19 virus in the sample.
  • 2. The method of claim 1, wherein prior to the harvesting step the method further comprises mixing the sample with an RNA stabilizer.
  • 3. The method of claim 1, wherein one or more of the at least one fluorescent labeled second primer pair is selective for a panel of target nucleotide sequences within a target region of a gene in the COVID-19 virus; andwherein the nucleic acid probes are specific to the target region of the gene, whereby the at least one fluorescent labeled COVID-19 virus amplicon generated is hybridized to the nucleic acid probe thereby discriminating the clade variants of the COVID-19 virus in the sample.
  • 4. The method of claim 3, further comprising: detecting the at least one fluorescent signal from the hybridized at least one fluorescent labeled COVID-19 virus amplicons associated with the panel of target nucleotide sequences within the target region of the gene; andgenerating an intensity distribution profile unique to each of the clade variants, whereby each of the clade variants is distinguishable from others.
  • 5. The method of claim 3, where the gene is a Spike gene.
  • 6. The method of claim 1, wherein the clade variants of the COVID-19 virus are Denmark, UK (B.1.1.7), South African (B.1.351), Brazil/Japan (P1), Brazil (B1.1.28), California USA, L452R (1.429), India (N440K), or Wuhan, or a combination thereof.
  • 7. The method of claim 1, wherein the first primer pair comprises the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, or SEQ ID NO: 3 and SEQ ID NO: 4, or SEQ ID NO: 5 and SEQ ID NO: 6, or SEQ ID NO: 7 and SEQ ID NO: 8, or a combination thereof.
  • 8. The method of claim 1, wherein the fluorescent labeled second primer pair comprises the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 10, or SEQ ID NO: 11 and SEQ ID NO: 12, or SEQ ID NO: 13 and SEQ ID NO: 14, or SEQ ID NO: 15 and SEQ ID NO: 16, or SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 24, or a combination thereof.
  • 9. The method of claim 1, wherein the nucleic acid probes comprise at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 30-129.
  • 10. The method of claim 1, wherein the sample comprises at least one of a nasopharyngeal swab, a nasal swab, a mouth swab, a mouthwash, an aerosol, or a swab from a hard surface.
  • 11. A method for detecting clade variants in a Coronavirus disease 2019 virus (COVID-19) in a sample, comprising: obtaining the sample;harvesting viruses from the sample;isolating total RNA from the harvested viruses;performing a combined reverse transcription and asymmetric PCR amplification reaction on the total RNA using at least one fluorescent labeled primer pair comprising an unlabeled primer and a fluorescently labeled primer, selective for a target sequence in all COVID-19 viruses to generate at least one fluorescent labeled COVID-19 virus amplicon;hybridizing the fluorescent labeled COVID-19 virus amplicons to a plurality of nucleic acid probes, each having a sequence corresponding to a sequence determinant that discriminates among the clade variants of the COVID-19 virus, said nucleic acid probes attached to a solid microarray support;washing the microarray at least once; andimaging the microarray to detect at least one fluorescent signal from the hybridized fluorescent labeled COVID-19 virus amplicons, thereby detecting the clade variants of the COVID-19 virus in the sample.
  • 12. The method of claim 11, wherein prior to the harvesting step the method further comprises mixing the sample with an RNA stabilizer.
  • 13. The method of claim 11, wherein one or more of the at least one fluorescent labeled second primer pair is selective for a panel of target nucleotide sequences within a target region of a gene in the COVID-19 virus; and wherein the nucleic acid probes are specific to the target region of the gene, whereby the at least one fluorescent labeled COVID-19 virus amplicon generated is hybridized to the nucleic acid probe thereby discriminating the clade variants of the COVID-19 virus in the sample.
  • 14. The method of claim 13, further comprising: detecting the at least one fluorescent signal from the hybridized at least one fluorescent labeled COVID-19 virus amplicons associated with the panel of target nucleotide sequences within the target region of the gene; andgenerating an intensity distribution profile unique to each of the clade variants, whereby each of the clade variants is distinguishable from others.
  • 15. The method of claim 13, where the gene is a Spike gene.
  • 16. The method of claim 11, wherein the clade variants of the COVID-19 virus are Denmark, UK (B.1.1.7), or South African (B.1.351), or Brazil/Japan (P1), or Brazil (B1.1.28), or California USA, or L452R (1.429), or India (N440K), or Wuhan, or a combination thereof.
  • 17. The method of claim 11, wherein the fluorescent labeled primer pair comprises the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 10, or SEQ ID NO: 11 and SEQ ID NO: 12, or SEQ ID NO: 13 and SEQ ID NO: 14, or SEQ ID NO: 15 and SEQ ID NO: 16, or SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 24, or a combination thereof.
  • 18. The method of claim 11, wherein the fluorescently labeled primer is in an excess of about 4-fold to about 8-fold over the unlabeled primer in the fluorescent labeled primer pair.
  • 19. The method of claim 11, wherein the nucleic acid probes comprise at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 30-129.
  • 20. The method of claim 11, wherein the sample comprises as least one of a nasopharyngeal swab, a nasal swab, a mouth swab, a mouthwash, an aerosol, or a swab from a hard surface.
CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional application claims the benefit of priority under 35 U.S.C. § 119(e) of provisional application U.S. Ser. No. 63/147,613, filed Feb. 9, 2021, hereby incorporated by reference in its entirety.

Provisional Applications (1)
Number Date Country
63147613 Feb 2021 US