Patent Grant
11149320
This application includes one or more Sequence Listings pursuant to 37 C.F.R. 1.821 et seq., which are disclosed in computer-readable media (file name: SARS-CoV-2_0400_0020US2_ST25.txt, created on Oct. 18, 2020, and having a size of 156,199 bytes), which file is herein incorporated by reference in its entirety.
The present invention is directed to methods for assaying for the presence of SARS-CoV-2 in a sample, including a clinical sample, and to oligonucleotides, reagents, and kits useful in such assays. In particular, the present invention is directed to such assays that are rapid, accurate and specific for the detection of SARS-CoV-2.
I. SARS-CoV-2
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a newly identified coronavirus species (the virus was previously provisionally named “2019 novel coronavirus” or “2019-nCoV”). SARS-CoV-2 infection is spread by human-to-human transmission via droplets or direct contact, and infection has been estimated to have a mean incubation period of 6.4 days and a Basic Reproduction Number of 2.24-3.58 (i.e., an epidemic doubling time of 6-8 days) (Fang, Y. et al. (2020) “Transmission Dynamics Of The COVID-19 Outbreak And Effectiveness Of Government Interventions: A Data-Driven Analysis,” J. Med. Virol. doi: 10.1002/jmv.25750; Zhao, W. M. et al. (2020) “The 2019 Novel Coronavirus Resource,” Yi Chuan. 42(2):212-221; Zhu, N. et al. (2020) “A Novel Coronavirus from Patients with Pneumonia in China, 2019,” New Engl. J. Med. 382(8):727-733).
Patients infected with SARS-CoV-2 exhibit COVID-19, a condition initially characterized by fever and cough (Kong, I. et al. (2020) “Early Epidemiological and Clinical Characteristics of 28 Cases of Coronavirus Disease in South Korea,” Osong Public Health Res Perspect. 11(1):8-14). In approximately 20% of patients, COVID-19 progresses to a severe respiratory disease and pneumonia that has a mortality of 5-10% (1-2% overall mortality). Bilateral lung involvement with ground-glass opacity are the most common finding from computed tomography images of the chest (Lai, C C. et al. (2020) “Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) And Coronavirus Disease-2019 (COVID-19): The Epidemic And The Challenges,” Int. J. Antimicrob. Agents. 55(3):105924). Since a cure for COVID-19 has not yet been identified, treatment presently consists of a “Four-Anti and Two-Balance” strategy included antivirus, anti-shock, anti-hyoxemia, anti-secondary infection, and maintaining water, electrolyte and acid-base balance and microecological balance (Xu, K. et al. (2020) “Management Of Corona Virus Disease-19 (COVID-19): The Zhejiang Experience,” Zhejiang Da Xue Bao Yi Xue Ban. 49(1):0).
Coronaviruses (CoVs) belong to the subfamily Orthocoronavirinae in the family Coronaviridae and the order Nidovirales. The Coronaviridae family of viruses are enveloped, single-stranded, RNA viruses that possess a positive-sense RNA genome of 26 to 32 kilobases in length. Four genera of coronaviruses have been identified, namely, Alphacoronavirus (αCoV), Betacoronavirus (βCoV), Deltacoronavirus (δCoV), and Gammacoronavirus (γCoV) (Chan, J. F. et al. (2013) “Interspecies Transmission And Emergence Of Novel Viruses: Lessons From Bats And Birds,” Trends Microbiol. 21(10):544-555). Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs.
Prior to 2019, only six coronavirus species were known to be pathogenic to humans. Four of these species were associated with mild clinical symptoms, but two coronaviruses, Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV) (Marra, M. A. et al. (2003) “The Genome Sequence of the SARS-Associated Coronavirus,” Science 300(5624):1399-1404) and Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV) (Mackay, I. M. (2015) “MERS Coronavirus: Diagnostics, Epidemiology And Transmission,” Virol. J. 12:222. doi: 10.1186/s12985-015-0439-5) were associated with human mortalities approaching 10% (Su, S. et al. (2016) “Epidemiology, Genetic Recombination, And Pathogenesis Of Coronaviruses,” Trends Microbiol. 24:490-502; Al Johani, S. et al. (2016) “MERS-CoV Diagnosis: An Update,” J. Infect. Public Health 9(3):216-219).
SARS-CoV-2 is closely related (88%) to two bat-derived Severe Acute Respiratory Syndrome-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, and is more distantly related to SARS-CoV (79%) and MERS-CoV (50%) (Xie, C. et al. (2020) “Comparison Of Different Samples For 2019 Novel Coronavirus Detection By Nucleic Acid Amplification Tests” Int. J. Infect. Dis. /doi.org/10.1016/j.ijid.2020.02.050; Mackay, I. M. (2015) “MERS Coronavirus: Diagnostics, Epidemiology And Transmission,” Virol. J. 12:222. doi: 10.1186/s12985-015-0439-5; Gong, S. R. et al. (2018) “The Battle Against SARS And MERS Coronaviruses: Reservoirs And Animal Models,” Animal Model Exp. Med. 1(2):125-133; Yin, Y. et al. (2018) “MERS, SARS And Other Coronaviruses As Causes Of Pneumonia,” Respirology 23(2):130-137). Phylogenetic analysis revealed that SARS-CoV-2 fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV (Drosten et al. (2003) “Identification Of A Novel Coronavirus In Patients With Severe Acute Respiratory Syndrome,” New Engl. J. Med. 348:1967-1976; Lai, C C. et al. (2020) “Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) And Coronavirus Disease-2019 (COVID-19): The Epidemic And The Challenges,” Int. J. Antimicrob. Agents. 55(3):105924; Lu, R. et al. (2020) “Genomic Characterisation And Epidemiology Of 2019 Novel Coronavirus: Implications For Virus Origins And Receptor Binding,” The Lancet 395(10224):565-574; Zhou, Y. et al. (2020) “Network-Based Drug Repurposing For Novel Coronavirus 2019-nCoV/SARS-CoV-2,” Cell Discov. 6(14): doi.org/10.1038/s41421-020-0153-3).
The SARS-CoV-2 genome has been sequenced from at least 170 isolates. The reference sequence is GenBank NC_045512 (Wang, C. et al. (2020) “The Establishment Of Reference Sequence For SARS-CoV-2 And Variation Analysis,” J. Med. Virol. doi: 10.1002/jmv.25762; Chan, J. F. et al. (2020) “Genomic Characterization Of The 2019 Novel Human-Pathogenic Coronavirus Isolated From A Patient With Atypical Pneumonia After Visiting Wuhan,” Emerg. Microbes. Infect. 9(1):221-236).
Comparisons of the sequences of multiple isolates of the virus (MN988668 and NC_045512, isolated from Wuhan, China, and MN938384.1, MN975262.1, MN985325.1, MN988713.1, MN994467.1, MN994468.1, and MN997409.1) reveal greater than 99.99% identity (Sah, R. et al. (2020) “Complete Genome Sequence of a 2019 Novel Coronavirus (SARS-CoV-2) Strain Isolated in Nepal,” Microbiol. Resource Announcements 9(11): e00169-20, pages 1-3; Brussow, H. (2020) “The Novel Coronavirus-A Snapshot of Current Knowledge,” Microbial Biotechnology 0:(0):1-6).
The SARS-CoV-2 genome is highly similar to that of human SARS-CoV, with an overall nucleotide identity of approximately 82% (Chan, J. F. et al. (2020) “Genomic Characterization Of The 2019 Novel Human-Pathogenic Corona Virus Isolated From A Patient With Atypical Pneumonia After Visiting Wuhan,” Emerg Microbes Infect 9:221-236; Chan, J. F. et al. (2020) “Improved Molecular Diagnosis Of COVID-19 By The Novel, Highly Sensitive And Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-Polymerase Chain Reaction Assay Validated In Vitro And With Clinical Specimens,” J Clin. Microbiol. JCM.00310-20. doi: 10.1128/JCM.00310-20). Based on its homology to related coronaviruses, SARS-CoV-2 is predicted to encode 12 open reading frame (ORFs) coding regions (ORF1ab, S (spike protein), 3, E (envelope protein), M (matrix), 7, 8, 9, 10b, N, 13 and 14. The arrangement of these coding regions is shown in
A. ORF1ab
ORF1ab is composed of 21290 nucleotides and encodes an open reading frame of 7096 amino acids in length. Via a −1 ribosomal frameshift, the encoded protein is a polyprotein (pp) composed of a first segment (pp1a) of 4401 amino acid residues, and a second segment (pp1ab) of 2695 amino acid residues (Chen, Y, et al. (2020) “Emerging Coronaviruses: Genome Structure, Replication, And Pathogenesis,” J. Med. Virol. 92:418-423; Lu, R. et al. (2020) “Genomic Characterisation And Epidemiology Of 2019 Novel Coronavirus: Implications For Virus Origins And Receptor Binding,” Lancet 395(10224):565-574). Both segments include the same 180 amino acid long leader sequence. The polyprotein includes multiple non-structural proteins (nsp): a 638 amino acid long nsp2 protein, a 1945 amino acid long nsp3 protein, a 500 amino acid long nsp4 protein, a 306 amino acid long nsp5 protein, a 290 amino acid long nsp6 protein, an 83 amino acid long nsp7 protein, a 198 amino acid long nsp8 protein, a 113 amino acid long nsp9 single-strand binding protein, a 139 amino acid long nsp10 protein, a 923 amino acid long nsp12 RNA-dependent RNA polymerase (RdRp), a 601 amino acid long nsp13 helicase, a 527 amino acid long nsp14a2 3→5′ exonuclease, a 346 amino acid long nsp15 endoRNAse, and a 298 amino acid long nsp16 2′-O-ribose-methyltransferase (Chen, Y, et al. (2020) “Emerging Coronaviruses: Genome Structure, Replication, And Pathogenesis,” J. Med. Virol. 92:418-423; Lu, R. et al. (2020) “Genomic Characterisation And Epidemiology Of 2019 Novel Coronavirus: Implications For Virus Origins And Receptor Binding,” Lancet 395(10224):565-574).
The sequence of the positive sense (“sense”) strand of the ORF1ab of SARS-CoV-2 of GenBank NC_045512 (SEQ ID NO:415) is shown in Table 1.
B. The S Gene
The S gene encodes the SARS-CoV-2 spike protein. The S protein of SARS-CoV is functionally cleaved into two subunits: the S1 domain and the S2 domain (He, Y. et al. (2004) “Receptor-Binding Domain Of SARS-CoV Spike Protein Induces Highly Potent Neutralizing Antibodies: Implication For Developing Subunit Vaccine,” Biochem. Biophys. Res. Commun. 324:773-781). The SARS-CoV S1 domain mediates receptor binding, while the SARS-CoV S2 domain mediates membrane fusion (Li, F. (2016) “Structure, Function, And Evolution Of Coronavirus Spike Proteins,” Annu. Rev. Virol. 3:237-261; He, Y. et al. (2004) “Receptor-Binding Domain Of SARS-CoV Spike Protein Induces Highly Potent Neutralizing Antibodies: Implication For Developing Subunit Vaccine, Biochem. Biophys. Res. Commun. 324:773-781). The S gene of SARS-CoV-2 may have a similar function. Thus, the spike protein of coronaviruses is considered crucial for determining host tropism and transmission capacity (Lu, G. et al. (2015) “Bat-To-Human: Spike Features Determining ‘Host Jump’ Of Coronaviruses SARS-CoV, MERS-CoV, And Beyond,” Trends Microbiol. 23:468-478; Wang, Q. et al. (2016) “MERS-CoV Spike Protein: Targets For Vaccines And Therapeutics,” Antiviral. Res. 133:165-177). In this regard, the S2 domain of the SARS-CoV-2 spike protein shows high sequence identity (93%) with bat-SL-CoVZC45 and bat-SL-CoVZXC21, but the SARS-CoV-2 S1 domain shows a much lower degree of identity (68%) with these bat-derived viruses (Lu, R. et al. (2020) “Genomic Characterisation And Epidemiology Of 2019 Novel Coronavirus: Implications For Virus Origins And Receptor Binding,” Lancet 395(10224):565-574). Thus, SARS-CoV-2 may bind to a different receptor than that bound by its related bat-derived viruses. It has been proposed that SARS-CoV-2 may bind to the angiotensin-converting enzyme 2 (ACE2) as a cell receptor (Lu, R. et al. (2020) “Genomic Characterisation And Epidemiology Of 2019 Novel Coronavirus: Implications For Virus Origins And Receptor Binding,” Lancet 395(10224):565-574).
The sequence of the positive sense (“sense”) strand of the S Gene of SARS-CoV-2 of GenBank NC_045512 (SEQ ID NO: 16) is shown in Table 2.
II. Assays for the Detection of SARS-CoV-2
SARS-CoV-2 was first identified in late 2019, and is believed to be a unique virus that had not previously existed. The first diagnostic test for SARS-CoV-2 used a real-time reverse transcription-PCR (rRT-PCR) assay that employed probes and primers of the SARS-CoV-2E, N and nsp2 (RNA-dependent RNA polymerase; RdRp) genes (the “SARS-CoV-2-RdRp-P2” assay) (Corman, V. M. et al. (2020) “Detection Of 2019 Novel Coronavirus (209-nCoV) By Real-Time RT-PCR,” Eurosurveill. 25(3):2000045; Spiteri, G. et al. (2020) “First Cases Of Coronavirus Disease 2019 (COVID-19) In The WHO European Region, 24 Jan. To 21 Feb. 2020,” Eurosurveill. 25(9) doi: 10.2807/1560-7917.ES.2020.25.9.2000178).
The probes employed in such assays were “TaqMan” oligonucleotide probes that were labeled with a fluorophore on the oligonucleotide's 5′ terminus and complexed to a quencher on the oligonucleotide's 3′ terminus. The “TaqMan” probe principle relies on the 5→3′ exonuclease activity of Taq polymerase (Peake, I. (1989) “The Polymerase Chain Reaction,” J. Clin. Pathol.; 42(7):673-676) to cleave the dual-labeled probe when it has hybridized to a complementary target sequence. The cleavage of the molecule separates the fluorophore from the quencher and thus leads to the production of a detectable fluorescent signal.
In the SARS-CoV-2-RdRp-P2 assay of Corman, V. M. et al. (2020), the RdRp Probe 2 and the probes of the E and N genes are described as being specific for SARS-CoV-2, whereas the RdRp Probe 2 is described as being a “PanSarbeco-Probe” that detects SARS-CoV and bat-SARS-related coronaviruses in addition to SARS-CoV-2. The assay is stated to have provided its best results using the E gene and nsp12 (RdRp) gene primers and probes (5.2 and 3.8 copies per 25 μL reaction at 95% detection probability, respectively). The resulting limit of detection (LoD) from replicate tests was 3.9 copies per 25 μL reaction (156 copies/mL) for the E gene assay and 3.6 copies per 25 μL reaction (144 copies/mL) for the nsp12 (RdRp) assay. The assay was reported to be specific for SARS-CoV-2 and to require less than 60 minutes to complete.
The US Center for Disease Control and Prevention (CDC) developed an rRT-PCR based assay protocol that targeted the SARS-CoV-2 N gene (Won, J. et al. (2020) “Development Of A Laboratory-Safe And Low-Cost Detection Protocol For SARS-CoV-2 Of The Coronavirus Disease 2019 (COVID-19),” Exp. Neurobiol. 29(2) doi: 10.5607/en20009).
Pfefferle, S. et al. (2020) (“Evaluation Of A Quantitative RT-PCR Assay For The Detection Of The Emerging Coronavirus SARS-CoV-2 Using A High Throughput System,” Eurosurveill. 25(9) doi: 10.2807/1560-7917.ES.2020.25.9.2000152) discloses the use of a custom-made primer/probe set targeting the E gene. The employed primers were modified with 2′-O-methyl bases in their penultimate base to prevent formation of primer dimers. ZEN double-quenched probe (IDT) were used to lower background fluorescence. The LoD was 689.3 copies/mL with 275.72 copies per reaction at 95% detection probability. The assay was reported to be specific for SARS-CoV-2 and to require less than 60 minutes.
Chan, J. F. et al. (2020) (“Improved Molecular Diagnosis Of COVID-19 By The Novel, Highly Sensitive And Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-Polymerase Chain Reaction Assay Validated In Vitro And With Clinical Specimens,” J. Clin. Microbiol. JCM.00310-20. doi: 10.1128/JCM.00310-20) explored the use of conserved and/or abundantly expressed SARS-CoV-2 genes as preferred targets of coronavirus RT-PCR assays. Such genes include the structural S and N genes, and the non-structural RdRp gene and ORF1ab. Chan, J. F. et al. (2020) describes the development of three real-time reverse transcriptase PCR (rRT-PCR) assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 and compares such assays to the RdRp-P2 assay of Corman, V. M. et al. The LoD of the SARS-CoV-2-RdRp/Hel assay, the SARS-CoV-2-S assay, and the SARS-CoV-2-N assay was 1.8 TCID50/ml, while the LoD of the SARS-CoV-2-RdRp-P2 assay was 18 TCID50/ml. The TCID50 is the median tissue culture infectious dose.
An rt-PCR-based assay protocol targeting the E, N, S and RdRp genes was designed for specimen self-collection from a subject via pharyngeal swab. The assay required Trizol-based RNA purification, and detection was accomplished via an RT-PCR assay using SYBR Green as a detection fluor. The assay was reported to require approximately 4 hours to complete (Won, J. et al. (2020) (“Development Of A Laboratory-Safe And Low-Cost Detection Protocol For SARS-CoV-2 Of The Coronavirus Disease 2019 (COVID-19),” Exp. Neurobiol. 29(2) doi: 10.5607/en20009).
Although prior rRT-PCR assays, such as the SARS-CoV-2-RdRp-P2 assay of Corman V. M. et al., are capable of detecting SARS-CoV-2, researchers have found them to suffer from major deficiencies. In use, such prior assays have been found to require laborious batch-wise manual processing and to not permit random access to individual samples (Cordes, A. K. et al. (2020) “Rapid Random Access Detection Of The Novel SARS-Coronavirus-2 (SARS-CoV-2, Previously 2019-nCoV) Using An Open Access Protocol For The Panther Fusion,” J. Clin. Virol. 125:104305 doi: 10.1016/j.jcv.2020.104305). Additionally, long turnaround times and complicated operations are required. These factors cause such assays to generally take more than 2-3 hours to generate results. Due to such factors, certified laboratories are required to process such assays. The need for expensive equipment and trained technicians to perform such prior rRT-PCR assays encumbers the use of such assays in the field or at mobile locations. Thus, researchers have found such prior assays to have limited suitability for use in the rapid and simple diagnosis and screening of patients required to contain an outbreak (Li, Z. et al. (2020) “Development and Clinical Application of A Rapid IgM-IgG Combined Antibody Test for SARS-CoV-2 Infection Diagnosis,” J. Med. Virol. doi: 10.1002/jmv.25727).
More significantly, prior rRT-PCR assays, such as the SARS-CoV-2-RdRp-P2 assay of Corman V. M. et al., have been found to lack specificity for SARS-CoV-2 (cross-reacting with SARS-CoV or other pathogens) (Chan, J. F. et al. (2020) “Improved Molecular Diagnosis Of COVID-19 By The Novel, Highly Sensitive And Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-Polymerase Chain Reaction Assay Validated In Vitro And With Clinical Specimens,” J. Clin. Microbiol. JCM.00310-20) and to provide a significant number of false negative results (Li, Z. et al. (2020) “Development and Clinical Application of A Rapid IgM-IgG Combined Antibody Test for SARS-CoV-2 Infection Diagnosis,” J. Med. Virol. doi: 10.1002/jmv.25727).
For example, in a retrospective analysis of 4880 clinically-identified COVID-19 patients, samples obtained from the respiratory tracts of the patients were subjected to rRT-PCR amplification of the SARS-CoV-2 open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) genes. Nasal and pharyngeal swabs of patients were evaluated for COVID-19 using a quantitative rRT-PCR (qRT-PCR) test. Only 38.42% (1875 of 4880) of actual COVID-19 patients were identified as positive using the rRT-PCR test. Of those testing positive, 39.80% were positive as determined by probes of the SARS-CoV-2 N gene and 40.98% were positive as determined by probes of the SARS-CoV-2 ORF1ab (Liu, R. et al. (2020) “Positive Rate Of RT-PCR Detection Of SARS-CoV-2 Infection In 4880 Cases From One Hospital In Wuhan, China, From January To February 2020,” Clinica Chimica Acta 505:172-175).
The study of Chan, J. F. et al. (2020) (“Improved Molecular Diagnosis Of COVID-19 By The Novel, Highly Sensitive And Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-Polymerase Chain Reaction Assay Validated In Vitro And With Clinical Specimens,” J. Clin. Microbiol. JCM.00310-20. doi: 10.1128/JCM.00310-20) found that of 273 specimens from 15 patients with laboratory-confirmed COVID-19, only 28% were SARS-CoV-2 positive by both the SARS-CoV-2-RdRp/Hel and RdRp-P2 assays. The SARS-CoV-2-RdRp/Hel assay was more sensitive, but still confirmed only 43.6% of the patients as having SARS-CoV-2 infection.
In a different study, RNA was extracted from 1070 clinical samples of 205 patients suffering from COVID-19. Real-time reverse transcription-PCR (rRT-PCR) was then used to amplify SARS-CoV-2 ORF1ab in order to confirm the COVID-19 diagnosis (Wang, W. et al. (2020) (“Detection of SARS-CoV-2 in Different Types of Clinical Specimens,” JAMA doi: 10.1001/jama.2020.3786). Bronchoalveolar lavage fluid specimens were reported to exhibit the highest positive rates (14 of 15; 93%), followed by sputum (72 of 104; 72%), nasal swabs (5 of 8; 63%), fibrobronchoscope brush biopsy (6 of 13; 46%), pharyngeal swabs (126 of 398; 32%), feces (44 of 153; 29%), and blood (3 of 307; 1%). None of the 72 urine specimens tested indicated a positive result. Thus, for example, pharyngeal swabs from such actual COVID-19 patients failed to accurately diagnose SARS-CoV-2 infection in 68% of those tested. Zhang, W. et al. (2020) (“Molecular And Serological Investigation Of 2019-nCoV Infected Patients: Implication Of Multiple Shedding Routes,” Emerg. Microbes Infect. 9(1):386-389) also discloses the presence of SARS-CoV-2 in feces of COVID-19 patients, however, its rRT-PCR assay results showed more anal swab positives than oral swab positives in a later stage of infection, suggesting viral shedding and the capacity of the infection to be transmitted through an oral-fecal route. A similar teaching is provided by Tang, A. et al. (2020) (“Detection of Novel Coronavirus by RT-PCR in Stool Specimen from Asymptomatic Child, China,” Emerg Infect Dis. 26(6). doi: 10.3201/eid2606.200301), which discloses that RT-PCR assays targeting ORF1ab and nucleoprotein N gene failed to detect SARS-CoV-2 in nasopharyngeal swab and sputum samples, but were able to detect virus in stool samples.
In a further study of individuals suffering from COVID-19, repeated assays for SARS-CoV-2 were also found to report negative results (Wu, X. et al. (2020) (“Co-infection with SARS-CoV-2 and Influenza A Virus in Patient with Pneumonia, China,” 26(6):pages 1-7. The publication teaches that existing assays for SARS-CoV-2 lack sufficient sensitivity, and thus lead to false negative diagnoses.
In light of the deficiencies encountered in using prior rRT-PCR assays, such as the SARS-CoV-2-RdRp-P2 assay of Corman V. M. et al., other approaches to assaying for SARS-CoV-2 have been explored. Li, Z. et al. (2020) (“Development and Clinical Application of A Rapid IgM-IgG Combined Antibody Test for SARS-CoV-2 Infection Diagnosis,” J. Med. Virol. doi: 10.1002/jmv.25727) teaches that a point-of-care lateral flow immunoassay could be used to simultaneously detect anti-SARS-CoV-2 IgM and IgG antibodies in human blood and thus avoid the problems of the RdRp-P2 assay of Corman, V. M. et al. Immunoassays, however, may fail to discriminate between individuals suffering from COVID-19 and individuals who were previously infected with SARS-CoV-2, but have since recovered.
In sum, despite all prior efforts a need remains for a method of rapidly and accurately assaying for the presence of SARS-CoV-2. The present invention is directed to this and other goals.
The present invention is directed to methods for assaying for the presence of SARS-CoV-2 in a sample, including a clinical sample, and to oligonucleotides, reagents, and kits useful in such assays. In particular, the present invention is directed to such assays that are rapid, accurate and specific for the detection of SARS-CoV-2.
In detail, the invention provides a detectably labeled oligonucleotide that is capable of specifically hybridizing to a SARS-CoV-2 polynucleotide, wherein the detectably labeled oligonucleotide has a nucleotide sequence that is able to specifically hybridize to an oligonucleotide having a nucleotide sequence that consists of the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7 or SEQ ID NO:8.
The invention additionally provides a kit for detecting the presence of SARS-CoV-2 in a clinical sample, wherein the kit comprises a detectably labeled oligonucleotide that is capable of specifically hybridizing to a SARS-CoV-2 polynucleotide, wherein the detectably labeled oligonucleotide has a nucleotide sequence that is able to specifically hybridize to an oligonucleotide having the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7 or SEQ ID NO:8.
The invention additionally provides the embodiment of such kit, wherein the detectably labeled oligonucleotide has a nucleotide sequence that is able to specifically hybridize to an oligonucleotide having the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4, and wherein the kit permits a determination of the presence or absence of the SARS-CoV-2 ORF1ab in a clinical sample.
The invention additionally provides the embodiment of such kit, wherein the detectably labeled oligonucleotide has a nucleotide sequence that is able to specifically hybridize to an oligonucleotide having the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:8, and wherein the kit permits a determination of the presence or absence of the SARS-CoV-2 S gene in a clinical sample.
The invention additionally provides the embodiment of such kits, wherein the kit comprises two detectably labeled oligonucleotides, wherein the detectable labels of the oligonucleotides are distinguishable, and wherein one of the two detectably labeled oligonucleotides has a nucleotide sequence that is able to specifically hybridize to an oligonucleotide having the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4, and the second of the two detectably labeled oligonucleotides has a nucleotide sequence that is able to specifically hybridize to an oligonucleotide having the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:8.
The invention additionally provides the embodiment of such kits, wherein the detectably labeled oligonucleotide is a TaqMan probe, a molecular beacon probe, a scorpion primer-probe, or a HyBeacon probe.
The invention additionally provides the embodiment of such kits, wherein the detectably labeled oligonucleotide is fluorescently labeled.
The invention additionally provides the embodiment of such kits, wherein the kit permits the detection of the D614G polymorphism of the S gene of SARS-CoV-2.
The invention additionally provides the embodiment of such kits, wherein the kit is a multi-chambered, fluidic device.
The invention additionally provides a method for detecting the presence of SARS-CoV-2 in a clinical sample, wherein the method comprises incubating the clinical sample in vitro in the presence of a detectably labeled oligonucleotide that is capable of specifically hybridizing to a SARS-CoV-2 polynucleotide, wherein the detectably labeled oligonucleotide has a nucleotide sequence that is able to specifically hybridize to an oligonucleotide having the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7 or SEQ ID NO:8; wherein the method detects the presence of SARS-CoV-2 in the clinical sample by detecting the presence of SARS-CoV-2 ORF1ab and/or SARS-CoV-2 S gene.
The invention additionally provides the embodiment of such method, wherein the detectably labeled oligonucleotide has a nucleotide sequence that is able to specifically hybridize to an oligonucleotide having the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4, and wherein the method detects the presence of SARS-CoV-2 in the clinical sample by detecting the presence of SARS-CoV-2 ORF1ab.
The invention additionally provides the embodiment of such method, wherein the detectably labeled oligonucleotide has a nucleotide sequence that is able to specifically hybridize to an oligonucleotide having the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:8, and wherein the method detects the presence of SARS-CoV-2 in the clinical sample by detecting the presence of SARS-CoV-2 S gene.
The invention additionally provides the embodiment of such methods, wherein the detectably labeled oligonucleotide is fluorescently labeled.
The invention additionally provides the embodiment of such methods, wherein the method comprises incubating the clinical sample in the presence of two detectably labeled oligonucleotides, wherein the detectable labels of the oligonucleotides are distinguishable, and wherein one of the two detectably labeled oligonucleotides has a nucleotide sequence that is able to specifically hybridize to an oligonucleotide having the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4, and the second of the two detectably labeled oligonucleotides has a nucleotide sequence that is able to specifically hybridize to an oligonucleotide having the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:8; wherein the method detects the presence of SARS-CoV-2 in the clinical sample by detecting the presence of both SARS-CoV-2 ORF1ab and SARS-CoV-2 S gene.
The invention additionally provides the embodiment of such method, wherein the detectably labeled oligonucleotide is fluorescently labeled.
The invention additionally provides the embodiment of such methods, wherein the method detects the presence or absence of the D614G polymorphism of the S gene of SARS-CoV-2.
The invention additionally provides the embodiment of such methods, wherein the method comprises a PCR amplification of the SARS-CoV-2 polynucleotide.
The invention additionally provides the embodiment of such methods, wherein the detectably labeled oligonucleotide is a TaqMan probe, a molecular beacon probe, a scorpion primer-probe, or a HyBeacon probe.
The invention additionally provides the embodiment of such methods, wherein the method comprises a LAMP amplification of the SARS-CoV-2 polynucleotide.
The invention additionally provides an oligonucleotide that comprises a 5′ terminus and a 3′ terminus, wherein the oligonucleotide has a SARS-CoV-2 oligonucleotide domain that has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:17-42, any of SEQ ID NOs:43-70, any of SEQ ID NOs:71-84, any of SEQ ID NOs:85-112, any of SEQ ID NOs:113-126, any of SEQ ID NOs:127-146, any of SEQ ID NOs:147-166, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, any of SEQ ID NOs:364-381, any of SEQ ID NOs:398-402, any of SEQ ID NOs:403-406, SEQ ID NO:411, or SEQ ID NO:412.
The invention additionally provides such an oligonucleotide wherein the oligonucleotide is detectably labeled and comprises a 5′ terminus and a 3′ terminus, wherein the oligonucleotide has a SARS-CoV-2 oligonucleotide domain that has a nucleotide sequence that consists of, consists essentially of, or comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:43-70, any of SEQ ID NOs:85-112, any of SEQ ID NOs:127-146, any of SEQ ID NOs:147-166, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, any of SEQ ID NOs:364-381, any of SEQ ID NOs:403-406, SEQ ID NO:411, or SEQ ID NO:412.
The invention additionally provides such an oligonucleotide wherein the oligonucleotide is detectably labeled and comprises a 5′ terminus and a 3′ terminus, wherein the oligonucleotide has a SARS-CoV-2 oligonucleotide domain that consists essentially of the nucleotide sequence of: SEQ ID NO:9, or SEQ ID NO:10.
The invention additionally provides such an oligonucleotide wherein the oligonucleotide is detectably labeled and comprises a 5′ terminus and a 3′ terminus, wherein the oligonucleotide has a SARS-CoV-2 oligonucleotide domain that consists essentially of the nucleotide sequence of: SEQ ID NO:11, or SEQ ID NO:12.
The invention additionally provides a TaqMan probe capable of detecting the presence of SARS-CoV-2, wherein the probe comprises an oligonucleotide, having a 5′ terminus and a 3′ terminus, that comprises a SARS-CoV-2 oligonucleotide domain whose nucleotide sequence consists of, consists essentially of, comprises, or is a variant of the nucleotide sequence of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:127-146, any of SEQ ID NOs:147-166, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, any of SEQ ID NOs:364-381, wherein the 5′ terminus of the oligonucleotide is labeled with a fluorophore and the 3′ terminus of the oligonucleotide is complexed to a quencher of such fluorophore.
The invention additionally provides such a TaqMan probe, wherein the probe is capable of detecting the SARS-CoV-2 ORF1ab, and wherein the SARS-CoV-2 oligonucleotide domain of the probe has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:9, SEQ ID NO:10, any of SEQ ID NOs:127-146, or any of SEQ ID NOs:147-166.
The invention additionally provides such a TaqMan probe, wherein the probe is capable of detecting the SARS-CoV-2 S gene, and wherein the SARS-CoV-2 oligonucleotide domain of the probe has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, or any of SEQ ID NOs:364-381.
The invention additionally provides such a TaqMan probe, wherein the probe is capable of detecting a polymorphism in the SARS-CoV-2 S gene, and wherein the SARS-CoV-2 oligonucleotide domain of the probe has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: any of SEQ ID NOs:167-252, or any of SEQ ID NOs:273-363.
The invention additionally provides a molecular beacon probe capable of detecting the presence of SARS-CoV-2, wherein the probe comprises an oligonucleotide, having a 5′ terminus and a 3′ terminus, that comprises a SARS-CoV-2 oligonucleotide domain whose nucleotide sequence consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:127-146, any of SEQ ID NOs:147-166, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, any of SEQ ID NOs:364-381, wherein such a SARS-CoV-2 oligonucleotide domain is flanked by a 5′ oligonucleotide and a 3′ oligonucleotide, wherein such 5′ oligonucleotide and such 3′ oligonucleotide are at least substantially complementary to one another, and wherein at least one of such 5′ oligonucleotide and such 3′ oligonucleotide is detectably labeled and another of such 5′ oligonucleotide and such 3′ oligonucleotide is complexed to a quencher or an acceptor of such detectable label.
The invention additionally provides such a molecular beacon probe, wherein the probe is capable of detecting the SARS-CoV-2 ORF1ab, and wherein the SARS-CoV-2 oligonucleotide domain of the probe has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:9, SEQ ID NO:10, any of SEQ ID NOs:127-146, or any of SEQ ID NOs:147-166.
The invention additionally provides such a molecular beacon probe, wherein the probe is capable of detecting the SARS-CoV-2 S gene, and wherein the SARS-CoV-2 oligonucleotide domain of the probe has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, or any of SEQ ID NOs:364-381.
The invention additionally provides such a molecular beacon probe, wherein the probe is capable of detecting a polymorphism in the SARS-CoV-2 S gene, and wherein the SARS-CoV-2 oligonucleotide domain of the probe has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: any of SEQ ID NOs:167-252, or any of SEQ ID NOs:273-363.
The invention additionally provides a scorpion primer-probe capable of detecting the presence of SARS-CoV-2, wherein the probe comprises an oligonucleotide, having a 5′ terminus and a 3′ terminus, that comprises a SARS-CoV-2 oligonucleotide domain whose nucleotide sequence consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:127-146, any of SEQ ID NOs:147-166, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, any of SEQ ID NOs:364-381, wherein such a SARS-CoV-2 oligonucleotide domain is flanked by a 5′ oligonucleotide and a 3′ oligonucleotide, wherein such 5′ oligonucleotide and such 3′ oligonucleotide are at least substantially complementary to one another, and wherein at least one of such 5′ oligonucleotide and such 3′ oligonucleotide is detectably labeled and the other of such 5′ oligonucleotide and such 3′ oligonucleotide is complexed to a quencher or an acceptor of such detectably label, and wherein such 3′ oligonucleotide further comprises a polymerization blocking moiety, and a PCR primer oligonucleotide positioned 3′ from said blocking moiety.
The invention additionally provides such a scorpion primer-probe, wherein the probe is capable of detecting the SARS-CoV-2 ORF1ab, and wherein the SARS-CoV-2 oligonucleotide domain of the probe has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:9, SEQ ID NO:10, any of SEQ ID NOs:127-146, or any of SEQ ID NOs:147-166.
The invention additionally provides such a scorpion primer-probe, wherein the probe is capable of detecting the SARS-CoV-2 S gene, and wherein the SARS-CoV-2 oligonucleotide domain of the probe has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, or any of SEQ ID NOs:364-381.
The invention additionally provides such a scorpion primer-probe, wherein the probe is capable of detecting a polymorphism in the SARS-CoV-2 S gene, and wherein the SARS-CoV-2 oligonucleotide domain of the probe has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: any of SEQ ID NOs:167-252, or any of SEQ ID NOs:273-363.
The invention additionally provides such a scorpion primer-probe, wherein the probe is capable of detecting a polymorphism in the SARS-CoV-2 S gene, and wherein the PCR primer oligonucleotide has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: any of SEQ ID NOs:43-70, or any of SEQ ID NOs:85-112.
The invention additionally provides a HyBeacon™ probe capable of detecting the presence of SARS-CoV-2, wherein the probe comprises an oligonucleotide, having a 5′ terminus and a 3′ terminus, that comprises a SARS-CoV-2 oligonucleotide domain whose nucleotide sequence consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:127-146, any of SEQ ID NOs:147-166, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, any of SEQ ID NOs:364-381, wherein at least one nucleotide residue of such SARS-CoV-2 oligonucleotide domain is detectably labeled.
The invention additionally provides such a HyBeacon™ probe, wherein the probe is capable of detecting a polymorphism in the SARS-CoV-2 S gene, and wherein the SARS-CoV-2 oligonucleotide domain of the probe has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: any of SEQ ID NOs:43-70, any of SEQ ID NOs:85-112, any of SEQ ID NOs:167-252, or any of SEQ ID NOs:273-363.
The invention additionally provides the embodiment of the above-described oligonucleotides, TaqMan probes, molecular beacon probes, scorpion primer-probes, or HyBeacon™ probes, wherein the detectable label is a fluorophore that has an excitation wavelength within the range of about 352-690 nm and an emission wavelength that is within the range of about 447-705 nm. The invention additionally provides the embodiment of such oligonucleotides, wherein the fluorophore is JOE or FAM.
The invention additionally provides an oligonucleotide primer capable of amplifying an oligonucleotide portion of a SARS-CoV-2 polynucleotide present in a sample, wherein such oligonucleotide primer has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: any of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, any of SEQ ID NOs:17-28, any of SEQ ID NOs:29-42, any of SEQ ID NOs:43-70, any of SEQ ID NOs:71-84, any of SEQ ID NOs:85-112, any of SEQ ID NOs:113-126, or any of SEQ ID NOs:398-410.
The invention additionally provides an oligonucleotide that has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:3 or SEQ ID NO:4.
The invention additionally provides an oligonucleotide that has a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:7 or SEQ ID NO:8.
The present invention is directed to methods for assaying for the presence of SARS-CoV-2 in a sample, including a clinical sample, and to oligonucleotides, reagents, and kits useful in such assays. In particular, the present invention is directed to such assays that are rapid, accurate and specific for the detection of SARS-CoV-2.
As used herein, an assay for the detection of SARS-CoV-2 is said to be “specific” for SARS-CoV-2 if it can be conducted under conditions that permit it to detect SARS-CoV-2 without exhibiting cross-reactivity to human DNA, or to DNA (or cDNA) of other pathogens, especially other coronavirus pathogens. The assays of the present invention detect SARS-CoV-2 by detecting the presence of a “SARS-CoV-2 polynucleotide” nucleic acid molecule in a clinical sample. As used herein, a SARS-CoV-2 polynucleotide nucleic acid molecule is an RNA or DNA molecule that comprises the genome of SARS-CoV-2 or a portion of a gene or open reading frame (ORF) thereof (i.e., at least 1,000 nucleotides, at least 2,000 nucleotides, at least 5,000 nucleotides, at least 10,000 nucleotides, or at least 20,000 nucleotides of the SARS-CoV-2 genome, or more preferably, the entire SARS-CoV-2 genome of 29,903 nucleotides).
In particular, an assay for the detection of SARS-CoV-2 is said to be specific for SARS-CoV-2 if it can be conducted under conditions that permit it to detect SARS-CoV-2 without exhibiting cross-reactivity to DNA (or cDNA) of Influenza A, Influenza B, Respiratory Syncytial Virus, Group A Streptococcus (Streptococcus pyogenes), Parainfluenza I, Parainfluenza III, Haemophilus parainfluenzae, Enterovirus or Adenovirus, or to SARS-CoV, MERS-CoV, or bat-derived Severe Acute Respiratory Syndrome-like coronaviruses, such as bat-SL-CoVZC45 or bat-SL-CoVZXC21. More preferably, an assay for the detection of SARS-CoV-2 is said to be specific for SARS-CoV-2 if it can be conducted under conditions that permit it to detect SARS-CoV-2 without exhibiting cross-reactivity to DNA (or cDNA) of Adenovirus 1, Bordetella pertussis, Chlamydophila pneumoniae, Coronavirus 229E, Coronavirus NL63, Coronavirus OC43, Enterovirus 68, Haemophilus influenzae, Human metapneumovirus (hMPV-9), Influenza A H3N2 (Hong Kong 8/68), Influenza B (Phuket 3073/2013), Legionella pneumophilia, MERS Coronavirus, Mycobacterium tuberculosis, Parainfluenza Type 1, Parainfluenza Type 2, Parainfluenza Type 3, Parainfluenza Type 4A, Rhinovirus B14, RSV A Long, RSV B Washington, SARS-Coronavirus, SARS-Coronavirus HKU39849, Streptococcus pneumoniae, Streptococcus pyogenes, human leukocytes, or pooled human nasal fluid.
As used herein, an assay for the detection of SARS-CoV-2 is said to be “accurate” for SARS-CoV-2 if it is capable of detecting a viral dose of 400 copies/ml of SARS-CoV-2 with an LoD of at least 80%, and of detecting a viral dose of 500 copies/ml of SARS-CoV-2 with an LoD of at least 90%.
As used herein, an assay for the detection of SARS-CoV-2 is said to be “rapid” for SARS-CoV-2 if it is capable of providing a determination of the presence or absence of SARS-CoV-2 within 2 hours, and more preferably within 90 minutes and most preferably, within 1 hour after the commencement of the assay.
III. Preferred Assays for the Detection of SARS-CoV-2
The present invention provides an assay for detecting the presence of SARS-CoV-2 in a “clinical sample”. Such detection may be accomplished in situ or in vitro, but is preferably conducted in vitro. The clinical samples that may be evaluated in accordance with the present invention include any that may contain SARS-CoV-2, and include blood samples, bronchoalveolar lavage fluid specimens, fecal samples, fibrobronchoscope brush biopsy samples, nasal swab samples, nasopharyngeal swab samples, pharyngeal swab sample, oral samples (including saliva samples, sputum samples, etc.) and urine samples. Preferably, however, the employed clinical sample will be a nasal swab sample, a nasopharyngeal swab sample, a pharyngeal swab sample, or a sputum sample, and most preferably, the employed clinical sample will be a nasopharyngeal swab sample. In one embodiment, the sample will be pre-treated to extract RNA that may be present in the sample. Alternatively, and more preferably, the sample will be evaluated without prior RNA extraction.
A. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) Assay Formats
In one embodiment, the present invention preferably uses a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay to detect the presence of SARS-CoV-2 in clinical samples. rRT-PCR assays are well known and widely deployed in diagnostic virology (see, e.g., Pang, J. et al. (2020) “Potential Rapid Diagnostics, Vaccine and Therapeutics for 2019 Novel Coronavirus (2019-nCoV): A Systematic Review,” J. Clin. Med. 26; 9(3)E623 doi: 10.3390/jcm9030623; Kralik, P. et al. (2017) “A Basic Guide to Real-Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything,” Front. Microbiol. 8:108. doi: 10.3389/fmicb.2017.00108).
To more easily describe the rRT-PCR assays of the present invention, such assays may be envisioned as involving multiple reaction steps:
It will be understood that such steps may be conducted separately (for example, in two or more reaction chambers, or with reagents for the different steps being added at differing times, etc.). However, it is preferred that such steps are to be conducted within the same reaction chamber, and that all reagents needed for the rRT-PCR assays of the present invention are to be provided to the reaction chamber at the start of the assay. It will also be understood that although the polymerase chain reaction (PCR) (see, e.g. Ghannam, M, G, et al. (2020) “Biochemistry, Polymerase Chain Reaction (PCR),” StatPearls Publishing, Treasure Is.; pp. 1-4; Lorenz, T. C. (2012) “Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting And Optimization Strategies,” J. Vis. Exp. 2012 May 22; (63):e3998; pp. 1-15) is the preferred method of amplifying SARS-CoV-2 cDNA produced via reverse transcription, other DNA amplification technologies could alternatively be employed.
Accordingly, in a preferred embodiment, the rRT-PCR assays of the present invention comprise incubating a clinical sample in the presence of a DNA polymerase, a reverse transcriptase, one or more pairs of SARS-CoV-2-specific primers, one or more SARS-CoV-2-specific probes (typically, at least one probe for each region being amplified by an employed pair of primers), deoxynucleotide triphosphates (dNTPs) and buffers. The conditions of the incubation are cycled to permit the reverse transcription of SARS-CoV-2 RNA, the amplification of SARS-CoV-2 cDNA, the hybridization of SARS-CoV-2-specific probes to such cDNA, the cleavage of the hybridized SARS-CoV-2-specific probes and the detection of unquenched probe fluorophores. The reverse transcriptase is needed only to produce a cDNA version of SARS-CoV-2 RNA.
The rRT-PCR assays of the present invention employ at least one set of at least one “Forward” primer that hybridizes to a polynucleotide domain of a first strand of a DNA molecule, and at least one “Reverse” primer that hybridizes to a polynucleotide domain of a second (and complementary) strand of such DNA molecule.
Preferably, such Forward and Reverse primers will permit the amplification of a region of ORF1ab, which encodes a non-structural polyprotein of SARS-CoV-2 and/or a region of the S gene, which encodes the virus spike surface glycoprotein and is required for host cell targeting. The SARS-CoV-2 spike surface glycoprotein is a key protein for specifically characterizing a coronavirus as being SARS-CoV-2 (Chen, Y. et al. (2020) “Structure Analysis Of The Receptor Binding Of 2019-Ncov,” Biochem. Biophys. Res. Commun. 525:135-140; Masters, P. S. (2006) “The Molecular Biology Of Coronaviruses,” Adv. Virus Res. 66:193-292). The amplification of either of such targets alone is sufficient for the specific determination of SARS-CoV-2 presence in clinical samples. It is, however, preferred to assay for SARS-CoV-2 by incubating nucleic acid molecules of a clinical sample under conditions sufficient to amplify both such targets, if present therein, and then determining whether both such amplified products are detectable.
The present invention encompasses methods, kits and oligonucleotides sufficient to amplify any portion of the SARS-CoV-2 ORF1ab. The nucleotide sequence of an exemplary ORF1ab region is provided as SEQ ID NO:415. The primers of the present invention thus include any two or more oligonucleotide SARS-CoV-2 ORF1ab primers, each being of 15, 16, 17, 18, 19, 20 or more than 20 nucleotide residues in length, that is capable of specifically hybridizing to SEQ ID NO:415, or its complement, and of mediating the amplification of an oligonucleotide region (for example, via PCR, Loop-Mediated Isothermal Amplification (LAMP), rolling circle amplification, ligase chain reaction amplification, strand-displacement amplification, bind-wash PCR, singing wire PCR, NASBA, etc.) thereof that is capable of specifically hybridizing to SEQ ID NO:415. Preferably, such amplified region of SEQ ID NO:415 will be greater than about 20 nucleotide residues in length, and preferably less than about 50 nucleotide residues in length, more preferably less than about 100 nucleotide residues in length, more preferably less than about 150 nucleotide residues in length, more preferably less than about 200 nucleotide residues in length, more preferably less than about 300 nucleotide residues in length, more preferably less than about 400 nucleotide residues in length, and most preferably less than about 500 nucleotide residues in length. The present invention further encompasses one or more detectably-labeled SARS-CoV-2 ORF1ab probe oligonucleotide(s) (and especially fluorophore labeled oligonucleotides, as discussed in detail below), that is capable of specifically hybridizing to such amplified region of SEQ ID NO:415, and of detecting the presence of such amplified region, for example, by comprising a molecular beacon probe, HyBeacon® probe, scorpion primer-probe, TaqMan probe, biotinylated oligoprobe, etc.
The present invention additionally encompasses methods, kits and oligonucleotides sufficient to amplify any portion of the SARS-CoV-2 S gene. The nucleotide sequence of an exemplary S gene is provided as SEQ ID NO:16. The primers of the present invention thus include any two or more oligonucleotide SARS-CoV-2 S gene primers, each being of 15, 16, 17, 18, 19, 20 or more than 20 nucleotide residues in length, that is capable of specifically hybridizing to SEQ ID NO:16, or its complement, and of mediating the amplification of an oligonucleotide region (for example, via PCR, Loop-Mediated Isothermal Amplification (LAMP), rolling circle amplification, ligase chain reaction amplification, strand-displacement amplification, bind-wash PCR, singing wire PCR, NASBA, etc.) thereof that is capable of specifically hybridizing to SEQ ID NO:16. Preferably, such amplified region of SEQ ID NO:16 will be greater than about 20 nucleotide residues in length, and preferably less than about 50 nucleotide residues in length, more preferably less than about 100 nucleotide residues in length, more preferably less than about 150 nucleotide residues in length, more preferably less than about 200 nucleotide residues in length, more preferably less than about 300 nucleotide residues in length, more preferably less than about 400 nucleotide residues in length, and most preferably less than about 500 nucleotide residues in length. The present invention further encompasses one or more detectably-labeled SARS-CoV-2 S gene probe oligonucleotide(s) (and especially fluorophore labeled oligonucleotides, as discussed in detail below), that is capable of specifically hybridizing to such amplified region of SEQ ID NO:16, and of detecting the presence of such amplified region, for example, by comprising a molecular beacon probe, HyBeacon® probe, scorpion primer-probe, TaqMan probe, biotinylated oligoprobe, etc.
The amplification of SARS-CoV-2 ORF1ab is preferably mediated using a “Forward ORF1ab Primer” and a “Reverse ORF1ab Primer,” whose sequences are suitable for amplifying a region of the SARS-CoV-2 ORF1ab. Although any Forward and Reverse ORF1ab Primers capable of mediating such amplification may be employed in accordance with the present invention, it is preferred to employ Forward and Reverse ORF1ab Primers that possess distinctive advantages. The preferred Forward ORF1ab Primer of the present invention comprises, consists essentially of, or consists of, the sequence (SEQ ID NO:1) atggtagagttgatggtcaa, which corresponds to the nucleotide sequence of nucleotides 19991-20010 of the sense-strand of the SARS-CoV-2 ORF1ab, or is a variant thereof. The preferred Reverse ORF1ab Primer of the present invention comprises, consists essentially of, or consists of, the sequence (SEQ ID NO:2) taagactagcttgtttggga, which corresponds to the nucleotide sequence of nucleotides 20088-20107 of the anti-sense-strand of SARS-CoV-2 ORF1ab, or is a variant thereof. Primers that consist essentially of the sequences of SEQ ID NO:1 and SEQ ID NO:2 amplify a double-stranded oligonucleotide having the sequence of nucleotides 19991-20107 of SARS-CoV-2 ORF1ab. Such preferred “Forward ORF1ab Primer” and preferred “Reverse ORF1ab Primer” have distinctive attributes for use in the detection of SARS-CoV-2.
The sequence of the “sense” strand of nucleotides 19991-20107 of the SARS-CoV-2 ORF1ab is SEQ ID NO:3; the sequence of the complement (“anti-sense”) strand is SEQ ID NO:4:
Such oligonucleotides illustrate the SARS-CoV-2 oligonucleotides that may be amplified using the ORF1ab primers of the present invention.
While it is preferred to detect the presence of the ORF1ab using primers that consist of the sequences of SEQ ID NO:1 and SEQ ID NO:2, the invention contemplates that other primers that consist essentially of the sequence of SEQ ID NO:1 or that consist essentially of the sequence of SEQ ID NO:2 (in that they possess 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional nucleotide residues, but retain the ability to specifically hybridize to DNA molecules having the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4, and more preferably retain the ability to specifically hybridize to DNA molecules having the nucleotide sequence of complement of the nucleotide sequence of SEQ ID NO:1 or the nucleotide sequence of complement of the nucleotide sequence of SEQ ID NO:2), or “variants” of such primers that retain the ability to specifically hybridize to DNA molecules having the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4, and more preferably retain the ability to specifically hybridize to DNA molecules having the nucleotide sequence of complement of the nucleotide sequence of SEQ ID NO:1 or the nucleotide sequence of complement of the nucleotide sequence of SEQ ID NO:2, could be employed in accordance with the principles and goals of the present invention. Such “Variant ORF1ab Primers” may, for example:
The alignment and relative orientation of the preferred Forward ORF1ab Primer (SEQ ID NO: 1) and Reverse ORF1ab Primer (SEQ ID NO:2) of the present invention and the region of SARS-CoV-2 ORF1ab that these primers amplify in a rRT-PCR assay of SARS-CoV-2 are shown in
The amplification of SARS-CoV-2 S gene is preferably mediated using a “Forward S Gene Primer” and a “Reverse S Gene Primer,” whose sequences are suitable for amplifying a region of the SARS-CoV-2 S gene. Although any Forward and Reverse S Gene Primers capable of mediating such amplification may be employed in accordance with the present invention, it is preferred to employ Forward and Reverse S Gene Primers that possess distinctive advantages. The preferred Forward S Gene Primer of the present invention comprises, consists essentially of, or consists of, the sequence (SEQ ID NO:5) ctaaccaggttgctgttctt, which corresponds to the nucleotide sequence of nucleotides 23376-23395 of the sense-strand of the SARS-CoV-2 S gene, or is a variant thereof. The preferred Reverse S Gene Primer comprises, consists essentially of, or consists of, the sequence (SEQ ID NO:6) cctgtagaataaacacgcca, which corresponds to the nucleotide sequence of nucleotides 23459-23478 of the anti-sense-strand of the SARS-CoV-2 S gene, or is a variant thereof. Primers that consist essentially of the sequences of SEQ ID NO:5 and SEQ ID NO:6 amplify a double-stranded oligonucleotide having the sequence of nucleotides 23376-23478 of the SARS-CoV-2 S gene. Such preferred “Forward S Gene Primer” and preferred “Reverse S Gene Primer” have distinctive attributes for use in the detection of SARS-CoV-2.
The sequence of the “sense” strand of nucleotides 23376-23478 of the SARS-CoV-2 S gene is SEQ ID NO:7; the sequence of the complement (“anti-sense”) strand is SEQ ID NO:8:
Such oligonucleotides illustrate the SARS-CoV-2 oligonucleotides that may be amplified using the S Gene Primers of the present invention.
The nucleotide residue that is responsible for the D614G single nucleotide polymorphism of the SARS-CoV-2 S gene is underlined. SARS-CoV-2 possessing the D614G mutation (in which the adenine residue present at position 28 of SEQ ID NO:7 (position 1841 of SEQ ID NO:16) is replaced with a guanine residue, and the thymine residue present at position 76 of SEQ ID NO:8 is replaced with a cytosine residue) has emerged as a predominant clade in Europe and is spreading worldwide and is associated with enhanced fitness and higher transmissibility (Haddad, H. et al. (2020) “Mirna Target Prediction Might Explain The Reduced Transmission Of SARS-CoV-2 In Jordan, Middle East,” Noncoding RNA Res. 5(3):135-143; Isabel, S. et al. (2020) “Evolutionary And Structural Analyses Of SARS-Cov-2 D614G Spike Protein Mutation Now Documented Worldwide,” Sci. Rep. 10(1):14031:1-9; Laamarti, M. et al. (2020) “Genome Sequences of Six SARS-CoV-2 Strains Isolated in Morocco, Obtained Using Oxford Nanopore MinION Technology,” Microbiol. Resour. Announc. 9(32):e00767-20:1-4; Omotuyi, I. O. et al. (2020) “Atomistic Simulation Reveals Structural Mechanisms Underlying D614G Spike Glycoprotein-Enhanced Fitness In SARS-CoV-2,” J. Comput. Chem. 41(24):2158-2161; Ogawa, J. et al. (2020) “The D614G Mutation In The SARS-Cov2 Spike Protein Increases Infectivity In An ACE2 Receptor Dependent Manner,” Preprint. bioRxiv. 2020; 2020.07.21.214932:1-10).
While it is preferred to detect the presence of the S gene using primers that consist of the sequences of SEQ ID NO:5 and SEQ ID NO:6, the invention contemplates that other primers that consist essentially of the sequence of SEQ ID NO:5 or that consist essentially of the sequence of SEQ ID NO:6 (in that they possess 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional nucleotide residues, but retain the ability to specifically hybridize to DNA molecules having the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:8, and more preferably retain the ability to specifically hybridize to DNA molecules having the nucleotide sequence of complement of the nucleotide sequence of SEQ ID NO:5 or the nucleotide sequence of complement of the nucleotide sequence of SEQ ID NO:6), or “variants” of such primers that retain the ability to specifically hybridize to DNA molecules having the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:8, and more preferably retain the ability to specifically hybridize to DNA molecules having the nucleotide sequence of complement of the nucleotide sequence of SEQ ID NO:5 or the nucleotide sequence of complement of the nucleotide sequence of SEQ ID NO:6, could be employed in accordance with the principles and goals of the present invention. Such “Variant S Gene Primers” may, for example:
The alignment and relative orientation of the Forward S Gene Primer (SEQ ID NO:5) and Reverse S Gene Primer (SEQ ID NO:6) of the present invention and the region of the SARS-CoV-2 S gene that these primers amplify in a rRT-PCR assay of SARS-CoV-2 are shown in
In accordance with the present invention, the presence or absence of SARS-CoV-2 in a sample, such as a clinical sample, is preferably accomplished using one or more detectably labeled oligonucleotides as probe(s). As used herein, the term “detectably labeled oligonucleotide” denotes a nucleic acid molecule that comprises at least 10 nucleotide residues and not more than 500 nucleotide residues, more preferably, not more than 200 nucleotide residues, still more preferably, not more than 100 nucleotide residues, and still more preferably, not more than 50 nucleotide residues, and that is capable of specifically hybridizing to the RNA or CDNA of SARS-CoV-2. As used herein, the term “specifically hybridizing” denotes the capability of a nucleic acid molecule to detectably anneal to another nucleic acid molecule under conditions in which such nucleic acid molecule does not detectably anneal to a non-complementary nucleic acid molecule. The probes of the present invention permit the detection of SARS-CoV-2-specific polynucleotides, and thus permit a diagnosis of COVID-19. Additionally, the variant probes of the present invention permit the detection of polymorphisms (such as single nucleotide polymorphisms (SNPs), e.g., the SNPs that cause the D614G, V515F, V622I, P631S polymorphisms in the SARS-CoV-2 S gene), that may be present in the SARS-CoV-2 polynucleotides of a clinical sample. SNPs may be advantageously detected using two probes having distinguishable labels.
Detection can be accomplished using any suitable method, e.g., molecular beacon probes, HyBeacon® probes, scorpion primer-probes, TaqMan probes, biotinylated oligoprobes in an enzyme-linked immunosorbent assay-based format, turbidity, radioisotopic-labeled oligoprobes, chemiluminescent detectors, amplification of the probe sequences using Q beta replicase, PNA-based detectors, LAMP, etc. (Bustin, S. A. et al. (2020) “RT-qPCR Testing of SARS-CoV-2: A Primer,” Intl. J. Molec. Sci. 21:3004:1-9; Chang, G.-J. J. et al. (1994) “An Integrated Target Sequence and Signal Amplification Assay, Reverse Transcriptase-PCR-Enzyme-Linked Immunosorbent Assay, To Detect and Characterize Flaviviruses,” J. Clin. Microbiol. 32(2):477-483; Navarro, E. et al. (2015) “Real-Time PCR Detection Chemistry,” Clin. Chim. Acta 439:231-250; Persing, D. H. et al. (1989) “In Vitro Amplification Techniques For The Detection Of Nucleic Acids: New Tools For The Diagnostic Laboratory,” Yale J. Biol. Med. 62(2):159-171; Schwab, K. J. et al. (2001) “Development Of A Reverse Transcription-PCR-DNA Enzyme Immunoassay For Detection Of “Norwalk-Like” Viruses And Hepatitis A Virus In Stool And Shellfish. Applied And Environmental Microbiology,” 67(2):742-749; Yuan, X. et al. (2019) “LAMP Real-Time Turbidity Detection For Fowl Adenovirus,” BMC Vet. Res. 15: 256:1-4; French, D. J. et al. (2001) “HyBeacon Probes: A New Tool For DNA Sequence Detection And Allele Discrimination,” Mol. Cell. Probes 15(6):363-374; French, D. J. et al. (2006) “HyBeacons®: A Novel DNA Probe Chemistry For Rapid Genetic Analysis,” Intl. Cong. Series 1288:707-709; French, D. J. et al. (2008) “HyBeacon Probes For Rapid DNA Sequence Detection And Allele Discrimination,” Methods Mol. Biol. 429:171-85; Notomi, T. et al. (2000) “Loop-Mediated Isothermal Amplification Of DNA,” Nucl. Acids Res. 28(12):E63:1-7; Zhang, H. et al. (2019) “LAMP-On-A-Chip: Revising Microfluidic Platforms For Loop-Mediated DNA Amplification,” Trends Analyt. Chem. 113:44-53; Eiken Chemical Co., Ltd. (2020) “Eiken Chemical Launches the Loopamp 2019 nCoV Detection Kit,” Press Release; pages 1-2; Zhang, H. et al. (2019) “LAMP-On-A-Chip: Revising Microfluidic Platforms For Loop-Mediated DNA Amplification,” Trends Analyt. Chem. 113:44-53; Yuan, X. et al. (2019) “LAMP Real-Time Turbidity Detection For Fowl Adenovirus,” BMC Vet. Res. 15: 256:1-4; U.S. Pat. Nos. 6,974,670; 7,175,985; 7,348,141; 7,399,588; 7,494,790; 7,998,673; and 9,909,168).
Preferably, the detection of the amplified SARS-CoV-2 polynucleotides of the present invention employs an oligonucleotide that is labeled with a fluorophore and complexed to a quencher of the fluorescence of that fluorophore (Navarro, E. et al. (2015) “Real-Time PCR Detection Chemistry,” Clin. Chim. Acta 439:231-250).
A wide variety of fluorophores and quenchers are known and are commercially available (e.g., Biosearch Technologies, Gene Link), and may be used in accordance with the methods of the present invention. Preferred fluorophores include the fluorophores Biosearch Blue, Alexa488, FAM, Oregon Green, Rhodamine Green-X, NBD-X, TET, Alexa430, BODIPY R6G-X, CAL Fluor Gold 540, JOE, Yakima Yellow, Alexa 532, VIC, HEX, and CAL Fluor Orange 560 (which have an excitation wavelength in the range of about 352-538 nm and an emission wavelength in the range of about 447-559 nm, and whose fluorescence can be quenched with the quencher BHQ1), or the fluorophores RBG, Alexa555, BODIPY 564/570, BODIPY TMR-X, Quasar 570, Cy3, Alexa 546, NED, TAMRA, Rhodamine Red-X, BODIPY 581/591, Redmond Red, CAL Fluor Red 590, Cy3.5, ROX, Alexa 568, CAL Fluor Red 610, BODIPY TR-X, Texas Red, CAL Fluor Red 635, Pulsar 650, Cy5, Quasar 670, CY5.5, Alexa 594, BODIPY 630/650-X, or Quasar 705 (which have an excitation wavelength in the range of about 524-690 nm and an emission wavelength in the range of about 557-705 nm, and whose fluorescence can be quenched with the quencher BHQ2). The preferred SARS-CoV-2-specific probes of the present invention are labeled with either the fluorophore 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (“JOE”) or the fluorophore 5(6)-carboxyfluorescein (“FAM”) on their 5′ termini. JOE is a xanthene fluorophore with an emission in yellow range (absorption wavelength of 520 nm; emission wavelength of 548 nm). FAM is a carboxyfluorescein molecule with an absorption wavelength of 495 nm and an emission wavelength of 517 nm; it is typically provided as a mixture of two isomers (5-FAM and 6-FAM). Quasar 670 is similar to cyanine dyes, and has an absorption wavelength of 647 nm and an emission wavelength of 670 nm.
The black hole quencher 1 (“BHQ1”) is a preferred quencher for FAM and JOE fluorophores. BHQ1 quenches fluorescent signals of 480-580 nm and has an absorption maximum at 534 nm.
The black hole quencher 2 (“BHQ2”) is a preferred quencher for Quasar 670. BHQ2 quenches fluorescent signals of 560-670 nm and has an absorption maximum at 579 nm.
JOE, FAM, Quasar 670, BHQ1 and BHQ2 are widely available commercially (e.g., Sigma Aldrich; Biosearch Technologies, etc.) and are coupled to oligonucleotides using methods that are well known (see, e.g., Zearfoss, N. R. et al. (2012) “End-Labeling Oligonucleotides with Chemical Tags After Synthesis,” Meth. Mol. Biol. 941:181-193). Oligonucleotide probes of any desired sequence labeled may be obtained commercially (e.g., ThermoFisher Scientific) already labeled with a desired fluorophore and complexed to a desired quencher.
As discussed above, the proximity of the quencher of a probe to the fluorophore of that probe results in a quenching of the fluorescent signal. Incubation of the probe in the presence of a double-strand-dependent 5→3′ exonuclease (such as the 5→3′ exonuclease activity of Taq polymerase) cleaves the probe when it has hybridized to a complementary target sequence, thus separating the fluorophore from the quencher and permitting the production of a detectable fluorescent signal.
In a preferred embodiment, such oligonucleotides are modified to be TaqMan probes by being detectably complexed to a fluorophore and a quencher, with the fluorophore being preferably complexed to a nucleotide residue within 5 nucleotides, within 4 nucleotides, within 3 nucleotides, or within 2 nucleotides of the 5′ terminus of the probe, and the quencher being preferably complexed to a nucleotide residue within 5 nucleotides, within 4 nucleotides, within 3 nucleotides, or within 2 nucleotides of the 3′ terminus of the probe. In one embodiment, the fluorophore is complexed to the 5′ terminal nucleotide residue of the probe and the quencher is complexed to the 3′ terminal nucleotide of the probe. Labeling for molecular beacon and scorpion primer-probes is similar, but the positions of the fluorophore and quencher are modified in order to account for the presence of stem oligonucleotides and/or a PCR primer oligonucleotide.
The preferred probe for detecting the region of ORF1ab that is amplified by the above-described preferred ORF1ab Primers (SEQ ID NO:1 and SEQ ID NO:2) comprises, consists essentially of, or consists of, the nucleotide sequence (SEQ ID NO:9) tgcccgtaatggtgttcttattacaga (the preferred “ORF1ab Probe”). Alternatively, an oligonucleotide that comprises, consists essentially of, or consists of, the complementary nucleotide sequence (SEQ ID NO:10) tctgtaataagaacaccattacgggca could be employed. The alignment and relative position of the preferred ORF1ab Probe of the present invention is shown in
While the preferred rRT-PCR assays of the present invention detect the presence of the ORF1ab using a probe that consists of the nucleotide sequence of SEQ ID NO:9 or a probe that consists of the nucleotide sequence of SEQ ID NO:10, the invention contemplates that other probes that comprise an oligonucleotide domain that consists essentially of the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:10 (in that they possess 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional nucleotide residues, but retain the ability to specifically hybridize to DNA molecules having the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4, and more that preferably retain the ability to specifically hybridize to DNA molecules having the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:10), or “variants” of such probes that comprise an oligonucleotide domain that exhibits the ability to specifically hybridize to DNA molecules having the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4, and more that preferably exhibits the ability to specifically hybridize to DNA molecules having the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:10 could be employed in accordance with the principles and goals of the present invention. Such “Variant ORF1ab Probes” may, for example:
The preferred probe for detecting the region of the S gene that is amplified by the above-described preferred S Gene Primers (SEQ ID NO:5 and SEQ ID NO:6) comprises, consists essentially of, or consists of, the sequence (SEQ ID NO:11) tgcacagaagtccctgttgct (the preferred “S Gene Probe”). Alternatively, an oligonucleotide that comprises, consists essentially of, or consists of, the complementary nucleotide sequence (SEQ ID NO:12) agcaacagggacttctgtgca could be employed. The alignment and relative position of the S Gene Probe of the present invention is shown in
While the preferred rRT-PCR assays of the present invention detect the presence of the S gene using a probe that consists of the nucleotide sequence of SEQ ID NO:11 or a probe that consists of the nucleotide sequence of SEQ ID NO:12, the invention contemplates that other probes that comprise an oligonucleotide domain that consists essentially of the nucleotide sequence of SEQ ID NO:11 or SEQ ID NO:12 (in that they possess 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional nucleotide residues, but retain the ability to specifically hybridize to DNA molecules having the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:8, and that more preferably retain the ability to specifically hybridize to DNA molecules having the nucleotide sequence of SEQ ID NO:11 or SEQ ID NO:12), or “variants” of such probes that comprise an oligonucleotide domain that exhibits the ability to specifically hybridize to DNA molecules having the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:8, and more that preferably exhibits the ability to specifically hybridize to DNA molecules having the nucleotide sequence of SEQ ID NO:11 or SEQ ID NO:12 could be employed in accordance with the principles and goals of the present invention. Such “Variant S Gene Probes” may, for example:
In a preferred embodiment, TaqMan probes are employed to detect amplified SARS-CoV-2 oligonucleotides in accordance with the present invention. As described above, such probes are labeled on their 5′ termini with a fluorophore, and are complexed on their 3′ termini with a quencher of the fluorescence of that fluorophore. In order to simultaneously detect the amplification of two polynucleotide domains of SARS-CoV-2, two TaqMan probes are employed that have different fluorophores (with differing and distinguishable emission wavelengths); the employed quenchers may be the same or different. The chemistry and design of “TaMan” probes is reviewed by Holland, P. M. et al. (1991) (“Detection Of Specific Polymerase Chain Reaction Product By Utilizing The 5′-3′ Exonuclease Activity Of Thermus Aquaticus DNA Polymerase,” Proc. Natl. Acad. Sci. (U.S.A.) 88(16):7276-7280), by Navarro, E. et al. (2015) (“Real-Time PCR Detection Chemistry,” Clin. Chim. Acta 439:231-250), and by Gasparic, B. M. et al. (2010) (“Comparison Of Nine Different Real-Time PCR Chemistries For Qualitative And Quantitative Applications In GMO Detection,” Anal. Bioanal. Chem. 396(6):2023-2029).
Suitable fluorophores and quenchers are as described above. In one embodiment of the invention, the 5′ terminus of the ORF1ab Probe is labeled with the fluorophore JOE, and the 3′ terminus of such probe is complexed to the quencher BHQ1 and the 5′ terminus of the S Gene Probe is labeled with the fluorophore FAM, and the 3′ terminus of such probe is complexed to the quencher BHQ1. In an alternative embodiment, the 5′ terminus of the ORF1ab Probe is labeled with the fluorophore FAM, and the 5′ terminus of the S Gene Probe is labeled with the fluorophore JOE. The use of such two fluorophores permits both probes to be used in the same assay.
Any of the SARS-CoV-2 oligonucleotide domains of the above-described ORF1ab probes may be employed to form TaqMan probes suitable for detecting the region of ORF1ab that is amplified by the above-described preferred ORF1ab Primers (e.g., SEQ ID NO:1, SEQ ID NO:2, any of SEQ ID NOs:17-28, any of SEQ ID NOs:29-42, any of SEQ ID NOs:398-399, any of SEQ ID NOs:403-406, and their respective variants).
Illustrative TaqMan ORF1ab probes may thus comprise any of the SARS-CoV-2 oligonucleotide domains of the above-described ORF1ab probes (e.g., SEQ ID NO:9, SEQ ID NO:10, any of SEQ ID NOs:127-146, or any of SEQ ID NOs:147-166, etc.)). As discussed above, the 5′ terminus of the TaqMan ORF1ab probe is labeled with a fluorophore, and the 3′ terminus of the probe is complexed to a quencher.
Similarly, any of the SARS-CoV-2 oligonucleotide domains of the above-described S gene probes may be employed to form TaqMan probes suitable for detecting the region of the S gene that is amplified by the above-described preferred S Gene Primers (e.g., SEQ ID NO:5, SEQ ID NO:6, any of SEQ ID NOs:43-70, any of SEQ ID NOs:71-84, any of SEQ ID NOs:85-112, any of SEQ ID NOs:113-126, or any of SEQ ID NOs:400-402, or any of SEQ ID NOs:407-410, and their respective variants).
Illustrative TaqMan S Gene probes may comprise any of the SARS-CoV-2 oligonucleotide domains of the above-described S gene probes (e.g., SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, or any of SEQ ID NOs:364-381, etc.)). As discussed above, the 5′ terminus of the TaqMan S Gene probe is labeled with a fluorophore, and the 3′ terminus of the probe is complexed to a quencher.
Molecular beacon probes can alternatively be employed to detect amplified SARS-CoV-2 oligonucleotides in accordance with the present invention. Molecular beacon probes are also labeled with a fluorophore and complexed to a quencher. However, in such probes, the quenching of the fluorescence of the fluorophore only occurs when the quencher is directly adjacent to the fluorophore. Molecular beacon probes are thus designed to adopt a hairpin structure while free in solution (thus bringing the fluorescent dye and quencher into close proximity with one another). When a molecular beacon probe hybridizes to a target, the fluorophore is separated from the quencher, and the fluorescence of the fluorophore becomes detectable. Unlike TaqMan probes, molecular beacon probes are designed to remain intact during the amplification reaction, and must re-anneal to the target nucleic acid molecule in every cycle for signal measurement. The chemistry and design of molecular beacon probes is reviewed by Han, S. X. et al. (2013) (“Molecular Beacons: A Novel Optical Diagnostic Tool,” Arch. Immunol. Ther. Exp. (Warsz). 61(2):139-148), by Navarro, E. et al. (2015) (“Real-Time PCR Detection Chemistry,” Clin. Chim. Acta 439:231-250), by Goel, G. et al. (2005) (“Molecular Beacon: A Multitask Probe,” J. Appl. Microbiol. 99(3):435-442), by Kitamura, Y. et al. (2020) (“Electrochemical Molecular Beacon for Nucleic Acid Sensing in a Homogeneous Solution,” Analyt. Sci. 36:959-964), and by Zheng, J. et al. (2015) (“Rationally Designed Molecular Beacons For Bioanalytical And Biomedical Applications,” Chem. Soc. Rev. 44(10):3036-3055). The use of molecular beacon probes to detect polymorphisms is reviewed by Peng, Q. et al. (2020) (“A Molecular-Beacon-Based Asymmetric PCR Assay For Detecting Polymorphisms Related To Folate Metabolism,” J. Clin. Lab. Anal. 34:e23337:1-7).
Additional nucleotides and/or linkers (e.g., oligo ethylene glycol linkers) may be interposed between the stem oligonucleotides and the loop oligonucleotide of the hairpin structure in order to provide improve the detection of single nucleotide polymorphisms (Farzan, V. M. et al. (2017) “Specificity Of SNP Detection With Molecular Beacons Is Improved By Stem And Loop Separation With Spacers,” Analyst 142:945-950). “Dumbbell” molecular beacon probes may be used to detect single nucleotide polymorphisms using a single label (Bengston, H. N. et al. (2014) “A Differential Fluorescent Receptor for Nucleic Acid Analysis,” Chembiochem. 15(2):228-231).
The design of molecular beacon probes can be assisted using software, such as Beacon Designer (Premier Biosoft) (Thorton, B. et al. (2011) “Real-Time PCR (qPCR) Primer Design Using Free Online Software,” Biochem. Molec. Biol. Educat. 39(2):145-154). However, common considerations are typically sufficient for acceptable results (Kolpashchikov, D. M. (2012) “An Elegant Biosensor Molecular Beacon Probe: Challenges And Recent Solutions,” Scientifica (Cairo). 2012:928783:1-17). Overall, to favor the formation of the probe-target complex, the melting temperature of the loop domain should be higher than that of the stem. The loop is typically 15-20 nucleotides long and fully complementary to the analyte. The stem should be C/G rich and contain 4-7 base pairs to ensure high stability and acceptable hybridization rates. Longer and more stable stems will reduce hybridization rates but may improve assay selectivity (Tsourkas, A. et al. (2003) “Hybridization Kinetics And Thermodynamics Of Molecular Beacons,” Nucleic Acids Research 31(4):1319-1330). The melting temperature of the stem should be at least 7° C. higher than the assay temperature to ensure efficient fluorescent quenching in the free MB probe. If the assay is SNP specific, the interrogated position should be complementary to a nucleotide close to the middle position of the loop sequence for better allele differentiation (Kolpashchikov, D. M. (2012) “An Elegant Biosensor Molecular Beacon Probe: Challenges And Recent Solutions,” Scientifica (Cairo). 2012:928783:1-17; Finetti-Sialer, M M. et al. (2005) “Isolate-Specific Detection of Grapevine fanleaf virus from Xiphinema index Through DNA-Based Molecular Probes,” Phytopathology 95(3):262-268).
Such probes thus comprise two small (e.g., 5-7 nucleotide long) complementary oligonucleotides positioned so as to flank the SARS-CoV-2 oligonucleotide and cause the probe to adopt a stem and loop-containing hairpin structure that positions a quencher adjacent to a fluorophore unless the probe's secondary structure is disrupted by hybridization to an oligonucleotide sequence that is complementary to the probe's loop sequence. The 5′ terminal potion of the complementary oligonucleotide that is positioned 5′ to the SARS-CoV-2 oligonucleotide is preferably labeled with a fluorophore, and the 3′ terminal domain of the complementary oligonucleotide that is positioned 3′ to the SARS-CoV-2 oligonucleotide is preferably complexed to a quencher of such fluorophore. Although it is preferred that such fluorophore be complexed to the 5′ terminal residue of the complementary oligonucleotide that is positioned 5′ to the SARS-CoV-2 oligonucleotide, it may be complexed within 5 nucleotides, within 4 nucleotides, within 3 nucleotides, or within 2 nucleotides of such 5′ terminal residue. Similarly, although it is preferred that such quencher be complexed to the 3′ terminal residue of the complementary oligonucleotide that is positioned 3′ to the SARS-CoV-2 oligonucleotide, it may be complexed within 5 nucleotides, within 4 nucleotides, within 3 nucleotides, or within 2 nucleotides of such 3′ terminal residue.
Examples of complementary oligonucleotides that may be added to the 3′ or 5′ termini of a SARS-CoV-2 oligonucleotide to form a molecular beacon probe include cggcgcc (SEQ ID NO:382) and its complement gcgccgg (SEQ ID NO:383); cggcgc (SEQ ID NO:384) and its complement gcgccg (SEQ ID NO:385); ccccccc (SEQ ID NO:386) and its complement ggggggg (SEQ ID NO:387); cccccc (SEQ ID NO:388) and its complement gggggg (SEQ ID NO:389); ccccc (SEQ ID NO:390) and its complement ggggg (SEQ ID NO:391); cgacc (SEQ ID NO:392) and its complement ggtcg (SEQ ID NO:393); ggcgc (SEQ ID NO:394) and its complement gcgcc (SEQ ID NO:395); gcgag (SEQ ID NO:396) and its complement ctcgc (SEQ ID NO:397).
Any of the SARS-CoV-2 oligonucleotide domains of the above-described ORF1ab probes may be employed as the loop domain of a molecular beacon probe suitable for detecting the region of ORF1ab that is amplified by the above-described preferred ORF1ab Primers (e.g., SEQ ID NO:1, SEQ ID NO:2, any of SEQ ID NOs:17-28, any of SEQ ID NOs:29-42, any of SEQ ID NOs:398-399, any of SEQ ID NOs:403-406, and their respective variants). Additional molecular beacon probes for the SARS-CoV-2 ORF1ab having shorter or longer loop regions can be readily constructed, for example by reducing or increasing the size of employed SARS-CoV-2 ORF1ab loop oligonucleotide, as desired.
Illustrative ORF1ab molecular beacon probes may comprise, from 5′ to 3′, a 5′ stem oligonucleotide (e.g., any of SEQ ID NOs:382-397, etc.), an ORF1ab oligonucleotide (e.g., any of the SARS-CoV-2 oligonucleotide domains of the above-described ORF1ab probes (e.g., SEQ ID NO:9, SEQ ID NO:10, any of SEQ ID NOs:127-146, or any of SEQ ID NOs:147-166, etc.)), which forms the loop domain of the molecular beacon probe, and a 3′ stem oligonucleotide whose sequence is complementary to that of the probe's 5′ stem oligonucleotide. As discussed above, the 5′ terminus of the ORF1ab molecular beacon probe is labeled with a fluorophore, and the 3′ terminus of the ORF1ab molecular beacon probe is complexed to a quencher. Additional molecular beacon probes for the SARS-CoV-2 ORF1ab having shorter or longer loop regions can be readily constructed, for example by reducing or increasing the size of employed SARS-CoV-2 ORF1ab loop oligonucleotide, as desired.
Similarly, any of the SARS-CoV-2 oligonucleotide domains of the above-described S gene probes may be employed as the loop domain of a molecular beacon probe suitable for detecting the region of the S gene that is amplified by the above-described preferred S Gene Primers (e.g., SEQ ID NO:5, SEQ ID NO:6, any of SEQ ID NOs:43-70, any of SEQ ID NOs:71-84, any of SEQ ID NOs:85-112, any of SEQ ID NOs:113-126, or any of SEQ ID NOs:400-402, or any of SEQ ID NOs:407-410, and their respective variants). Additional molecular beacon probes for the SARS-CoV-2 ORF1ab having shorter or longer loop regions can be readily constructed, for example by reducing or increasing the size of employed SARS-CoV-2 ORF1ab loop oligonucleotide, as desired.
Illustrative S Gene molecular beacon probes may comprise, from 5′ to 3′, a 5′ stem oligonucleotide (e.g., any of SEQ ID NOs:382-397, etc.), an S Gene oligonucleotide (e.g., any of the SARS-CoV-2 oligonucleotide domains of the above-described S gene probes (e.g., SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, or any of SEQ ID NOs:364-381, etc.)) which forms the loop domain of the molecular beacon probe, and a 3′ stem oligonucleotide whose sequence is complementary to that of the probe's 5′ stem oligonucleotide. As discussed above, the 5′ terminus of the S Gene molecular beacon probe is labeled with a fluorophore, and the 3′ terminus of the ORF1ab molecular beacon probe is complexed to a quencher. Additional molecular beacon probes for the SARS-CoV-2 S gene having shorter or longer loop regions can be readily constructed, for example by reducing or increasing the size of employed SARS-CoV-2 S gene loop oligonucleotide, as desired. Suitable fluorophores and quenchers are as described above.
Scorpion primer-probes (Whitcombe, D. et al. (1999) “Detection Of PCR Products Using Self-Probing Amplicons And Fluorescence,” Nat. Biotechnol. 17(8):804-807; Thelwell, N. et al. (2000) “Mode Of Action And Application Of Scorpion Primers To Mutation Detection,” Nucleic Acids Res. 28(19):3752-3761; Finetti-Sialer, M. M. et al. (2005) “Isolate-Specific Detection of Grapevine fanleaf virus from Xiphinema index Through DNA-Based Molecular Probes,” Phytopathology 95(3):262-268; Solinas, A. et al. (2001) “Duplex Scorpion Primers In SNP Analysis And FRET Applications,” Nucl. Acids Res. 29(20):E96:1-9) can alternatively be employed to detect amplified SARS-CoV-2 oligonucleotides in accordance with the present invention. Scorpion primer-probes comprise 3′ and 5′ complementary oligonucleotides that are separated by an intervening loop oligonucleotide so as to adopt a stem and loop hairpin structure while free in solution. The 5′ terminus of the 5′ stem oligonucleotide is labeled with a fluorophore. The 3′ terminus of the 3′ stem oligonucleotide is complexed to a quencher, so that upon formation of a hairpin structure with the 5′ stem oligonucleotide, fluorescence is quenched. Scorpion primer-probes differ from molecular beacon probes in that the 3′ terminus of the 3′ stem oligonucleotide is additionally complexed to a blocker of polymerase-mediated primer extension (e.g., a hexaethylene glycol (HEG) blocker (Ma, M. Y. X. et al. (1993) “Design And Synthesis Of RNA Miniduplexes Via A Synthetic Linker Approach,” Biochemistry 32(7):1751-1758; Ma, M. Y. X. et al. (1993) “Design And Synthesis Of RNA Miniduplexes Via A Synthetic Linker Approach. 2. Generation Of Covalently Closed, Double-Stranded Cyclic HIV-1 TAR RNA Analogs With High Tat-Binding Affinity,” Nucleic Acids Res. 21(11):2585-2589)), and additionally comprises a 3′ PCR primer oligonucleotide that is complementary to a sequence of a target oligonucleotide. Thus, scorpion primer-probes have the overall structure (5′ to 3′): [fluorophore]-[5′ stem oligonucleotide]-[loop oligonucleotide]-[complementary 3′ stem oligonucleotide]-[quencher]-[blocker]-PCR primer oligonucleotide.
Upon hybridizing to a target oligonucleotide, the 3′ terminus of the PCR primer oligonucleotide is extended; however, the presence of the blocker prevents the polymerase-mediated extension of the 3′ terminus of the target hybridized target oligonucleotide. The sequences of the PCR primer oligonucleotide and the loop oligonucleotide are selected such that the sequence of the loop oligonucleotide is the same as a sequence of the target molecule approximately 11 bases or less downstream from the base of the target molecule that is hybridized to the 3′ terminus of the PCR primer oligonucleotide. Thus, extension of the PCR primer forms a oligonucleotide domain of the scorpion primer-probe that is complementary to the sequence of the loop oligonucleotide. In the next denaturation step of the PCR process, the loop sequence of the scorpion primer-probe hybridizes to the extended PCR product, thus opening the probe's hairpin structure. This separates the scorpion primer-probe's fluorophore from its quencher and permits fluorescence to be detected.
Any of the SARS-CoV-2 oligonucleotide domains of the above-described ORF1ab probes may be employed as the loop domain of a scorpion primer-probe suitable for detecting the region of ORF1ab that is amplified by the above-described preferred ORF1ab Primers (e.g., SEQ ID NO:1, SEQ ID NO:2, any of SEQ ID NOs:17-28, any of SEQ ID NOs:29-42, any of SEQ ID NOs:398-399, any of SEQ ID NOs:403-406, and their respective variants). As discussed above, such probes are similar to molecular beacon probes, but comprise a blocker moiety, typically positioned 3′ to the probe's quencher moiety, and a 3′ PCR primer oligonucleotide.
Illustrative ORF1ab scorpion primer-probes would comprise, from 5′ to 3′, a 5′ stem oligonucleotide (e.g., any of SEQ ID NOs:382-398, etc.), an ORF1ab oligonucleotide (e.g., any of the SARS-CoV-2 oligonucleotide domains of the above-described ORF1ab probes (e.g., SEQ ID NO:9, SEQ ID NO:10, any of SEQ ID NOs:127-146, or any of SEQ ID NOs:147-166, etc.), a 3′ stem oligonucleotide whose sequence is complementary to that of the probe's 5′ stem oligonucleotide, and a PCR primer oligonucleotide domain whose sequence is selected so that it is capable of hybridizing to a region of ORF1ab that is approximately 7 bases, 8 bases, 9 bases, 10 bases, or more preferably 11 bases upstream of an ORF1ab sequence that is the same as the sequence of the probe's ORF1ab oligonucleotide domain (or differs from such sequence by 5, 4, 3, 2 or 1 nucleotide residues), such that extension of the PCR primer oligonucleotide domain forms an extension product whose sequence is complementary to the probe's ORF1ab oligonucleotide domain.
To illustrate the structure of such ORF1ab scorpion primer-probes, the nucleotide sequences of an ORF1ab scorpion primer-probe whose loop polypeptide domain has the sequence of the preferred ORF1ab Probe tgcccgtaatggtgttcttattacaga (SEQ ID NO:9) could have the sequence, from 5′ to 3′, of a 5′ stem oligonucleotide (e.g., any of SEQ ID NOs:382-397, etc.), the preferred ORF1ab Probe (SEQ ID NO:9), a 3′ stem oligonucleotide whose sequence is complementary to that of the probe's 5′ stem oligonucleotide, and a PCR primer oligonucleotide having the sequence gagttgatggtcaagtagac (SEQ ID NO:398, corresponding to residues 12-26 of SEQ ID NO:3). After extension of the primer by 38 bases, the primer extension product contains a domain complementary to the sequence of the preferred ORF1ab Probe. Denaturation occurring in a subsequent step of the PCR process denatures the hybridized, complementary stem oligonucleotides, thereby permitting such oligonucleotides to separate from one another. Such separation attenuates the quenching of the fluorophore and thereby causes the fluorescent signal to become detectable. During the subsequent annealing stage of the PCR process, hybridization occurs between the loop domain of the probe and the complementary primer extension product of the probe. Such hybridization prevents the complementary stem oligonucleotides of the scorpion probe from re-hybridizing to one another, and thus causes the detectable fluorescent signal to be maintained.
Similarly, an ORF1ab scorpion primer-probe whose loop polypeptide domain has the sequence ttcttattacagaaggtagt (SEQ ID NO:141, corresponding to residues 52-73 of SEQ ID NO:3) could have the sequence, from 5′ to 3′, of a 5′ stem oligonucleotide (e.g., any of SEQ ID NOs:382-397, etc.), the ORF1ab oligonucleotide (SEQ ID NO:141), a 3′ stem oligonucleotide whose sequence is complementary to that of the probe's 5′ stem oligonucleotide, and a PCR primer oligonucleotide having the sequence gtagacttatttagaaatgc (SEQ ID NO:399, corresponding to residues 21-40 of SEQ ID NO:3).
Similarly, illustrative S Gene Scorpion Primer-Probes would comprise, from 5′ to 3′, a 5′ stem oligonucleotide (e.g., any of SEQ ID NOs:382-397, etc.), an S Gene oligonucleotide (e.g., any of the SARS-CoV-2 oligonucleotide domains of the above-described ORF1ab probes (e.g., SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, or any of SEQ ID NOs:364-381, etc.)), a 3′ stem oligonucleotide whose sequence is complementary to that of the probe's 5′ stem oligonucleotide, and a PCR primer oligonucleotide domain whose sequence is selected so that it is capable of hybridizing to a region of the S gene that is approximately 7 bases, 8 bases, 9 bases, 10 bases, or more preferably 11 bases upstream of an S gene sequence that is the same as the sequence of the probe's S gene oligonucleotide domain (or differs from such sequence by 5, 4, 3, 2 or 1 nucleotide residues), such that extension of the PCR primer oligonucleotide domain forms an extension product whose sequence is complementary to the probe's S gene oligonucleotide domain.
To illustrate the structure of such S gene scorpion primer-probes, the nucleotide sequences of an S gene scorpion primer-probe whose loop polypeptide domain has the sequence of the preferred S gene probe tgcacagaagtccctgttgct (SEQ ID NO:11) could have the sequence, from 5′ to 3′, of a 5′ stem oligonucleotide (e.g., any of SEQ ID NOs:382-397, etc.), the preferred S gene probe (SEQ ID NO:11), a 3′ stem oligonucleotide whose sequence is complementary to that of the probe's 5′ stem oligonucleotide, and a PCR primer oligonucleotide having the sequence ccaggttgctgttctttatc (SEQ ID NO:400, corresponding to residues 5-24 of SEQ ID NO:7). After extension of the primer by 32 bases, the primer extension product contains a domain complementary to the sequence of the preferred S gene probe. Denaturation occurring in a subsequent step of the PCR process denatures the hybridized, complementary stem oligonucleotides, thereby permitting such oligonucleotides to separate from one another. Such separation attenuates the quenching of the fluorophore and thereby causes the fluorescent signal to become detectable. During the subsequent annealing stage of the PCR process, hybridization occurs between the loop domain of the probe and the complementary primer extension product of the probe. Such hybridization prevents the complementary stem oligonucleotides of the scorpion probe from re-hybridizing to one another, and thus causes the detectable fluorescent signal to be maintained.
Similarly, an S gene scorpion primer-probe whose loop polypeptide domain has the sequence cagaagtccctgttgctatt (SEQ ID NO:257, corresponding to residues 40-59 of SEQ ID NO:7) could have the sequence, from 5′ to 3′, of a 5′ stem oligonucleotide (e.g., any of SEQ ID NOs:382-297, etc.), the S gene oligonucleotide (SEQ ID NO:257), a 3′ stem oligonucleotide whose sequence is complementary to that of the probe's 5′ stem oligonucleotide, and a PCR primer oligonucleotide having either the sequence gttgctgttctttatcagga (SEQ ID NO:401, corresponding to residues 9-28 of SEQ ID NO:7) or the sequence gttgctgttctttatcaggg (SEQ ID NO:402). The nucleotide residue that is responsible for the D614G single nucleotide polymorphism of the SARS-CoV-2 S gene is underlined. The use of S gene scorpion primer-probes having such PCR primer oligonucleotides would distinguish SARS-CoV-2 genomes having the single nucleotide polymorphism responsible for the D614G variation from SARS-CoV-2 S genomes lacking such polymorphism.
As discussed above, the 5′ terminus of the 5′ stem oligonucleotide of such scorpion primer-probes is labeled with a fluorophore, and the 3′ terminus of the 3′ stem oligonucleotide of such scorpion primer-probes is complexed to a quencher, which is separated from the 5′ terminus of the probe's PCR primer oligonucleotide by a blocker moiety. Suitable fluorophores and quenchers are as described above.
As discussed above, the invention additionally contemplates rRT-PCR assays in which detection is mediated through the use of HyBeacon™ probes (LGC Limited). HyBeacon™ probes comprise oligonucleotides that lack significant secondary structure and possess a fluorophore moiety attached to an internal nucleotide, and are typically modified at their 3′ terminus to prevent polymerase-mediated extension (U.S. Pat. Nos. 7,348,141 and 7,998,673; French, D. J. et al. (2001) “HyBeacon Probes: A New Tool For DNA Sequence Detection And Allele Discrimination,” Mol. Cell. Probes 15(6):363-374; French, D. J. et al. (2006) “HyBeacons: A Novel DNA Probe Chemistry For Rapid Genetic Analysis,” Intl. Cong. Series 1288:707-709; French, D. J. et al. (2008) “HyBeacon Probes For Rapid DNA Sequence Detection And Allele Discrimination,” Methods Mol Biol. 429:171-85). Such probes do not rely on probe secondary structures, enzymatic digestion or interaction with additional oligonucleotides for target detection and sequence discrimination, but instead emit greater amounts of fluorescence when hybridized to complementary target oligonucleotides than when present in a non-hybridized single-stranded conformation. This shift in the quantity of fluorescence emission occurs as a direct result of target hybridization and, therefore, permits the detection and discrimination of DNA sequences by real-time PCR and melting curve analysis methodologies. Sequences differing by as little as a single nucleotide may be distinguished by measuring and exploiting the variation in Tm that occurs between different probe/target duplexes. HyBeacon™ Probes do not rely on probe secondary structures, enzymatic digestion or interaction with additional oligonucleotides for target detection and sequence discrimination. Typically, the HyBeacon™ probes of the present invention comprise 20 nucleotides or more in length. Suitable fluorophores and quenchers are as described above. Exemplary fluorophores that may be employed as the fluorophore of such probes include FAM, HEX, and TET.
Any of the SARS-CoV-2 oligonucleotide domains of the above-described ORF1ab probes (e.g., SEQ ID NO:9, SEQ ID NO:10, any of SEQ ID NOs:127-146, or any of SEQ ID NOs:147-166, etc.) may be employed to form a HyBeacon™ probe suitable for detecting the region of ORF1ab that is amplified by the above-described preferred ORF1ab Primers (e.g., SEQ ID NO:1, SEQ ID NO:2, any of SEQ ID NOs:17-28, any of SEQ ID NOs:29-42, any of SEQ ID NOs:398-399, any of SEQ ID NOs:403-406, and their respective variants). Additional HyBeacon™ probes for the SARS-CoV-2 ORF1ab having shorter or longer ORF1ab regions can be readily constructed, for example by reducing or increasing the size of employed SARS-CoV-2 ORF1ab oligonucleotide, as desired.
Illustrative ORF1ab HyBeacon™ probes thus comprise, from 5′ to 3′, an oligonucleotide capable of hybridizing to a domain of the SARS-CoV-2 ORF1ab (e.g., any of the SARS-CoV-2 oligonucleotide domains of the above-described ORF1ab probes (e.g., SEQ ID NO:9, SEQ ID NO:10, any of SEQ ID NOs:127-146, or any of SEQ ID NOs:147-166, etc.). As discussed above, an internal residue of the ORF1ab HyBeacon™ probe is labeled, preferably with a fluorophore, and the 3′ terminus of the probe is preferably modified terminus to prevent its polymerase-mediated extension when annealed to a complementary target molecule.
Similarly, any of the SARS-CoV-2 oligonucleotide domains of the above-described S Gene probes (e.g., SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, or any of SEQ ID NOs:364-381, etc.) may be employed to form a HyBeacon™ probe suitable for detecting the region of the S gene that is amplified by the above-described preferred S Gene Primers (e.g., SEQ ID NO:5, SEQ ID NO:6, any of SEQ ID NOs:43-70, any of SEQ ID NOs:71-84, any of SEQ ID NOs:85-112, any of SEQ ID NOs:113-126, or any of SEQ ID NOs:400-402, or any of SEQ ID NOs:407-410, and their respective variants). Additional HyBeacon™ probes for the SARS-CoV-2 S Gene having shorter or longer S Gene regions can be readily constructed, for example by reducing or increasing the size of employed SARS-CoV-2 S Gene oligonucleotide, as desired.
Illustrative S Gene HyBeacon™ probes thus comprise, from 5′ to 3′, an oligonucleotide capable of hybridizing to a domain of the SARS-CoV-2 S Gene (e.g., any of the SARS-CoV-2 oligonucleotide domains of the above-described S gene probes (e.g., SEQ ID NO:11, SEQ ID NO:12, any of SEQ ID NOs:167-252, any of SEQ ID NOs:253-272, any of SEQ ID NOs:273-363, or any of SEQ ID NOs:364-381, etc.). As discussed above, an internal residue of the S Gene HyBeacon™ probe is labeled with a fluorophore, and the 3′ terminus of the probe is preferably modified terminus to prevent its polymerase-mediated extension when annealed to a complementary target molecule. HyBeacon™ probes are particularly suitable for detecting single nucleotide polymorphisms (SNPs) in the S gene of SARS-CoV-2 viruses of a clinical sample (such as SNPs that cause the D614G, V515F, V622I, or P631S S gene polymorphisms). Particularly preferred are HyBeacon™ probes that are capable of detecting the A1841G single nucleotide polymorphism that causes the S gene D614G polymorphism. Examples of such probes include oligonucleotides that have the sequence of: any of SEQ ID NOs:43-70, any of SEQ ID NOs:85-112, any of SEQ ID NOs:167-252, or any of SEQ ID NOs:273-363, etc.
The assays of the present invention possess particular distinctive attributes that distinguish such assays from the assays of the prior art. One characteristic of the present invention relates to the use of at least two SARS-CoV-2 target regions as a basis for detection in an rRT-PCR assay. Thus, the rRT-PCR assays of the present invention preferably employ at least two sets of Forward and Reverse primers so as to be capable of specifically and simultaneously amplifying two oligonucleotide regions of SARS-CoV-2 RNA. In preferred embodiments, the primers of one of such two sets of primers have sequences that are capable of specifically amplifying a region of ORF1ab, and the primers of the second of such two sets of primers have sequences that are capable of specifically amplifying a region of the S gene.
The use of two amplification targets increases the accuracy of the assays of the present invention since they help ensure that such assays will continue to detect SARS-CoV-2 even if one target becomes eliminated from clinical isolates (for example by spontaneous mutation). The use of two amplification targets also increases the sensitivity of the assay because it is possible that the amplification of a particular target might not provide a detectable concentration of amplified product, for example due to processing or handling issues. By having two targets, the assays of the present invention are more likely to avoid such “false negative” results.
The selection of ORF1ab and the S genes as targets is a further characteristic of the assays of the present invention. These genes are particularly characteristic of SARS-CoV-2, and indeed the targeted region of the SARS-CoV-2 S gene (i.e., its S1 domain) exhibits relatively low homology (only 68%) to the S genes of other coronaviruses (by comparison the ORF1a of SARS-CoV-2 exhibits about 90% homology to the ORF1a of SARS-CoV; the ORF1b of SARS-CoV-2 exhibits about 86% homology to the ORF1b of SARS-CoV (Lu, R. et al. (2020) “Genomic Characterisation And Epidemiology Of 2019 Novel Coronavirus: Implications For Virus Origins And Receptor Binding,” The Lancet 395(10224):565-574). Thus, it is more likely that the assays of the present invention will not inaccurately amplify sequences of non-SARS-CoV-2 pathogens. Thus, the assays of the present invention are more likely to avoid “false positive” results.
The assays of the present invention employ probes that are unique to SARS-CoV-2 and detect SARS-CoV-2 under conditions in which non-SARS-CoV-2 pathogens are not detected. In a further attribute, the assays of the present invention employ very fast system primers that are designed to mediate the same degree of amplification under the same reaction parameters and temperatures.
The melting temperatures (Tm) of PCR primers determine their kinetics of denaturation from complementary oligonucleotides and their kinetics of annealing to complementary oligonucleotides (see, SantaLucia, J. (1998) A Unified View Of Polymer, Dumbbell, And Oligonucleotide DNA Nearest-Neighbor Thermodynamics,” Proc. Natl. Acad. Sci. (U.S.A.) 95:1460-1465; von Ahsen, N. et al. (1999) “Application Of A Thermodynamic Nearest-Neighbor Model To Estimate Nucleic Acid Stability And Optimize Probe Design: Prediction Of Melting Points Of Multiple Mutations Of Apolipoprotein B-3500 And Factor V With A Hybridization Probe Genotyping Assay On The Lightcycler,” Clin. Chem. 45(12):2094-2101). Primer pairs that exhibit “substantially identical melting temperatures” (i.e., ±2° C., more preferably, ±1° C., still more preferably ±0.5° C., and most preferably ±0.1° C., as calculated using the method of SantaLucia, J. (1998)) maximize the overall yield of the products that they amplify, and the rate at which such products are produced.
Significantly, the preferred Forward and Reverse ORF1ab Primers of the present invention exhibit such substantially identical melting temperatures, which is a further distinction of the present invention. The preferred Forward ORF1ab Primer has a base-stacking Tm of 58.2° C., whereas the preferred Reverse ORF1ab Primer has a base-stacking Tm of 58.1° C. Thus, the use of the preferred Forward and Reverse ORF1ab Primers of the present invention serves to maximize the overall yield of the amplified ORF1ab product, and the rate at which such product is produced.
The preferred Forward and Reverse S Gene Primers of the present invention also exhibit substantially identical melting temperatures, which is a further distinction of the present invention. The preferred Forward S Gene Primer has a base-stacking Tm of 60° C., whereas the preferred Reverse S Gene Primer has a base-stacking Tm of 59.9° C. Thus, the use of the preferred Forward and Reverse S Gene Primers of the present invention serves to maximize the overall yield of the amplified S Gene product, and the rate at which such product is produced.
Significantly, the melting temperatures of the Forward and Reverse ORF1ab Primers of the present invention are substantially similar to the melting temperature of the preferred Forward and Reverse S Gene Primers of the present invention. Thus, these two sets of preferred primers are extremely well-matched, which is a further distinction of the present invention. Their combined use serves to equalize the overall yield of the amplified ORF1ab and S gene products, which are of similar length (117 nucleotides vs. 103 nucleotides). The substantially similar melting temperatures of the employed sets of primers and the similar lengths of the two amplified products are further distinctions of the present invention.
In designing an rRT-PCR assay, it is desirable for the employed probe to have a Tm that is 5-10° C. higher than the employed amplification primers. The employed ORF1ab Probe has a base-stacking Tm of 66.2° C., an 8° C. difference from the Tm of the preferred ORF1ab Primers of the present invention. The employed S Gene Probe has a matching base-stacking Tm of 66.6° C., a 6.6° C. difference from the Tm of the preferred S Gene Primers of the present invention. Thus, each of the preferred probes of the present invention exhibit a desired Tm and the two preferred probes of the present invention exhibit substantially identical Tms. These are further distinctions of the present invention.
C. Other Amplification Assay Formats
Although the invention's assays for the detection of SARS-CoV-2 have been described in terms of rRT-PCR assays, the invention additionally contemplates the use of other assay formats, such as Loop-Mediated Isothermal Amplification (LAMP), rolling circle amplification, ligase chain reaction amplification, strand-displacement amplification, bind-wash PCR, singing wire PCR, NASBA (Fakruddin, M. et al. (2013) “Nucleic Acid Amplification: Alternative Methods Of Polymerase Chain Reaction,” J. Pharm. Bioallied Sci. 5(4):245-252; Zhang, H. et al. (2019) “LAMP-On-A-Chip: Revising Microfluidic Platforms For Loop-Mediated DNA Amplification,” Trends Analyt. Chem. 113:44-53; Bodulev, O. L. et al. (2020) “Isothermal Nucleic Acid Amplification Techniques and Their Use in Bioanalysis,” Biochemistry (Mosc) 85(2):147-166; Dunbar, S. et al. (2019) “Amplification Chemistries In Clinical Virology,” J. Clin. Virol. 115:18-31; Daher, R. K. et al. (2016) “Recombinase Polymerase Amplification for Diagnostic Applications,” Clin. Chem. 62(7):947-958; Goo, N. I. et al. (2016) “Rolling Circle Amplification As Isothermal Gene Amplification In Molecular Diagnostics,” Biochip J. 10(4):262-271; PCT Publication No. WO 2018/073435; U.S. Pat. No. 10,619,151; US Patent Publication No. US 2020/0063173; US 2019/0249168; US 2018/0237842), etc.).
For example, loop-mediated isothermal amplification (LAMP) may be used to detect SARS-CoV-2 in accordance with the present invention. The LAMP process amplifies DNA using four primers to amplify a target DNA oligonucleotide that is present in a double-stranded DNA molecule whose strands comprise the following domains: 3′ F3c-F2c-F1c-target oligonucleotide-B1-B2-B3 5′ and 5′ F3-F2-F1-complement of target oligonucleotide-B1c-B2c-B3c 3′, wherein F3 and F3c, F2 and F2c, F1 and F1c, B3 and B3c, B2 and B2c, and B and B1c have complementary sequences. The four LAMP primers are:
The selection of appropriate primers may be facilitated through the use of primer selection software (e.g., PrimerExplorerV5, NEB LAMP Primer Design Tool, etc.). Illustrative sets of LAMP primers for amplifying domains of the SARS-CoV-2 ORF1ab and S gene are shown in Table 11.
The illustrative ORF1ab LAMP primers mediate the amplification of a domain of ORF1ab between the F2/F2c domains and the B2/B2c domains (SEQ ID NO:411) (residues 10-126 of which correspond to SEQ ID NO:3):
and its complement (SEQ ID NO:412) (residues 19-135 of which correspond to SEQ ID NO:4):
The illustrative S Gene LAMP primers mediate the amplification of a domain of the S gene between the F2/F2c domains and the B2/B2c domains (SEQ ID NO:413) (residues 28-130 of which correspond to SEQ ID NO:7) (the nucleotide residue that is responsible for the D614G single nucleotide polymorphism of the SARS-CoV-2 S gene is underlined):
and its complement (SEQ ID NO:414) (residues 25-127 of which correspond to SEQ ID NO:8):
In a preferred embodiment, detection of LAMP amplification is accomplished using one or two loop-primers, i.e., a Loop Primer B and/or a Loop Primer F (which contain sequences complementary to the single-stranded domain located between the above-described B1 and B2 domains or between the above-described F1 and F2 domains (PCT Publication No. WO 2017/108663). Either the Loop Primer F or the Loop Primer B, if present, is labeled at its 5′-end with at least one acceptor fluorophore. A further oligonucleotide probe, which is labeled at its 3′-end with at least one donor fluorophore is also employed. Especially preferred is the donor/acceptor pair BODIPY FL/ATTO647N. The further oligonucleotide probe has a sequence that is capable of hybridizing to the target nucleic acid sequence at a position which is 5′ to the labeled Loop Primer F or Loop Primer B so that, when hybridized to the target nucleic acid sequence, the 3′-end of the oligonucleotide probe is brought into close proximity to the 5′-end of the labeled Loop Primer F or Loop Primer B.
D. Nested and Multiplexed Amplification Reactions
In one embodiment, the specificity and efficiency of the SARS-CoV-2 detection assays of the present invention are increased through the use of pairs of nested primers (see, e.g., U.S. Pat. Nos. 4,683,202 and 8,906,622; Basiri, A. et al. (2020) “Microfluidic Devices For Detection Of RNA Viruses,” Rev Med Virol. e2154:1-11; Ratcliff, R. M. et al. (2007) “Molecular diagnosis of medical viruses,” Curr. Issues Mol. Biol. 9(2):87-102; Hu, Y. et al. (2009) “Nested Real-Time PCR For Hepatitis A Detection,” Lett. Appl. Microbiol. 49(5):615-619).
In one embodiment, the SARS-CoV-2 detection assays of the present invention are multiplexed reactions (Elnifro, E. M. et al. (2000) “Multiplex PCR: Optimization And Application In Diagnostic Virology,” Clin. Microbiol. Rev. 13(4):559-570; Lam, W. Y. et al. (2007) “Rapid Multiplex Nested PCR For Detection Of Respiratory Viruses,” J. Clin. Microbiol. 45(11):3631-3640; Ratcliff, R. M. et al. (2007) “Molecular diagnosis of medical viruses,” Curr. Issues Mol. Biol. 9(2):87-102).
In one such embodiment the amplification of SARS-CoV-2 ORF1ab and S gene sequences is concurrently achieved in the same reaction chamber. The invention also pertains to multiplexed amplification reactions, in which the amplification and/or detection of two or more different SARS-CoV-2 target sequences of the same gene (e.g., one or more different SARS-CoV-2 ORF1ab target sequences in addition to the SARS-CoV-2 ORF1ab target sequences described above, one or more different SARS-CoV-2 S gene target sequences in addition to the SARS-CoV-2 S gene target sequences described above, etc.) is concurrently achieved through the use of additional sets of primer and probe molecules specific for such other target sequences. In one embodiment, such additional SARS-CoV-2 target sequences encompass polymorphisms that distinguish different SARS-CoV-2 clades. Exemplary polymorphisms of the SARS-CoV-2 S gene that may be detected in such embodiments of the invention are shown in Table 12.
In one embodiment, the SARS-CoV-2 detection assays of the present invention are multiplexed reactions in which the amplification and/or detection of one or more SARS-CoV-2 target sequences other than ORF1a or the S gene is concurrently achieved through the use of additional sets of primer and probe molecules specific for such other target sequences. Such sequences could be sequences of the 3, E (envelope protein), M (matrix), 7, 8, 9, 10b, N, 13 and 14 genes, or sequences that encode the nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp12, nsp13, nsp14a2, nsp15, and/or nsp16 proteins, etc.
In one embodiment, the SARS-CoV-2 detection assays of the present invention are multiplexed reactions in which the amplification and/or detection of one or more SARS-CoV-2 target sequences and the amplification and/or detection of one or more target sequences of a pathogen other than SARS-CoV-2 (and especially a respiratory pathogen other than SARS-CoV-2) is concurrently achieved through the use of additional sets of primer and probe molecules specific for such other target sequences. Examples of such other pathogens include Streptococcus pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, Haemophilus influenzae, Neisseria meningitidis, influenza virus (e.g., influenza A, influenza B, etc.), rhinovirus, non-SARS-CoV-2 pathogenic coronavirus, parainfluenza virus, human metapneumovirus (hMPV), respiratory syncytial virus (RSV), adenovirus, etc. (see, e.g., Basile, K. et al. (2018) “Point-Of-Care Diagnostics For Respiratory Viral Infections,” Exp. Rev. Molec. Diagnos. 18(1):75-83; Mahony, J. B. et al. (2011) “Molecular Diagnosis Of Respiratory Virus Infections,” Crit. Rev. Clin. Lab. Sci. 48(5-6):217-249; Ieven, M. (2007) “Currently Used Nucleic Acid Amplification Tests For The Detection Of Viruses And Atypicals In Acute Respiratory Infections,” J. Clin. Virol. 40(4):259-276).
A. Detection of the SARS-CoV-2 ORF1ab
In accordance with the methods of the present invention, the detection of the presence of SARS-CoV-2 ORF1ab oligonucleotides in a clinical sample may be achieved using a TaqMan ORF1ab Probe by:
In accordance with the methods of the present invention, the detection of the presence of SARS-CoV-2 ORF1ab oligonucleotides in a clinical sample may alternatively be achieved using a Molecular Beacon ORF1ab Probe by:
In accordance with the methods of the present invention, the detection of the presence of SARS-CoV-2 ORF1ab oligonucleotides in a clinical sample may alternatively be achieved using an ORF1ab Scorpion Primer-Probe by:
Suitable Forward (or sense strand) ORF1ab Primers for such assays include oligonucleotides having a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:1 or any of SEQ ID NOs:17-28. Suitable Reverse (or antisense strand) ORF1ab Primers for such assays include oligonucleotides having a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:2 or any of SEQ ID NOs:29-42.
B. Detection of the SARS-CoV-2 S Gene
In accordance with the methods of the present invention, the detection of the presence of SARS-CoV-2 S Gene oligonucleotides in a clinical sample may be achieved using a TaqMan S Gene Probe by:
In accordance with the methods of the present invention, the detection of the presence of SARS-CoV-2 S gene oligonucleotides in a clinical sample may be achieved using a Molecular Beacon S Gene Probe by:
In accordance with the methods of the present invention, the detection of the presence of SARS-CoV-2 S gene oligonucleotides in a clinical sample may alternatively be achieved using an S Gene Scorpion Primer-Probe by:
Suitable Forward (or sense strand) S Gene Primers include oligonucleotides having a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:5 or any of SEQ ID NOs:43-70, or any of SEQ ID NOs:71-84. Suitable Reverse (or antisense strand) S Gene Primers include oligonucleotides having a nucleotide sequence that consists of, consists essentially of, comprises, or is a variant of, the nucleotide sequence of: SEQ ID NO:6, or any of SEQ ID NOs:85-112, or any of SEQ ID NOs:113-126.
As discussed above, the region of the SARS-CoV-2 S gene amplified by the primers of the present invention comprises the nucleotide residue (position 1841 of SEQ ID NO:16) that is responsible for the D614G polymorphism of the SARS-CoV-2 S gene. In accordance with the methods of the present invention, the detection of the presence of the D614G polymorphism may be achieved using primers whose 3′ termini distinguish the nucleotide residue present at such position. Exemplary primers having this characteristic include primers having the nucleotide sequence of any of SEQ ID NOs:43-70 or any of SEQ ID NOs:85-112.
In accordance with the methods of the present invention, the detection of the presence of the D614G polymorphism may alternatively be achieved using molecular beacon probes, HyBeacon™ probes or scorpion primer-probes whose sequences comprise the position 1841 nucleotide. Exemplary oligonucleotides having this characteristic include: any of SEQ ID NOs:43-70, any of SEQ ID NOs:85-112, any of SEQ ID NOs:167-252, or any of SEQ ID NOs:273-363
In a preferred embodiment, the above-described preferred primers and probes assay the presence of SARS-CoV-2 using a Direct Amplification Disc (DiaSorin Molecular LLC) and SIMPLEXA® Direct Chemistry (DiaSorin Molecular LLC), as processed by a LIAISON® MDX (DiaSorin Molecular LLC) rRt-PCR platform. The operating principles of DiaSorin Molecular LLC's LIAISON® MDX rRt-PCR platform, SIMPLEXA® Direct Chemistry and Direct Amplification Disc are disclosed in U.S. Pat. No. 9,067,205, US Patent Publn. No. 2012/0291565 A1, EP 2499498 B1, EP 2709760 B1, all herein incorporated by reference in their entireties.
In brief, the LIAISON® MDX (DiaSorin) rRt-PCR platform is a compact and portable thermocycler that additionally provides centrifugation and reaction processing capabilities. The device is capable of mediating sample heating (>5° C./sec) and cooling (>4° C./sec), and of regulating temperature to 0.5° C. (in the range from room temperature to 99° C.). The LIAISON® MDX rRt-PCR platform has the ability to excite fluorescent labels at 475 nm, 475 nm, 520 nm, 580 nm, and 640 nm, and to measure fluorescence at 520 nm, 560 nm, 610 nm, and 682 nm, respectively.
The Direct Amplification Disc is radially oriented, multi-chambered, fluidic device that is capable of processing the amplification of target sequences (if present) in up to 8 (50 μL) clinical samples at a time. The samples may be provided directly to the Direct Amplification Disc, as cellular material or lysates, without any prior DNA or RNA extraction.
In brief, an aliquot of the clinical sample and reaction reagents (i.e., a DNA polymerase, a reverse transcriptase, one or more pairs of SARS-CoV-2-specific primers (preferably, the above-discussed preferred Forward and Reverse ORF1ab Primers and the above-discussed preferred Forward and Reverse S Gene Primers, two or more SARS-CoV-2-specific probes (preferably, the above-discussed preferred ORF1ab Probe and the above-discussed preferred S Gene Probe), and deoxynucleotide triphosphates (dNTPs) and buffers) are separately provided to a provision area of the Direct Amplification Disc (see, U.S. Pat. No. 9,067,205, US Patent Publn No. 2012/0291565 A1, EP 2709760 B1). Preferably, the reaction reagents required for rRT-PCR are provided using “master mixes,” which are widely available commercially (Applied Biosystems; ThermoFisher Scientific, etc.). Primers may be provided at a concentration of between 0.1 and 0.5 μM (5-25 pmol/per 50 μl reaction). Probe molecules may be provided at a concentration of between 0.05 and 0.25 μM (2.5-12.5 pmol/per 50 μl reaction).
The LIAISON® MDX device centrifuges the Direct Amplification Disc to thereby force a domain of the sample and reagents to be separately moved into reservoirs for a reaction chamber. The centrifugation moves any excess sample or reagents to a holding chamber. A laser within the LIAISON® MDX device then opens a first valve permitting the sample to flow into the reaction chamber. The chamber is then heated (for example to 95° C.); the high temperature and centrifugation serves to lyse cells that may be present in the sample. The laser within the LIAISON® MDX device then opens a second valve permitting reagents sample to flow into the reaction chamber and mix with the sample. The LIAISON® MDX device then commences to subject the reaction to PCR thermocycling. An internal control may be used to monitor successful instrument and sample processing and to detect RT-PCR failure and/or inhibition.
An internal control may be employed in order to confirm that the reaction conditions are suitable for target amplification and detection. A suitable internal control, for example, is one that amplifies MS2 phage sequences. A suitable Forward MS2 Phage Internal Control Primer has the sequence (SEQ ID NO:13 tgctcgcggatacccg); a suitable Reverse MS2 Phage Internal Control Primer has the sequence (SEQ ID NO:14 aacttgcgttctcgagcgat). Amplification mediated by such internal control primers may be detected using a TaqMan probe (MS2 Phage Internal Control Probe) having the sequence (SEQ ID NO:15 acctcgggtttccgtcttgctcgt. Alternatively, other MS2 internal control primers may be employed (Dreier, J. et al. (2005) “Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays,” J. Clin. Microbiol. 43(9):4551-4557). The probe may be labeled with the Quasar 670 fluorophore and complexed to the BHQ2 quencher, or with any other fluorophore and any quencher capable of quenching the fluorescence of such fluorophore.
The LIAISON MDX Software runs a pre-heating cycle to denature the SARS-CoV-2 viral coat protein and thereby release the SARS-CoV-2 RNA. This step is followed by reverse transcription and subsequent amplification. During the extension phase of the PCR cycle, the 5′ nuclease activity of DNA polymerase degrades any probe that has hybridized to amplified product in the reaction, thereby causing the fluorescent label of the probe to separate from the quencher of the probe. Such separation permits a fluorescent signal to be detected. With each cycle, additional fluorescent label molecules are cleaved from their respective probes, increasing the fluorescence intensity.
Reaction results are monitored and presented to users via LIAISON® MDX's software. Such software provides easy to understand results with the ability to check amplification curves after a run. The software also plots QC Charts and can be bi-directionally interfaced with LIS for easy integration into lab workflow. The LIAISON® MDX permit random access to individual samples, and thus allows users to start the analysis of new samples without waiting for previously-started analyses to complete. Assay results can be obtained in one hour or less. Table 13 shows the Diagnostic Algorithm of the assay.
Accordingly, if the ORF1ab and the S gene CT values are both ≤40 for a patient specimen, the result is reported as “Detected” for the SARS-CoV-2 RNA. The internal control is not applicable. If the ORF1ab CT value is ≤40 and the S gene CT value is 0 for a patient specimen, the result is reported as “Detected” for the SARS-CoV-2 RNA. The internal control is not applicable. If the ORF1ab CT value is 0 and the S gene CT value is ≤40 for a patient specimen, the result is reported as “Detected” for the SARS-CoV-2 RNA. The internal control is not applicable. If the ORF1ab and the S gene CT values are both 0 for a patient specimen and the internal control CT is non-zero and ≤45, the result is reported as “Not Detected” for the SARS-CoV-2 RNA. If the ORF1ab and the S gene CT values are both 0 for a patient specimen and the internal control CT is also 0, the result is reported as “Invalid.” This specimen should be re-assayed. If the internal control is still 0 for the repeated assay, the test should be repeated with a new sample, if clinically warranted.
The invention additionally includes kits for conducting the above-described assays. In one embodiment, such kits will include one or more containers containing reagents for specifically detecting the SARS-CoV-2 ORF1ab (e.g., a Forward ORF1ab Primer, a Reverse ORF1ab Primer, and an ORF1ab Probe, that is preferably detectably labelled) and instructions for the use of such reagents to detect SARS-CoV-2. Such kits may comprise a Variant Forward ORF1ab Primer, a Variant Reverse ORF1ab Primer, and/or a Variant ORF1ab Probe. Most preferably, such kits will comprise the above-described preferred ORF1ab Forward Primer, the above-described preferred ORF1ab Reverse Primer and the above-described preferred ORF1ab Probe.
In a second embodiment, such kits will include one or more containers containing reagents for specifically detecting the SARS-CoV-2 S gene (e.g., a Forward S Gene Primer, a Reverse S Gene Primer, and an S Gene Probe, that is preferably detectably labelled) and instructions for the use of such reagents to detect SARS-CoV-2. Such kits may comprise a Variant Forward S Gene Primer, a Variant Reverse S Gene Primer, and/or a Variant S Gene Probe. Most preferably, such kits will comprise the above-described preferred S Gene Forward Primer, the above-described preferred S Gene Reverse Primer, and the above-described preferred S Gene Probe.
In a third embodiment, such kits will include one or more containers containing reagents for specifically detecting both the SARS-CoV-2 ORF1ab and the SARS-CoV-2 S gene (e.g., a Forward ORF1ab Primer, a Reverse ORF1ab Primer, an ORF1ab Probe, a Forward S Gene Primer, a Reverse S Gene Primer, and an S Gene Probe) and instructions for the use of such reagents to detect SARS-CoV-2, and will most preferably comprise the above-described preferred ORF1ab Forward Primer, the above-described preferred ORF1ab Reverse Primer, the above-described preferred ORF1ab Probe, the above-described preferred S Gene Forward Primer, the above-described preferred S Gene Reverse Primer and the above-described preferred S Gene Probe.
The containers of such kits will be vials, tubes, etc. and the reagents may be in liquid form or may be lyophilized. Alternatively, such containers will be a multi-chambered, fluidic device that is capable of processing the amplification of such primers. For example, the kits of the present invention may be a Direct Amplification Disc (U.S. Pat. No. 9,067,205) that has been preloaded with reagents for amplifying the above-described SARS-CoV-2 gene sequences.
VII. Embodiments of the Invention
Having now generally described the invention, the same will be more readily understood through reference to the following numbered Embodiments (“E”), which are provided by way of illustration and are not intended to be limiting of the present invention unless specified:
Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless specified.
Two sets of primers and probes were designed for the specific detection of SARS-CoV-2. Each primer/probe set on its own has been shown to provide sensitive and specific detection of SARS-CoV-2 with no detection or cross-reactivity to other coronaviruses. The SARS-CoV-2 Reference Sequence (NC_045512.2; Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, complete genome) was used to design such primers and probes.
The genome alignment of CoVs shows 58% identity of non-structural protein-coding region and 43% identity of structural proteins-coding region among different coronaviruses, with 54% identity at the whole genome level. This suggests that the non-structural proteins are more conserved and that the structural proteins exhibit greater diversity to fit their different environments (Chen, Y, et al. (2020) “Emerging Coronaviruses: Genome Structure, Replication, And Pathogenesis,” J. Med. Virol. 92:418-423).
An analysis was conducted comparing the sequence of SARS-CoV-2 to the sequences of six other CoVs that can infect humans and cause respiratory diseases, in order to select a region that would be able to detect and specifically discriminate SARS-CoV-2 from such other CoVs. The analysis focused on genomic regions coding for structural proteins that are unique to this virus (Ji, W. et al. (2020) “Cross-Species Transmission Of The Newly Identified Coronavirus 2019-nCoV,” J Med. Virol. 92:433-440). However, since it is possible that such regions might frequently recombine, in parallel, primers were designed against genomic regions coding for non-structural proteins.
Regarding the selection of the S gene, the SARS-CoV-2 may be generated by a homologous recombination within a region spanning between position 21500 and 24000 (2500 bp), which covers most of the S gene sequence (Chen, Y, et al. (2020) “Emerging Coronaviruses: Genome Structure, Replication, And Pathogenesis,” J. Med. Virol. 92:418-423). In particular, inside the 2500 bp region, Chen, Y, et al. (2020) identified a unique sequence corresponding to the first 783 nucleotides at the 5′ end of the S gene. BLAST analysis of a 783 nucleotide fragment provided no match with any sequence present in NCBI database, apart from the Wuhan seafood market pneumonia virus isolate Wuhan-Hu-14.
Regarding the selection of the ORF1ab sequence, the SARS-CoV-2 has a characteristic non-structural protein-coding region, covering about two-thirds of its genome length, and encoding 16 non-structural proteins (nsp1-16); the sequence shows 58% identity to the sequences of other CoVs (Chen, Y, et al. (2020) “Emerging Coronaviruses: Genome Structure, Replication, And Pathogenesis,” J. Med. Virol. 92:418-423). This approximately 20 kb region was chosen for the design of different primer sets specific for SARS-CoV-2.
All primer sets designed to target ORF1ab and the S gene have been tested on the SARS-CoV2 complete genome sequences available in the Global Initiative on Sharing All Influenza Data (GISAID) database, using Geneious Prime software. Sequences were mapped to the Reference Sequence of SARS-CoV-2 (NC_045512.2), and the identified primers and probes were tested against the consensus. The analysis showed that all regions recognized by the identified primers and probes have a homology of 100% with all available SARS-CoV-2 sequences.
In addition to verifying the specificity of the design, the sequences of the six CoVs that can infect humans causing respiratory diseases (i.e., HCoV-229E, HCoV-OC43, HCoV-NL63, HKU1, SARS-CoV and MERS-CoV) were examined. The accession numbers for such sequences are: NC_002645.1 (Human coronavirus 229E); NC_006213.1 (Human coronavirus OC43 strain ATCC VR-759); NC_005831.2 (Human Coronavirus NL63), NC_006577.2 (Human coronavirus HKU1), NC_004718.3 (SARS-coronavirus), and NC_019843.3 (Middle East Respiratory Syndrome coronavirus).
The sequences of the above-described preferred Forward and Reverse ORF1ab Primers (SEQ ID NO:1 and SEQ ID NO:2, respectively), the above-described preferred Forward and Reverse S Gene Primers (SEQ ID NO:5 and SEQ ID NO:6, respectively), the above-described preferred ORF1ab Probe (SEQ ID NO:9) and the above-described preferred S Gene Probe (SEQ ID NO:11) were identified through such an analysis.
Upon in silico analysis, a SIMPLEXA® SARS-CoV-2 Direct assay using the above-described preferred Forward and Reverse ORF1ab and S Gene Primers and the above-described preferred ORF1ab and S Gene Probes were found to detect all SARS-CoV-2 virus strains and to exhibit no cross-reactivity with non-SARS-CoV-2 species.
In addition to the in silico analysis, an in vitro analysis of specificity was performed. The results of the in vitro specimen testing are presented in Table 14.
Bordetella
pertussis
Chlamydophila
pneumoniae
Haemophilus
influenzae
Legionella
pneumophilia
Mycobacterium
tuberculosis
Streptococcus
pneumoniae
Streptococcus
pyogenes
The assay was also found to demonstrate 100% specificity on a negative matrix (Universal Transport Medium (UTM); Copan Diagnostics). No not-specific signals were observed.
In conclusion, the above-described preferred Forward and Reverse ORF1ab Primers and the above-described preferred ORF1ab Probe were found to be capable of detecting SARS-CoV-2 without exhibiting cross-reactivity to human DNA, or to DNA (or cDNA) of other pathogens. Additionally, the above-described preferred Forward and Reverse S Gene Primers and the above-described preferred S Gene Probe were found to be capable of detecting SARS-CoV-2 without exhibiting cross-reactivity to human DNA, or to DNA (or cDNA) of other pathogens. The assay is thus specific for SARS-CoV-2.
The observation that the assay of the present invention reports the detection of SARS-CoV-2 when only one of such sets of primers and probes is employed (i.e., either a probe and primer set that targets ORF1ab or a probe and primer set that targets the S gene) indicates that by using both such sets of probes and primers, one can increase assay sensitivity in cases of low viral loads and that the accuracy of the assay will not be jeopardized by any point mutation which may occur during COVID-19 spread across the population.
To demonstrate the improvement in assay sensitivity obtained using both sets of preferred primers and probes, a preparation of SARS-CoV2 viral particles (from isolate 2019nCoV/italy-INMI1) in an oral swab-UTM matrix was tested at doses ranging from 10−5 to 10−8 TCID50/mL. As reported in Table 15 and Table 16, relative to the detection of either ORF1ab sequences or S gene sequences, the use of both sets of preferred primers and probes was found to increase the sensitivity of the assay, achieving the detection of the 10−8 TCID50/mL dose instead of 10−7 TCID50/mL.
The results obtained at 10−8TCID50/mL (400 copies/mL) are summarized in Table 16.
The data used in Table 16 was based on a viral dose of 10−8 TCID50/mL (400 copies/mL). When the samples contained 500 viral RNA copies/mL, the assays of the present invention exhibited a 100% ability to detect SARS-CoV-2 (Table 17).
This level of sensitivity (determined with genomic viral RNA) reflects the type of results one would obtain using clinical samples containing SARS-CoV-2. The assays of the present invention thus will provide healthcare workers with analytical indications that will enable them to better interpret the results of the assay in clinical practice.
In a comparison between the methods of the present invention and the reference method of Corman, V. M. et al. (2020) (“Detection Of 2019 Novel Coronavirus (2019-nCoV) By Real-Time RT-PCR,” Eurosurveill. 25(3):2000045), the lower limit of detection (LoD) for both target genes was found to be the same: 3.2 (CI: 2.9-3.8) log 10 cp/mL and 0.40 (CI: 0.2-1.5) TCID50/mL for S gene while 3.2 log 10 (CI: 2.9-3.7) log 10 cp/mL and 0.4 (CI: 0.2-1.3) TCID50/mL for ORF1ab. The LoD obtained with extracted viral RNA for both S gene or ORF1ab was 2.7 log 10 cp/mL. Crossreactive analysis performed in 20 nasopharyngeal swabs confirmed a 100% of clinical specificity of the assay. Clinical performances of the SIMPLEXA® COVID-19 Direct assay were assessed in 278 nasopharyngeal swabs tested in parallel with Corman's method. Concordance analysis showed an “almost perfect” agreement in SARS-CoV-2 RNA detection between the two assays, being x=0.938; SE=0.021; 95% CI=0.896-0.980, with the SIMPLEXA® COVID-19 Direct assay showing a slightly higher sensitivity relative to the reference Corman's method, identifying nearly 3% additional positive samples, and detecting SARS-CoV-2 in BAL samples that had been found to give invalid results with the reference method (Bordi, L. et al. (2020) “Rapid And Sensitive Detection Of SARS-Cov-2 RNA Using The SIMPLEXA® COVID-19 Direct Assay,” J. Clin. Virol. 128:104416:1-5).
The methods of the present invention were found to have the lowest LoD (39±23 copies/ml) in a comparative study of different SARS-CoV-2 assays (Zhen, W. et al. (2020) “Comparison of Four Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2 in Nasopharyngeal Specimens,” J. Clin. Microbiol. 58(8):e00743-20:1-8).
Similar findings that the methods of the present invention were more sensitive than other laboratory tests for SARS-CoV-2 have been reported by other research groups (Lieberman, J. A. et al. (2020) “Comparison of Commercially Available and Laboratory-Developed Assays for In Vitro Detection of SARS-CoV-2 in Clinical Laboratories,” J. Clin. Microbiol. 58(8):e00821-20:1-6; Rhoads, D. D. et al. (2020) “Comparison Of Abbott ID NOW™, DiaSorin SIMPLEXA®, And CDC FDA Emergency Use Authorization Methods For The Detection Of SARS-CoV-2 From Nasopharyngeal And Nasal Swabs From Individuals Diagnosed With COVID-19,” J. Clin. Microbiol. 58(8):e00760-20:1-2).
Cradic, K. et al. (2020) (“Clinical Evaluation and Utilization of Multiple Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2,” Am. J. Clin. Pathol. 154(2):201-207) found that the methods of the present invention were more sensitive than the Abbott ID NOW™ test, and as sensitive as the Roche COBAS® SARS-CoV-2 assay, despite not requiring sample processing steps of the Roche COBAS® assay or the Roche COBAS® assay's larger sample volume.
Fung, B. et. al. (2020) (“Direct Comparison of SARS-CoV-2 Analytical Limits of Detection across Seven Molecular Assays,” J. Clin. Microbiol. 58(9):e01535-20:) found that the Roche COBAS® assay was more sensitive than the assays of the present invention, but required more time to produce diagnostic results; the study did not evaluate the impact of specimen matrix on the ability to detect virus or compatibility with different media types.
Liotti, F. M. et al. (2020) (“Evaluation Of Three Commercial Assays For SARS-CoV-2 Molecular Detection In Upper Respiratory Tract Samples,” Eur. J. Clin. Microbiol. Infect. Dis. 10.1007/s10096-020-04025-0:1-9), likewise found that the methods of the present invention provided an accurate diagnostic test for SARS-CoV-2.
All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102020000006754 | Mar 2020 | IT | national |
This application is a continuation-in-part of, and claims priority to, U.S. patent application Ser. No. 16/837,364 (filed on Apr. 1, 2020; pending), which application claims priority to Italian Patent Application No. 102020000006754, filed on Mar. 31, 2020 (pending), each of which applications is herein incorporated by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4683202 | Mullis | Jul 1987 | A |
| 6974670 | Notomi et al. | Dec 2005 | B2 |
| 7175985 | Kanda et al. | Feb 2007 | B1 |
| 7348141 | French et al. | Mar 2008 | B2 |
| 7393638 | Chou | Jul 2008 | B2 |
| 7399588 | Minekawa et al. | Jul 2008 | B2 |
| 7494790 | Notomi et al. | Feb 2009 | B2 |
| 7527967 | Chao et al. | May 2009 | B2 |
| 7566533 | Jacobs et al. | Jul 2009 | B2 |
| 7582740 | Briese et al. | Sep 2009 | B2 |
| 7622112 | Berry et al. | Nov 2009 | B2 |
| 7638280 | Kanda et al. | Dec 2009 | B2 |
| 7709188 | Kostrikis | May 2010 | B2 |
| 7736850 | Van Der Werf et al. | Jun 2010 | B2 |
| 7998673 | French et al. | Aug 2011 | B2 |
| 8142997 | Scholl et al. | Mar 2012 | B2 |
| 8343718 | Van Der Werf et al. | Jan 2013 | B2 |
| 8445650 | Simpson et al. | Mar 2013 | B2 |
| 8541003 | Anderson et al. | Sep 2013 | B2 |
| 8784829 | Morsey et al. | Jul 2014 | B2 |
| 8906622 | Morley et al. | Dec 2014 | B2 |
| 9067205 | Ludowise et al. | Jun 2015 | B2 |
| 9132423 | Battrell et al. | Sep 2015 | B2 |
| 9909168 | Notomi et al. | Mar 2018 | B2 |
| 9945856 | van der Hoek | Apr 2018 | B2 |
| 10619151 | Stehr et al. | Apr 2020 | B2 |
| 10689716 | Daunert et al. | Jun 2020 | B1 |
| 20050095618 | Tsui et al. | May 2005 | A1 |
| 20050136480 | Brahmachari et al. | Jun 2005 | A1 |
| 20050208066 | Chao et al. | Sep 2005 | A1 |
| 20060286124 | Burt et al. | Dec 2006 | A1 |
| 20070092938 | Kwang et al. | Apr 2007 | A1 |
| 20070099178 | Minekawa et al. | May 2007 | A1 |
| 20080081047 | Berry et al. | Apr 2008 | A1 |
| 20080090224 | Yu et al. | Apr 2008 | A1 |
| 20100233250 | Baras et al. | Sep 2010 | A1 |
| 20100279273 | Bergeron et al. | Nov 2010 | A1 |
| 20100285457 | Peiris et al. | Nov 2010 | A1 |
| 20110159001 | Lanzavecchia | Jun 2011 | A1 |
| 20110262892 | Aoyagi et al. | Oct 2011 | A1 |
| 20120045469 | Baras et al. | Feb 2012 | A1 |
| 20120291565 | Ludowise et al. | Nov 2012 | A1 |
| 20160238601 | Baric et al. | Aug 2016 | A1 |
| 20180237842 | Buersgens et al. | Aug 2018 | A1 |
| 20180326044 | Carter | Nov 2018 | A1 |
| 20180371534 | Minnucci et al. | Dec 2018 | A1 |
| 20190249168 | Stehr et al. | Aug 2019 | A1 |
| 20200063173 | Buersgens et al. | Feb 2020 | A1 |
| Number | Date | Country |
|---|---|---|
| 111057798 | Apr 2020 | CN |
| 111273026 | Jun 2020 | CN |
| 1020534 | Jul 2000 | EP |
| 1642978 | Apr 2006 | EP |
| 1694829 | Aug 2010 | EP |
| 1690207 | Sep 2010 | EP |
| 2275534 | Jan 2011 | EP |
| 2361974 | Aug 2011 | EP |
| 1697507 | Sep 2012 | EP |
| 2597162 | May 2013 | EP |
| 2709760 | Jun 2019 | EP |
| 2499498 | Jul 2019 | EP |
| WO 2004097021 | Nov 2004 | WO |
| WO 2005054469 | Jun 2005 | WO |
| WO 2005056584 | Jun 2005 | WO |
| WO 2005057464 | Jun 2005 | WO |
| WO 2006068663 | Jun 2006 | WO |
| WO 2008155316 | Dec 2008 | WO |
| WO 2009085025 | Jul 2009 | WO |
| WO 2009128963 | Oct 2009 | WO |
| WO 2010063685 | Jun 2010 | WO |
| WO 2011059443 | May 2011 | WO |
| WO 2017108663 | Jun 2017 | WO |
| Entry |
|---|
| Al Johani, S. et al. (2016) “MERS-CoV Diagnosis: An Update,” J. Infect. Public Health 9(3):216-219. |
| Basile, K. et al. (2018) “Point-Of-Care Diagnostics for Respiratory Viral Infections,” Exp. Rev. Molec. Diagnos. 18(1):75-83. |
| Basiri, A. et al. (2020) “Microfluidic Devices for Detection of RNA Viruses,” Rev Med Virol. e2154:1-11. |
| Bengston, H.N. et al. (2014) “A Differential Fluorescent Receptor for Nucleic Acid Analysis,” Chembiochem. 15(2):228-231. |
| Bodulev, O.L. et al. (2020) “Isothermal Nucleic Acid Amplification Techniques and Their Use in Bioanalysis,” Biochemistry (Mosc) 85(2):147-166. |
| Bordi, L. et al. (2020) “Rapid and Sensitive Detection of SARS-Cov-2 RNA Using the Simplexa™ COVID-19 Direct Assay,” J. Clin. Virol. 128:104416:1-5. |
| Brüssow, H. (2020) “The Novel Coronavirus—A Snapshot of Current Knowledge,” Microbial Biotechnology 0:(0):1-6. |
| Bustin, S.A. et al. (2020) “RT-qPCR Testing of SARS-CoV-2: A Primer,” Intl. J. Molec. Sci. 21:3004:1-9; Chang, G.-J.J. et al. (1994) “An Integrated Target Sequence and Signal Amplification Assay, Reverse Transcriptase-PCR-Enzyme-Linked Immunosorbent Assay, To Detect and Characterize Flaviviruses,” J. Clin. Microbiol. 32(2):477-483. |
| Chan, J.F. et al. (2013) “Interspecies Transmission and Emergence of Novel Viruses: Lessons From Bats and Birds,” Trends Microbiol. 21(10):544-555. |
| Chan, J.F. et al. (2020) “Genomic Characterization of the 2019 Novel Human-Pathogenic Coronavirus Isolated From a Patient With Atypical Pneumonia After Visiting Wuhan,” Emerg. Microbes. Infect. 9(1):221-236. |
| Chan, J.F. et al. (2020) “Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-Polymerase Chain Reaction Assay Validated In Vitro and With Clinical Specimens,” J Clin. Microbiol. JCM.00310-20. doi: 10.1128/JCM.00310-20; pp. 1-33. |
| Chan, J.F. et al. (2020) “A Familial Cluster of Pneumonia Associated With the 2019 Novel Coronavirus Indicating Person-To-Person Transmission: A Study of a Family Cluster,” Lancet 2020; 395: 514-23. |
| Chang, G.-J.J. et al. (1994) “An Integrated Target Sequence and Signal Amplification Assay, Reverse Transcriptase-PCR-Enzyme-Linked Immunosorbent Assay, To Detect and Characterize Flaviviruses,” J. Clin. Microbiol. 32(2):477-483. |
| Chen, Y, et al. (2020) “Emerging Coronaviruses: Genome Structure, Replication, and Pathogenesis,” J. Med. Virol. 92:418-423. |
| Chen, Y. et al. (2020) “Structure Analysis of the Receptor Binding of 2019-Ncov,” Biochem. Biophys. Res. Commun. 525:135-140. |
| Cordes, A.K. et al. (2020) “Rapid Random Access Detection of the Novel SARS-Coronavirus-2 (SARS-CoV-2, Previously 2019-nCoV) Using an Open Access Protocol for the Panther Fusion,” J. Clin. Virol. 125:104305 doi: 10.1016/j.jcv.2020.104305; pp. 1-2. |
| Corman, V.M. et al. (2020) “Detection of 2019 Novel Coronavirus (2019-nCoV) By Real-Time RT-PCR,” Eurosurveill. 25(3):2000045; pp. 1-8. |
| Cradic, K. et al. (2020) (“Clinical Evaluation and Utilization of Multiple Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2,” Am. J. Clin. Pathol. 154(2):201-207. |
| Daher, R.K. et al. (2016) “Recombinase Polymerase Amplification for Diagnostic Applications,” Clin. Chem. 62(7):947-958. |
| DHHS Press Release (Mar. 13, 2020) “HHS Funds Development of COVID-19-19 Diagnostic Tests,” https://www.hhs.gov/about/news/2020/03/13/hhs-funds-development-covid-19-diagostic-tests.html 2 pages. |
| DiaSorin Liaison MDX Product Brochure (2018); pp. 1-4. |
| DiaSorin Press Release (Mar. 30, 2020) “DiaSorin Molecular COVID-19 Test Has Received FDA Emergency Use Authorization,” https://molecular.diasorin.com/international/wp-content/uploads/2020/03/DiaSorin-Molecular-COVID-19-EUA-APPROVED.pdf (3 pages). |
| Dreier, J. et al. (2005) “Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays,” J. Clin. Microbiol. 43(9):4551-4557. |
| Drosten et al. (2003) “Identification of a Novel Coronavirus in Patients With Severe Acute Respiratory Syndrome,” New Engl. J. Med. 348:1967-1976. |
| Dunbar, S. et al. (2019) “Amplification Chemistries in Clinical Virology,” J. Clin. Virol. 115:18-31. |
| Eiken Chemical Co., Ltd. (2020) “Eiken Chemical Launches the Loopamp 2019 nCoV Detection Kit,” Press Release; pp. 1-2. |
| Fakruddin, M. et al. (2013) “Nucleic Acid Amplification: Alternative Methods of Polymerase Chain Reaction,” J. Pharm. Bioallied Sci. 5(4):245-252. |
| Fang, Y. et al. (2020) “Transmission Dynamics of the COVID-19 Outbreak and Effectiveness of Government Interventions: A Data-Driven Analysis,” J. Med. Virol. doi: 10.1002/jmv.25750. |
| Farzan, V.M. et al. (2017) “Specificity of SNP Detection With Molecular Beacons Is Improved by Stem and Loop Separation With Spacers,” Analyst 142:945-950. |
| Finetti-Sialer, M.M. et al. (2005) “Isolate-Specific Detection of Grapevine fanleaf virus from Xiphinema index Through DNA-Based Molecular Probes,” Phytopathology 95(3):262-268. |
| Fluorophores and BHQ (2019) Biosearch Technologies; 1 page. |
| French, D.J. et al. (2001) “HyBeacon Probes: A New Tool for DNA Sequence Detection and Allele Discrimination,” Mol. Cell. Probes 15(6):363-374. |
| French, D.J. et al. (2006) “HyBeacons®: A Novel DNA Probe Chemistry for Rapid Genetic Analysis,” Intl. Cong. Series 1288:707-709. |
| French, D.J. et al. (2008) “HyBeacon Probes for Rapid DNA Sequence Detection and Allele Discrimination,” Methods Mol Biol. 429:171-85. |
| Gasparic, M.B et al. (2010) “Comparison of Nine Different Real-Time PCR Chemistries for Qualitative and Quantitative Applications in GMO Detection,” Anal. Bioanal. Chem. 396(6):2023-2029. |
| GenBank Accession No. NC_002645.1 (Human coronavirus 229E) (2018) 12 pages. |
| GenBank Accession No. NC_004718.3 (SARS-coronavirus) (2018) 18 pages. |
| GenBank Accession No. NC_005831.2 (Human Coronavirus NL63) (2018) 11 pages. |
| GenBank Accession No. NC_006213.1 (Human coronavirus OC43 strain ATCC VR-759) (2019) 13 pages. |
| GenBank Accession No. NC_006577.2 (Human coronavirus HKU1) (2018) 14 pages. |
| GenBank Accession No. NC_019843.3 (Middle East Respiratory Syndrome coronavirus) (2018) 16 pages. |
| GenBank Accession No. NC_045512.2 (SARS-CoV-2) (2020) 16 pages. |
| Ghannam, M,G, et al. (2020) “Biochemistry, Polymerase Chain Reaction (PCR),” StatPearls Publishing, Treasure Is.; pp. 1-4. |
| Goel, G. et al. (2005) “Molecular Beacon: A Multitask Probe,” J. Appl. Microbiol. 99(3):435-442. |
| Gong, S.R. et al. (2018) “The Battle Against SARS and MERS Coronaviruses: Reservoirs and Animal Models,” Animal Model Exp. Med. 1(2):125-13. |
| Goo, N.I. et al. (2016) “Rolling Circle Amplification As Isothermal Gene Amplification in Molecular Diagnostics,” Biochip J. 10(4):262-271. |
| Haddad, H. et al. (2020) “Mirna Target Prediction Might Explain the Reduced Transmission of SARS-CoV-2 in Jordan, Middle East,” Noncoding RNA Res. 5(3):135-143. |
| Han, S.X. et al. (2013) “Molecular Beacons: A Novel Optical Diagnostic Tool,” Arch. Immunol. Ther. Exp. (Warsz). 61(2):139-148. |
| He, Y. et al. (2004) Receptor-Binding Domain of SARS-CoV Spike Protein Induces Highly Potent Neutralizing Antibodies: Implication for Developing Subunit Vaccine, Biochem. Biophys. Res. Commun. 324:773-781. |
| Holland, P.M. et al. (1991) “Detection of Specific Polymerase Chain Reaction Product by Utilizing the 5′→3′ Exonuclease Activity of Thermus Aquaticus DNA Polymerase,” Proc. Natl. Acad. Sci. (U.S.A.) 88(16):7276-7280. |
| Hu, Y. et al. (2009) “Nested Real-Time PCR for Hepatitis A Detection,” Lett. Appl. Microbiol. 49(5):615-619. |
| Ieven, M. (2007) “Currently Used Nucleic Acid Amplification Tests for the Detection of Viruses and Atypicals in Acute Respiratory Infections,” J. Clin. Virol. 40(4):259-276. |
| Isabel, S. et al. (2020) “Evolutionary and Structural Analyses of SARS-Cov-2 D614G Spike Protein Mutation Now Documented Worldwide,” Sci. Rep. 10(1):14031:1-9. |
| Ji, W. et al. (2020) “Cross-Species Transmission of the Newly Identified Coronavirus 2019-nCoV,” J Med. Virol. 92:433-440. |
| Jung, Y.J. et al. (2020) “Comparative Analysis of Primer-Probe Sets for the Laboratory Confirmation of SARS-Cov-2,” bioRxiv 2020.02.25.964775:1-13. |
| Kitamura, Y. et al. (2020) “Electrochemical Molecular Beacon for Nucleic Acid Sensing in a Homogeneous Solution,” Analyt. Sci. 36:959-964. |
| Kolpashchikov, D.M. (2012) “An Elegant Biosensor Molecular Beacon Probe: Challenges and Recent Solutions,” Scientifica (Cairo). 2012:928783:1-17. |
| Kong, I. et al. (2020) “Early Epidemiological and Clinical Characteristics of 28 Cases of Coronavirus Disease in South Korea,” Osong Public Health Res Perspect. 11(1):8-14. |
| Kralik, P. et al. (2017) “A Basic Guide to Real-Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything,” Front. Microbiol. 8:108; pp. 1-9. |
| Laamarti, M et al. (2020) “Genome Sequences of Six SARS-CoV-2 Strains Isolated in Morocco, Obtained Using Oxford Nanopore MinION Technology,” Microbiol. Resour. Announc. 9(32):e00767-20:1-4. |
| Lai, C.C. et al. (2020) “Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Coronavirus Disease—2019 (COVID-19): The Epidemic and The Challenges,” Int. J. Antimicrob. Agents. 55(3):105924; pp. 1-9. |
| Lam, W.Y. et al. (2007) “Rapid Multiplex Nested PCR for Detection of Respiratory Viruses,” J. Clin. Microbiol. 45(11):3631-3640. |
| Li, C. et al. (2020) “High Sensitivity Detection of SARS-CoV-2 using Multiplex PCR and a Multiplex-PCR-Based Metagenomic Method,” bioRxiv:1-24. |
| Li, F. (2016) “Structure, Function, and Evolution of Coronavirus Spike Proteins,” Annu. Rev. Virol. 3:237-261. |
| Li, Q. et al. (2020) “Early Transmission Dynamics in Wuhan, China, Of Novel Coronavirus-Infected Pneumonia,” New Engl. J. Med. DOI: 10.1056/NEJMoa2001316; pp. 1-9. |
| Li, Z. et al. (2020) “Development and Clinical Application of a Rapid IgM-IgG Combined Antibody Test for SARS-CoV-2 Infection Diagnosis,” J. Med. Virol. doi: 10.1002/jmv.25727; pp. 1-16. |
| Lieberman, J.A. et al. (2020) “Comparison of Commercially Available and Laboratory-Developed Assays for In Vitro Detection of SARS-CoV-2 in Clinical Laboratories,” J. Clin. Microbiol. 58(8):e00821-20:1-6. |
| Liotti, F.M. et al. (2020) “Evaluation of Three Commercial Assays for SARS-CoV-2 Molecular Detection in Upper Respiratory Tract Samples,” Eur. J. Clin. Microbiol. Infect. Dis. 10.1007/s10096-020-04025-0:1-9. |
| Liu, R. et al. (2020) “Positive Rate of RT-PCR Detection of SARS-CoV-2 Infection in 4880 Cases From One Hospital in Wuhan, China, From Jan. To Feb. 2020,” Clinica Chimica Acta 505:172-175. |
| Lorenz, T.C. (2012) “Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies,” J. Vis. Exp. May 22, 2012;(63):e3998; pp. 1-15. |
| Lu, G. et al. (2015) “Bat-To-Human: Spike Features Determining ‘Host Jump’ Of Coronaviruses SARS-CoV, MERS-CoV, and Beyond,” Trends Microbiol. 23:468-478. |
| Lu, R. et al. (2020) “Genomic Characterisation and Epidemiology of 2019 Novel Coronavirus: Implications for Virus Origins and Receptor Binding,” Lancet 395(10224):565-574. |
| Lu, R. et al. (2020) “SARS-Cov-2 Detection Using Digital PCR for COVID-19 Diagnosis, Treatment Monitoring and Criteria for Discharge,” medRxiv 2020.03.24.20042689:1-21. |
| Ma, M.Y.X. et al. (1993) “Design and Synthesis of RNA Miniduplexes Via a Synthetic Linker Approach,” Biochemistry 32(7):1751-1758. |
| Ma, M.Y.X. et al. (1993) “Design and Synthesis of RNA Miniduplexes Via a Synthetic Linker Approach. 2. Generation of Covalently Closed, Double-Stranded Cyclic HIV-1 TAR RNA Analogs With High Tat-Binding Affinity,” Nucleic Acids Res. 21(11):2585-2589. |
| Mackay, I.M. (2015) “MERS Coronavirus: Diagnostics, Epidemiology and Transmission,” Virol. J. 12:222. doi: 10.1186/s12985-015-0439-5; pp. 1-21. |
| Mahony, J.B. et al. (2011) “Molecular Diagnosis of Respiratory Virus Infections,” Crit. Rev. Clin. Lab. Sci. 48(5-6):217-249. |
| Marra, M.A. et al. (2003) “The Genome Sequence of the SARS-Associated Coronavirus,” Science 300(5624):1399-1404. |
| Masters, P.S. (2006) “The Molecular Biology of Coronaviruses,” Adv. Virus Res. 66:193-292. |
| Navarro, E. et al. (2015) “Real-Time PCR Detection Chemistry,” Clin. Chim. Acta 439:231-250. |
| Northill, J. et al. (2020) Severe Acute Respiratory Syndrome Coronavirus 2 (Sarscov-2) Real-Time RT-PCR ORF lab 2020 (Wuhan-ORF lab; 2019-Ncov-Related Test) V.3, dx.doi.org/w10.17504/protocols.io.bchvit66, pp. 1-5. |
| Notomi, T. et al. (2000) “Loop-Mediated Isothermal Amplification of DNA,” Nucl. Acids Res. 28(12):E63:1-7. |
| Ogawa, J. et al. (2020) “The D614G Mutation in the SARS-Cov2 Spike Protein Increases Infectivity in an ACE2 Receptor Dependent Manner,” Preprint. bioRxiv. 2020;2020.07.21.214932:1-10. |
| Omotuyi, I.O. et al. (2020) “Atomistic Simulation Reveals Structural Mechanisms Underlying D614G Spike Glycoprotein-Enhanced Fitness in SARS-CoV-2,” J. Comput. Chem. 41(24):2158-2161. |
| Pang, J. et al. (2020) “Potential Rapid Diagnostics, Vaccine and Therapeutics for 2019 Novel Coronavirus (2019-nCoV): A Systematic Review,” J. Clin. Med. 26;9(3)E623; pp. 1-33. |
| Peake, I. (1989) “The Polymerase Chain Reaction,” J. Clin. Pathol. ;42(7):673-676. |
| Peng, Q. et al. (2020) “A Molecular-Beacon-Based Asymmetric PCR Assay for Detecting Polymorphisms Related to Folate Metabolism,” J. Clin. Lab. Anal. 34:e23337:1-7. |
| Persing, D.H. et al. (1989) “In Vitro Amplification Techniques for the Detection of Nucleic Acids: New Tools for the Diagnostic Laboratory,” Yale J. Biol. Med. 62(2):159-171. |
| Pfefferle, S. et al. (2020) “Evaluation of a Quantitative RT-PCR Assay for the Detection of the Emerging Coronavirus SARS-CoV-2 Using a High Throughput System,” Euro. Surveill. 25(9) doi: 10.2807/1560-7917.ES.2020.25.9.2000152; pp. 1-5. |
| Quenchers Design and Protocols (2020) Gene Link Web Brochure; 3 pages. |
| Reid, A. (2020) “WHOinHouseAssays,” World Health Organization (WHO) Publication, https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf?sfvrsn=de3a76aa_2 pp. 1-82. |
| Rhoads, D.D. et al. (2020) “Comparison of Abbott ID Now™, DiaSorin Simplexa®, and CDC FDA Emergency Use Authorization Methods for the Detection of SARS-CoV-2 From Nasopharyngeal and Nasal Swabs From Individuals Diagnosed With COVID-19,” J. Clin. Microbiol. 58(8):e00760-20:1-2. |
| Sah, R. et al. (2020) “Complete Genome Sequence of a 2019 Novel Coronavirus (SARS-CoV-2) Strain Isolated in Nepal,” Microbiol. Resource Announcements 9(11): e00169-20, pp. 1-3. |
| SantaLucia, J. (1998) A Unified View of Polymer, Dumbbell, and Oligonucleotide DNA Nearest-Neighbor Thermodynamics, Proc. Natl. Acad. Sci. (U.S.A.) 95:1460-1465. |
| Schwab, K.J. et al. (2001) “Development of a Reverse Transcription-PCR-DNA Enzyme Immunoassay for Detection of “Norwalk-Like” Viruses and Hepatitis A Virus in Stool and Shellfish. Applied and Environmental Microbiology,” 67(2):742-749. |
| Sigma Aldrich (2014) “Primers and Fluorescent Probes for Real-Time PCR and Other Applications,” Product Brochure; pp. 1-20. |
| Solinas, A. et al. (2001) “Duplex Scorpion Primers in SNP Analysis and FRET Applications,” Nucl. Acids Res. 29(20):E96:1-9. |
| Spiteri, G. et al. (2020) “First Cases of Coronavirus Disease 2019 (COVID-19) In the WHO European Region, Jan. 24 To Feb. 21, 2020,” Eurosurveill. 25(9) doi: 10.2807/1560-7917.ES.2020.25.9.2000178; pp. 1-11. |
| Su, S. et al. (2016) “Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses,” Trends Microbiol. 24:490-502. |
| Tang, A. et al. (2020) “Detection of Novel Coronavirus by RT-PCR in Stool Specimen from Asymptomatic Child, China,” Emerg Infect Dis. 26(6):pp. 1-7. |
| Thelwell, N. et al. (2000) “Mode of Action and Application of Scorpion Primers to Mutation Detection,” Nucleic Acids Res. 28(19):3752-3761. |
| Thorton, B. et al. (2011) “Real-Time PCR (qPCR) Primer Design Using Free Online Software,” Biochem. Molec. Biol. Educat. 39(2):145-154. |
| Tsourkas, A. et al. (2003) “Hybridization Kinetics and Thermodynamics of Molecular Beacons,” Nucleic Acids Research 31(4):1319-1330. |
| Von Ahsen, N. et al. (1999) “Application of a Thermodynamic Nearest-Neighbor Model to Estimate Nucleic Acid Stability and Optimize Probe Design: Prediction of Melting Points of Multiple Mutations of Apolipoprotein B-3500 and Factor V With A Hybridization Probe Genotyping Assay on the Lightcycler,” Clin. Chem. 45(12):2094-2101. |
| Wang, C. et al. (2020) “The Establishment of Reference Sequence for SARS-CoV-2 and Variation Analysis,” J. Med. Virol. doi: 10.1002/jmv.25762; pp. 1-8. |
| Wang, Q. et al. (2016) “MERS-CoV Spike Protein: Targets for Vaccines and Therapeutics,” Antiviral. Res. 133:165-177. |
| Wang, W. et al. (2020) “Detection of SARS-CoV-2 in Different Types of Clinical Specimens,” JAMA doi: 10.1001/jama.2020.3786; pp. 1-2. |
| Whitcombe, D. et al. (1999) “Detection of PCR Products Using Self-Probing Amplicons and Fluorescence,” Nat. Biotechnol. 17(8):804-807. |
| Won, J. et al. (2020) “Development of a Laboratory-Safe and Low-Cost Detection Protocol for SARS-CoV-2 of the Coronavirus Disease 2019 (COVID-19),” Exp. Neurobiol. 29(2):pp. 1-13. |
| Wu, X. et al. (2020) “Co-Infection with SARS-CoV-2 and Influenza A Virus in Patient with Pneumonia, China,” Emerg Infect Dis. 26(6 26(6); pp. 1-7. |
| Xie, C. et al. (2020) “Comparison of Different Samples for 2019 Novel Coronavirus Detection by Nucleic Acid Amplification Tests” Int. J. Infect. Dis. /doi.org/10.1016/j.ijid.2020.02.050; pp. 1-12. |
| Xu, K. et al. (2020) “Management of Corona Virus Disease-19 (Covid-19): The Zhejiang Experience,” Zhejiang Da Xue Bao Yi Xue Ban. 49(1):0; Abstract Only; pp. 1-2. |
| Yin, Y. et al. (2018) “MERS, SARS and Other Coronaviruses As Causes of Pneumonia,” Respirology 23(2):130-137. |
| Yuan, X. et al. (2019) “LAMP Real-Time Turbidity Detection for Fowl Adenovirus,” BMC Vet. Res. 15: 256:1-4. |
| Zearfoss, N.R. et al. (2012) “End-Labeling Oligonucleotides with Chemical Tags After Synthesis,” Meth. Mol. Biol. 941:181-193. |
| Zhang, H. et al. (2019) “LAMP-On-A-Chip: Revising Microfluidic Platforms for Loop-Mediated DNA Amplification,” Trends Analyt. Chem. 113:44-53. |
| Zhang, W. et al. (2020) “Molecular and Serological Investigation of 2019-nCoV Infected Patients: Implication of Multiple Shedding Routes,” Emerg. Microbes Infect. 9(1):386-389. |
| Zhao, W.M. et al. (2020) “The 2019 Novel Coronavirus Resource,” Yi Chuan. 42(2):212-221; Abstract Only; pp. 1-2. |
| Zhen, W. et al. (2020) “Comparison of Four Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2 in Nasopharyngeal Specimens,” J. Clin. Microbiol. 58(8):e00743-20:1-8. |
| Zheng, J. et al. (2015) “Rationally Designed Molecular Beacons for Bioanalytical and Biomedical Applications,” Chem. Soc. Rev. 44(10):3036-3055. |
| Zhou, Y. et al. (2020) “Network-Based Drug Repurposing for Novel Coronavirus 2019-nCoV/SARS-CoV-2,” Cell Discov. 6(14): doi.org/10.1038/s41421-020-0153-3; pp. 1-18. |
| Zhu, N. et al. (2020) “A Novel Coronavirus from Patients with Pneumonia in China, 2019,” New Engl. J. Med. 382(8):727-733. |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16837364 | Apr 2020 | US |
| Child | 17078249 | US |
