Patent Application
20230416846
The present invention relates to digital CRISPR-based methods for detecting and quantitating target nucleic acids in a sample, a kit for use in such methods, and methods for detecting the presence and/or severity of disease in a subject.
DNA and RNA are usually used as detection targets to indicate the presence of a biological entity. Technologies for nucleic acid quantification are needed in diverse areas, ranging from biomedical research to clinical diagnostics to environmental protection. The widely used RT-qPCR, as a gold standard for COVID-19 diagnosis, offer advantages in speed and sensitivity but require precise thermal cycling and high PCR efficiency. Quantification via RT-qPCR relies on the use of external standards or references, and the results can be variable, with a 20-30% variability reported even within trained laboratories [Sedlak, R. H. and Jerome, K. R. Diagnostic microbiology and infectious disease 75: 1-4 (2013)]. Thus, an absolute quantification method with improved precision and accuracy is vital for virus research.
Digital PCR is increasingly used as a highly accurate and sensitive method for absolute quantification of nucleic acids [Salipante, S. J. and Jerome, K. R. Clinical chemistry 66: 117-123 (2020); Sedlak, R. H. and Jerome, K. R. Diagnostic microbiology and infectious disease 75: 1-4 (2013)]. In a digital PCR reaction, the PCR mixture is separated into thousands of individual reactions, resulting in either zero or one of the nucleic acid target molecules present in each partition. After independent PCR amplification and endpoint fluorescence detection of each partition, the copy number of the sample is determined based on the proportion of positive partitions. Since the PCR reaction in each partition proceeds independently, absolute quantification by digital PCR is more precise, more tolerant to inhibitors and overcomes poor amplification efficiency [Whale, A. S. et al. Nucleic acids research 40: e82-e82 (2012)]. The sensitivity and precision of digital PCR-based virus detection has been demonstrated in quantitative detection and viral load analysis of, for example, SARS-CoV-2-infected patient samples with a limit of detection (LOD) at ˜2 copies/reaction and fewer false negatives and fewer false positives compared with RT-PCR [Alteri, C. et al. PloS one 15: e0236311 (2020); Liu, X. et al. Emerging microbes & infections 9: 1175-1179 (2020); Suo, T. et al. Emerging microbes & infections 9(1): 1259-1268 (2020); Yu, F. et al. Clinical Infectious Diseases 71: 793-798 (2020)]. In addition to its application in viral diagnostics, digital PCR has also been successfully used in other areas of virus research, including the study of aerodynamic transmission of SARS-CoV-2 and in quantifying residual SARS-CoV-2 load in pulmonary tissues of a virus-negative patient by nasopharyngeal swab-qPCR test [Liu, Y. et al. Nature 582: 557-560 (2020); Yao, X.-H. et al. Cell research 30: 541-543 (2020)]. The main drawback of digital PCR, however; is the relatively long reaction time (˜4 hours) needed as a result of the 1-2° C./s ramp rate for efficient inter-partition heat transfer during thermal cycling, compared to that of qPCR which requires 1 hour. Reducing the reaction time of digital PCR is therefore crucial in enabling the adoption of the technology in rapid virus detection.
Isothermal amplification methods, which amplify the nucleic acid target molecule at a constant temperature and thereby reduce the reaction time have also been used in virus detection. These include methods that employ recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP) [Notomi, T. et al. Nucleic acids research 28: E63-E63 (2000); Piepenburg, O. et al. PLOS Biology 4: e204 (2006); Tomita, N. et al. Nature protocols 3: 877-882 (2008)]. More recently, innovative diagnostic methods using RNA-guided CRISPR/Cas system have been developed to detect nucleic acids. In the RNA-guided CRISPR/Cas system, Cas effectors such as Cas12a, Cas12b and Cas13a are exploited for their “collateral cleavage activity”: once the Cas protein finds and cleaves a specific DNA/RNA target, it binds and degrades other nonspecific DNA/RNA oligos such as fluorescently-tagged reporter oligos [Chen, J. S. et al. Science 360: 436-439 (2018); Gootenberg, J. S. et al. Science 356: 438-442 (2017); Li, S. Y. et al. Cell discovery 4: 20 (2018)]. By combining RPA- or LAMP-mediated isothermal amplification of the target molecule with the CRISPR/Cas biosensing system, methods such as SHERLOCK and DETECTR have successfully demonstrated the detection of dengue virus, human papillomavirus as well as SARS-CoV-2 in clinical samples [Broughton, J. P. et al. Nature biotechnology (2020); Ding, X. et al. Nature communications 11: 4711 (2020); Gootenberg, J. S. et al. Science 356: 438-442 (2017); Li, S. Y. et al. Cell discovery 4: 20 (2018)]. However, as CRISPR-based methods are not quantitative and require multiple manipulations between the amplification and detection steps, there remains a need for a quantitative, rapid and robust virus detection method.
There is a need for an improved molecular platform to enable rapid, visual and modular detection and quantification of nucleic acids and other target molecules.
The present invention provides a nucleic acid detection and quantification system comprising: a CRISPR system comprising an effector protein and one or more guide RNAs designed to bind to corresponding target molecules; a Nucleic Acids-based masking construct; a sample partitioning step which subdivides the sample into a plurality of compartments, and optionally, nucleic acid amplification reagents to amplify target molecules in a sample. This method combines the advantages of quantitative digital PCR, rapid isothermal amplification and specific CRISPR detection into a one-pot reaction system which partitions the individual reactions into a plurality of small compartments in a high-density chip. In this study, we demonstrate a digital CRISPR method (also called RApid Digital Crispr Approach, RADICA) that allows for absolute quantification of nucleic acids at a constant temperature in one hour. We validated this method using DNA containing the N (nucleoprotein) gene of SARS-CoV-2, and showed a linear signal-to-input response of R2 value>0.99. We further compared our digital CRISPR detection system against the traditional digital PCR method and show superior speed using the digital CRISPR system (1 h vs 4 h) while demonstrating comparable sensitivity and accuracy to that of traditional digital PCR. Further, we successfully used digital CRISPR in the absolute quantification of Epstein-Barr virus from human B cells, human adenovirus and herpes simplex virus (R2 value>0.98) as well multiplex detection of several targets in a reaction. Together, our rapid and sensitive digital CRISPR method allows for accurate detection and absolute quantification of nucleic acids.
In a first aspect there is provided a method for detecting and quantitating target nucleic acids in a sample comprising:
In some embodiments, the Cas effector is Cas12a or Cas12b.
In some embodiments, the method is used to detect and/or quantify a pathogen, gene expression, gene copy number variation or adventitious agents in a sample.
In some embodiments, the at least one guide polynucleotide is crRNA.
In some embodiments, the amplification is selected from the group comprising nucleic-acid sequence-based amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, strand-displacement amplification, exonuclease III-assisted signal amplification, hybridization chain reaction, helicase-dependent amplification, isothermal circular strand displacement polymerization, multiple displacement amplification, primase-based whole genome amplification, rolling circle amplification and whole genome amplification.
In some embodiments, the amplification coupled with Cas12a is selected from the group comprising recombinase polymerase amplification, strand-displacement amplification, rolling circle amplification and multiple displacement amplification.
In some embodiments, the amplification coupled with Cas12b is selected from the group comprising loop-mediated isothermal amplification, helicase-dependent amplification, strand-displacement amplification and rolling circle amplification.
In some embodiments, the amplification coupled with Cas13 is selected from the group comprising nucleic-acid sequence-based amplification recombinase polymerase amplification and strand-displacement amplification.
In some embodiments, the amplification coupled with Cas14 is selected from the group comprising recombinase polymerase amplification, strand-displacement amplification and rolling circle amplification.
In some embodiments, the isothermal amplification is recombinase polymerase amplification or loop-mediated isothermal amplification.
In some embodiments, the masking construct suppresses generation of a detectable positive signal until cleaved, or masks a detectable positive signal until the masking construct is cleaved.
In some embodiments, the masking construct comprises a quenched fluorescent nucleic acids probe, such as a ssDNA probe, dsDNA or RNA probe.
In some embodiments, the target is DNA or RNA.
In some embodiments, the target is virus DNA or RNA.
In some embodiments, the virus is SARS-CoV-2 virus, human adenovirus (HAdV), herpes simplex virus (HSV) or Epstein-Barr virus (EBV).
In some embodiments, the partitioning is microfluidics-based, droplets-based or membrane-based, preferably chip-based.
In some embodiments, the mixture is partitioned into at least 1,000 compartments, preferably at least 10,000 compartments.
In some embodiments, the guide has a sequence comprising a mismatch to the one or more target sequences.
In some embodiments, the mismatch is up or downstream of a single nucleotide variation in the guide sequence.
In some embodiments, the isothermal amplification is recombinase polymerase amplification (RPA). A schematic of a recombinase polymerase amplification RADICA according to the invention is shown in
In some embodiments, the RPA reaction is at 42° C. in step c).
In some embodiments, the Cas effector is Cas12a, In some embodiments, the Cas12a homolog is from Lachnospiraceae bacterium ND2006 (LbCas12a).
It was found that using Cas12a at 42° C. in step c), a 40 min incubation was enough for qualitative detection and 60 min incubation is enough for quantitative detection (Example 3).
In some embodiments, the isothermal amplification is a warm-start LAMP or RT-LAMP reaction.
In some embodiments, the warm-start RT-LAMP reaction is at 60° C. in step c).
In some embodiments, the Cas effector is Cas12b, which has been shown to be compatible with the one-pot RT-LAMP reaction with high sensitivity). A schematic of a warm-start RADICA according to the invention is shown in
In some embodiments, the Cas effector is Cas12b from Alicyclobacillus acidiphilus (AapCas12b).
In some embodiments, WarmStart RTx reverse transcriptase, WarmStart DNA polymerase, and Cas12b/crRNA are combined. In some embodiments Bst 2.0 WarmStart DNA polymerase is used.
In some embodiments, the nucleic acids-based masking construct is a quenched fluorescent reporter comprising 5 thymine (T) bases. Advantageously, this masking construct shows high signal-to-noise ratio and reaction rate.
In some embodiments, taurine is added to the reaction mixture to improve reaction kinetics.
In some embodiments, 40 min incubation is enough for qualitative detection and 60 min incubation is enough for quantitative detection by warm-start RADICA.
In some embodiments, the isothermal amplification reaction is a multiplex reaction.
In some embodiments, the multiplex reaction detects a target nucleic acid and a human nucleic acid (DNA) control.
In some embodiments, the multiplex reaction detects a wildtype target nucleic acid and a variant or mutant thereof. For example, a multiplex reaction is used to detect wildtype SARS-CoV-2 N (or other) gene and a SARS-CoV-2 mutant variant thereof. The reaction may comprise multiplex primers to detect a wildtype target nucleic acid and at least one variant thereof, as exemplified in Examples 17 and 18 and
In some embodiments, the target nucleic acids are SARS-CoV-2, HAdV, HSV, or Epstein-Barr virus nucleic acids;
In some embodiments, the amplification reaction is warm-start.
In a second aspect there is provided a method for detecting the presence and/or severity of a disease in a subject, comprising the steps of:
In some embodiments, the disease is a pathogen infection.
In some embodiments, the disease is a virus infection.
In some embodiments, the Cas effector is Cas12a and/or Cas12b.
In some embodiments, the method is used to detect and/or quantify a pathogen, gene expression or gene copy number variation.
In some embodiments, the at least one guide polynucleotide is crRNA.
In some embodiments, the amplification is selected from the group comprising nucleic-acid sequence-based amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, strand-displacement amplification, exonuclease III-assisted signal amplification, hybridization chain reaction, helicase-dependent amplification, isothermal circular strand displacement polymerization, multiple displacement amplification, primase-based whole genome amplification, rolling circle amplification and whole genome amplification.
In some embodiments, the isothermal amplification is recombinase polymerase amplification or loop-mediated isothermal amplification.
In some embodiments, the masking construct suppresses generation of a detectable positive signal until cleaved, or masks a detectable positive signal until the masking construct is cleaved.
In some embodiments, the masking construct comprises a quenched fluorescent nucleic acids probe.
In some embodiments, the target is DNA or RNA.
In some embodiments, the target is virus DNA or RNA.
In some embodiments, the virus is SARS-CoV-2, human adenovirus (HAdV), herpes simplex virus (HSV), or Epstein-Barr virus.
In some embodiments, the partitioning is microfluidics-based, droplets-based or membrane-based, preferably chip-based.
In some embodiments, the mixture is partitioned into at least 1,000 compartments.
In some embodiments, the isothermal amplification is a warm-start RT-LAMP reaction.
In some embodiments, the isothermal amplification reaction is a multiplex reaction.
In some embodiments, the method further comprises administering a treatment that is efficacious for the severity of the disease in said subject.
In a third aspect there is provided a kit to quantitate target nucleic acids in a sample comprising:
In some embodiments of the kit, the target nucleic acids are SARS-CoV-2, human adenovirus (HAdV), herpes simplex virus (HSV), or Epstein-Barr virus nucleic acids; the isothermal amplification reaction reagents are:
In some embodiments, the partitioning device or substrate comprises at least 10,000 compartments.
Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the Examples. The whole content of such bibliographic references is herein incorporated by reference.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention belongs. Certain terms employed in the specification, examples and appended claims are collected here for convenience.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a target sequence” includes a plurality of such target sequences, and a reference to “an enzyme” is a reference to one or more enzymes and equivalents thereof known to those skilled in the art, and so forth.
The phrases “nucleic acid” or “nucleic acid sequence,” as used herein, refer to an oligonucleotide, nucleotide, polynucleotide, or any fragment thereof, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
As used herein, the term “oligonucleotide”, refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray. As used herein, the term “oligonucleotide” is substantially equivalent to the terms “amplimers,” “primers,” “oligomers,” and “probes,” as these terms are commonly defined in the art. The recognition nanostructure may comprise an inverter oligonucleotide.
The term “sample,” as used herein, is used in its broadest sense. For example, a biological sample suspected of containing human adenovirus, HSV, EBV or SARS-CoV-2 genome sequences may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA (in solution or bound to a solid support); a tissue; a tissue print; and the like.
It would be understood that oligonucleotides used in the present invention may be structurally and/or chemically modified to, for example, prolong their activity in samples potentially containing nucleases, during performance of methods of the invention, or to improve shelf-life in a kit. Thus, the aptamer and/or inverter and/or signaling nanostructure or any oligonucleotide primers or probes used according to the invention may be chemically modified. In some embodiments, said structural and/or chemical modifications include the addition of tags, such as fluorescent tags, radioactive tags, biotin, a 5′ tail, the addition of phosphorothioate (PS) bonds, 2′-O-Methyl modifications and/or phosphoramidite C3 Spacers during synthesis.
As used herein, the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in context with the present disclosure, the term “comprising” or “including” also includes “consisting of”. The variations of the word “comprising”, such as “comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.
Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, New York (2012).
The sequences of primers, crRNA, and FQ reporters were synthesised by Integrated DNA Technologies. Plasmids containing the N gene from each virus genomes (SARS-CoV-2, SARS-CoV, and MERS-CoV) were purchased from Integrated DNA Technologies. The synthetic RNA covering 99.9% of the bases of the SARS-CoV-2 viral genome was purchased from Twist Bioscience (Genbank ID: MN908947.3). The DNA and RNA concentrations were measured by dPCR or RT-dPCR assay. TwistAmp® Basic were from TwistDx. EnGen® Lba Cas12a were from New England Biolabs. Bst 2.0 WarmStart polymerase, WarmStart RTx Reverse Transcriptase and RNase Inhibitor were from New England Biolabs. Cas12b was from Magigen Biotechnology. Clarity JN solution and Clarity digital chip were from JN Medsys. QIAcuity digital nanoplates were from QIAGEN. The USCDC N2 assay for SARS-CoV-2 detection was from Integrated DNA Technologies. TaqMan™ Fast Virus 1-Step Master Mix was from Applied Biosystems.
crRNA Preparation for Cas12a:
Constructs were ordered as DNA from Integrated ssDNA Technologies with an appended T7 promoter sequence. crRNA ssDNA was annealed to a short T7 primer (T7-3G IVT primer [Kellner, M. J. et al. Nature protocols 14: 2986-3012 (2019)] or T7-Cas12scaffold-F [Lucia, C. et al. bioRxiv 2020.2002.2029.971127 (2020)] and treated with fill-in PCR (Platinum™ SuperFi II PCR Master Mix) to generate the DNA templates. These DNA were used as DNA templates to synthesize crRNA using the HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs) according to published protocols [Kellner, M. J. et al. Nature protocols 14: 2986-3012 (2019); Lucia, C. et al. bioRxiv 2020.2002.2029.971127 (2020)]. The synthesized crRNA was purified using Monarch® RNA Cleanup Kit (New England Biolabs) after treatment with DNase I (RNase-free, New England Biolabs), Thermolabile Exonuclease I (New England Biolabs), and T5 Exonuclease (New England Biolabs).
crRNA Preparation for Cas12b:
The DNA template for crRNA synthesis was obtained by first annealing the Cas12b crRNA universal scaffold oligo (DNA oligo) with the respective DNA oligo (containing the target region and the region complementary to Cas12b crRNA scaffold and filling in both ends using Platinum™ SuperFi II PCR Master Mix. The resulted DNA was used as a template for crRNA synthesis using HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs). The DNA template was then removed by DNase I (RNase-free, New England Biolabs), Thermolabile Exonuclease I (New England Biolabs), and T5 Exonuclease (New England Biolabs), and the crRNA was purified using Monarch® RNA Cleanup Kit (New England Biolabs).
The one-pot reaction combining RPA-DNA amplification and Cas12a detection was performed as follows: 300 nM forward primer, 300 nM reverse primer, 500 nM FQ reporter, 1×RPA rehydration buffer containing 1×RPA Pellet (TwistDx), 200 nM EnGen® Lba Cas12a (New England Biolabs), 200 nM crRNA, were prepared followed by adding various amounts of DNA input, and 14 mM magnesium acetate. When RNA was used as a target, 300 nM reverse primer 2 was used with 10 U/μL PhotoScript Reverse transcriptase (New England Biolabs) or 10 U/μL SuperScript™ IV Reverse Transcriptase (Invitrogen) and 0.5 U/μL RNase H (Invitrogen or New England Biolabs), as indicated. Commercial chips for sample partitioning and matched fluorescence reader for endpoint detection were used in RADICA. The RADICA reaction was prepared by adding 1× Clarity™ JN solution (JN Medsys) to the RPA-Cas12a bulk reactions stated above. To prevent spontaneous target amplification by RPA at room temperature, the RPA-CRISPR reaction was prepared without the addition of Mg2+, which is required for the polymerase activity. All reactions were prepared on ice and samples were loaded within one minute to prevent premature target amplification. 15 μL of the mixture was loaded on the chip by a Clarity™ autoloader for sample partitioning. The reaction partitions were sealed with the Clarity™ Sealing Enhancer and 230 μL Clarity™ Sealing Fluid. The partitioned reactions were incubated in water baths or heat blocks at 42° C. for 1 hour, unless otherwise indicated. After incubation, a Clarity™ Reader was used to read the fluorescent signal in the partitions, and Clarity™ software was used to calculate input DNA copy numbers.
The DNA/RNA target samples were mixed with 1.6 μM FIP primers, 1.6 μM BIP primers, 0.2 μM F3 primers, 0.2 μM B3 primers, 0.4 μM LoopF primers, 0.4 μM LoopB primers, 1.4 mM dNTPs, 8 mM MgSO4, 2 μM FQ-5T reporters, 0.96 U/μL Bst 2.0 WarmStart polymerase, 0.3 U/μL WarmStart RTx Reverse Transcriptase (for RNA), 1 U/μL RNase Inhibitor, 50 mM Taurine, 50 nM Cas12b, and 50 nM crRNA in 1× isothermal amplification buffer, unless otherwise indicated. RADICA reaction was prepared by adding 1× Clarity JN solution to the RT-LAMP-Cas12b bulk reactions described above and partitioned on Clarity digital chip (˜1.336 nL partition volume, ˜10,000 partitions per reaction). 15 μL reaction mixtures were loaded onto the digital chip followed by treatment with the Clarity sealing enhancer and sealing with 230 μL Clarity sealing fluid. The tube containing the digital chip was warmed in a water bath at 60° C. for 1 hour, unless otherwise indicated. After incubation, the end-point fluorescence in the 10,000 partitions was detected by Clarity Reader. Using Clarity software, the threshold was determined based on the florescence distribution of the partitions and positive partition percentages and input nucleic acids concentration were then calculated based on the threshold.
The DNA/RNA target samples were mixed with 1.6 μM FIP primers, 1.6 μM BIP primers, 0.2 μM F3 primers, 0.2 μM B3 primers, 0.4 μM LoopF primers, 0.4 μM LoopB primers, 1.4 mM dNTPs, 8 mM MgSO4, 2 μM FQ-5T reporters, 0.96 U/μL Bst 2.0 WarmStart polymerase, 0.3 U/μL WarmStart RTx Reverse Transcriptase (for RNA), 1 U/μL RNase Inhibitor, 50 mM Taurine, 50 nM Cas12b, and 50 nM crRNA in 1× isothermal amplification buffer, unless otherwise indicated. The RADICA reaction was prepared by adding 250 nM Cyanine 680 succinimidyl ester (biotium) as reference dye to the RT-LAMP-Cas12b bulk reactions stated above and partitioned on QIAcuity digital nanoplates (˜0.91 nL partition volume, 26,000 partitions per reaction). 40 μL reaction mixtures were loaded onto the QIAcuity digital nanoplate and into the QIAcuity Digital PCR System. In the QIAcuity machine, the reactions were automatically partitioned into 26,000 microwells followed by incubation for 1 hour and end-point fluorescence detection. To get the best signal to noise ratio, exposure duration (200 ms to 800 ms) and gain (2 to 8) were adjusted for each experiment. Usually, a 600 ms/6 will be used for the sample reading. Positive partition percentages, as well as nucleic acid concentration, were calculated by QIAcuity software.
One example of RADICA design schematic is illustrated in
To increase the detection sensitivity, we added an isothermal amplification step using RPA, whose reaction temperature (25° C. to 42° C.) is compatible with that of Cas12a (25° C. to 48° C.). To avoid Cas12a-mediated cleavage of the target molecule before amplification, we designed crRNA to target single-stranded DNA (ssDNA) that is generated only after the amplification of the target molecule. This allowed for a one-step digital RPA-CRISPR absolute quantification method that eliminates multiple operations inherent in two-step CRISPR-based detection methods such as SHERLOCK, HOLMES and DETECTR [Chen, J. S. et al. Science 360: 436-439 (2018); Gootenberg, J. S. et al. Science 356: 438-442 (2017); Li, S. Y. et al. Cell discovery 4: 20 (2018)]. It is easier to design ssDNA-targeting crRNA than traditional dsDNA-targeting crRNA, because the nuclease activity of Cas12a in ssDNA is independent of the presence of a protospacer adjacent motif (PAM) [Li, S. Y. et al. Cell research 28: 491-493 (2018)]. We showed that Cas12a increased the signal to noise ratio of the partitions as it further amplifies the fluorescent signals in the positive partitions (
Primers and crRNAs specific for dsDNA containing the SARS-CoV-2 N (nucleoprotein) gene were designed as described previously [Ding, X. et al. Nature communications 11: 4711 (2020)]. The target regions overlap those of the China CDC assay (N gene region) with some modification to meet the primer and crRNA design requirements. The method was optimized with the primers and crRNA shown in Table 1.
Cas12a Bulk Assay without Preamplification:
Unless otherwise indicated, 50 nM EnGen® Lba Cas12a (New England Biolabs), 50 nM crRNA, and 250 nM FQ reporter were incubated with dsDNA dilution series in NEB buffer 2.1 at 37° C., and fluorescence signals were measured every 5 min.
SARS-CoV-2 N gene-containing G-Block dsDNA, SARS-CoV-2, SARS-CoV, and MERS N gene-containing plasmids were purchased from Integrated DNA Technologies. The SARS-CoV-2 N gene-containing plasmid (IDT) was linearized using FastDigest Scal (Thermo Scientific) and then used as DNA targets. The SARS-CoV-2 N gene-containing plasmid was used as a template to amplify the N gene using primer N-RNA-F/N-RNA-R by Platinum™ SuperFi II PCR Master Mix (Invitrogen). The PCR product was purified by QIAquick PCR Purification Kit (QIAGEN) and used as RNA synthesis templates.
Since N-RNA-F has a T7 promoter sequence, the amplified DNA using N-RNA-F/R primer will contain a T7 promoter upstream of gene N. The T7 tagged N gene dsDNA was transcribed into SARS-CoV-2 RNA using HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs) according to the manufacturer's protocol. The synthesized RNA (N gene) was purified using the Monarch® RNA Cleanup Kit (New England Biolabs) after treatment with DNase I (RNase-free, New England Biolabs). The synthetic RNA covering 99.9% of the bases of the SARS-CoV-2 viral genome were purchased from Twist Bioscience (Genbank ID: MN908947.3).
The one-pot reaction combining RPA-DNA amplification and Cas12a detection was performed as follows: 300 nM forward primer, 300 nM reverse primer, 500 nM FQ reporter, 1×RPA rehydration buffer containing 1×RPA Pellet (TwistDx), 200 nM EnGen® Lba Cas12a (New England Biolabs), 200 nM crRNA, were prepared followed by adding various amounts of DNA input, and 14 mM magnesium acetate. When detecting RPA signal is needed, 250 mM SYTO-82 fluorescent nucleic acids stain was added into the reaction. The reaction mixture was incubated at 42° C. unless otherwise indicated and fluorescence kinetics were monitored every 1 min.
Commercial chips for sample partitioning and matched fluorescence reader for endpoint detection were used in RADICA. The RADICA reaction was prepared by adding 1× Clarity™ JN solution (JN Medsys) to the RPA-Cas12a bulk reactions stated above. To prevent spontaneous target amplification by RPA at room temperature, the RPA-CRISPR reaction was prepared without the addition of Mg2+, which is required for the polymerase activity. All reactions were prepared on ice and samples were loaded within one minute to prevent premature target amplification. 15 μL of the mixture was loaded on the chip by a Clarity™ autoloader for sample partitioning. The reaction partitions were sealed with the Clarity™ Sealing Enhancer and 230 μL Clarity™ Sealing Fluid. The partitioned reactions were incubated in water baths or heat blocks at 42° C. for 1 hour, unless otherwise indicated. After incubation, a Clarity™ Reader was used to read the fluorescent signal in the partitions, and Clarity™ software was used to calculate input DNA copy numbers.
When a constant amount of dsDNA was used as a target in bulk reaction, 50 nM to 250 nM Cas12a/crRNA concentration has no influence on the fluorescence intensities and reaction rates (
We combined the RPA and Cas12a reactions in a one-pot reaction. We performed the bulk reaction at 25° C., 37° C., and 42° C., which fall within the reaction temperature ranges of RPA (25° C. to 42° C.) and Cas12a (25° C. to 48° C.). With serial dilutions of plasmid DNA, the reaction proceeded at 25° C. and 42° C., with a limit of detection of 9.4 copies/μL (
We next investigated the earliest time that the reaction completes in all the partitions. The reaction proceeded quickly with an increase in fluorescence signals detected in some compartments at 20 min, but with a low signal-to-noise ratio at this time point (
We characterized the performance of RADICA in detecting and quantifying SARS-CoV-2 and compared it to that of dPCR. Linearized plasmids containing the SARS-CoV-2 N gene were serially diluted and used as the target DNA in the aforementioned optimized RADICA or dPCR reactions. The method was optimized with the primers and crRNA shown in Table 2 for SARS-CoV-2 DNA detection.
Primer and crRNA Design for RADICA Targeting (SARS-CoV-2):
SARS-CoV-2 primers and crRNA were designed based on previously published papers [Ding, X. et al. Nature communications 11: 4711 (2020)] or 264 SARS-CoV-2 genome sequences from GISAID [Shu, Y. and McCauley, J. Eurosurveillance 22: 30494 (2017)]. Other human-related coronavirus sequences were downloaded from NCBI. UGENE software was used to analyze and align viral genomes (MUSCLE or Kalign). Consensus sequences (threshold: 90%) of 264 SARS-CoV-2 genomes, 328 SARS-CoV, 572 MERS-CoV, 70 Human-CoV-229E genomes, 48 Human-CoV-HKU1 genomes, 71 Human-CoV-NL63, and 178 Human-CoV-0C43 were exported separately from UGENE and used for specificity analysis.
The G-block dsDNA, plasmid, dsDNA and RNA concentrations were quantified by dPCR. Serial dilutions of targets were mixed with 500 nM CHNCDC-geneN-F, 500 nM CHNCDC-geneN-R, 250 nM CHNCDC-geneN-P, 1× TaqMann™ Fast Virus 1-Step Master Mix (for RNA, Applied Biosystems) or TaqMann™ Fast Advanced Master Mix (for DNA, Applied Biosystems), 1× Clarity™ JN solution (JN Medsys). For RNA samples, the reaction mixture was incubated at 55° C. 5 min before partitioning the reaction mix on Clarity™ autoloader. Then the reaction partitions were sealed with the Clarity™ Sealing Enhancer and 230 μL Clarity™ Sealing Fluid, followed by thermal cycling using the following parameters: 95° C. for 15 min (one cycle), 95° C. 50 s and 56° C. 90 s (40 cycles, ramp rate=1° C./s), 70° C. 5 min. The endpoint fluorescence of the partitions was detected using Clarity™ Reader and the final DNA copy numbers were analyzed by Clarity™ software.
The RADICA reaction mix for SARS-CoV-2 DNA was prepared by mixing 300 nM forward primer (N-AIOD-F), 300 nM reverse primer (N-AIOD-R), 500 nM FQ reporter, 1× RPA rehydration buffer containing 1×RPA Pellet (TwistDx), 200 nM EnGen® Lba Cas12a (New England Biolabs), 200 nM crRNA (synthesized by N-AIOD-crRNA-IVT-F and T7-3G IVT primer), 1× Clarity™ JN solution (JN Medsys), followed by adding various amounts of DNA input, and 14 mM magnesium acetate. All reactions were prepared on ice and samples were loaded within one minute to prevent premature target amplification. 15 μL of the mixture was loaded on the chip by a Clarity™ autoloader for sample partitioning. The reaction partitions were sealed with the Clarity™ Sealing Enhancer and 230 μL Clarity™ Sealing Fluid. The partitioned reactions were incubated in water baths or heat blocks at 42° C. for 1 hour, unless otherwise indicated. After incubation, a Clarity™ Reader was used to read the fluorescent signal in the partitions, and Clarity™ software was used to calculate input DNA copy numbers.
A proportional increase in the number of positive partitions with increasing concentrations of the target DNA was detected by RADICA (
The RADICA reaction mix for SARS-CoV-2 DNA was prepared by mixing 300 nM forward primer (N-AIOD-F), 300 nM reverse primer (N-AIOD-R), 500 nM FQ reporter, 1×RPA rehydration buffer containing 1×RPA Pellet (TwistDx), 200 nM EnGen® Lba Cas12a (New England Biolabs), 200 nM crRNA (synthesized by N-AIOD-crRNA-IVT-F and T7-3G IVT primer), 1× Clarity™ JN solution (JN Medsys), followed by adding various amounts of circular plasmid or linearized plasmid, and 14 mM magnesium acetate. All reactions were prepared on ice and samples were loaded within one minute to prevent premature target amplification. 15 μL of the mixture was loaded on the chip by a Clarity™ autoloader for sample partitioning. The reaction partitions were sealed with the Clarity™ Sealing Enhancer and 230 μL Clarity™ Sealing Fluid. The partitioned reactions were incubated in water baths or heat blocks at 42° C. for 1 hour, unless otherwise indicated. After incubation, a Clarity™ Reader was used to read the fluorescent signal in the partitions, and Clarity™ software was used to calculate input DNA copy numbers.
Plasmids are routinely used as reference DNA or standards; and conformational changes in supercoiled DNA can have a profound effect on PCR-based quantification. Single-molecule amplification of non-linearized plasmids was unsuccessful in a PCR-based study, resulting in an underestimation for circular plasmid quantification in some dPCR machines [Dong, L. et al. Scientific reports 5: 13174 (2015)].To test whether plasmid conformation affects the accuracy of RADICA, undigested plasmids containing SARS-CoV-2 N gene were serially diluted and used for digital PCR or RADICA reactions. Concentrations of non-linearized plasmids measured by dPCR were half of those detected for linearized plasmids (
The specificity of RADICA is analysed by 300 nM forward primer (N-AIOD-F), 300 nM reverse primer (N-AIOD-R), 500 nM FQ reporter, lx RPA rehydration buffer containing 1×RPA Pellet (TwistDx), 200 nM EnGen® Lba Cas12a (New England Biolabs), 200 nM crRNA (synthesized by N-AIOD-crRNA-IVT-F and T7-3G IVT primer), followed by adding plasmid containing the SARS-CoV-2 or SARS-CoV or MERS-CoV gene, and 14 mM magnesium acetate. Monitor the fluorescent signal of the reaction at 42° C.
Primer and crRNA designs are key in determining the specificity of CRISPR-based nucleic acids detection. RPA tolerates up to nine nucleotide base-pair mismatches across primer and probe binding sites [Li, J. et al. The Analyst 144: 31-67 (2018)]. To specifically detect SARS-CoV-2 with RADICA, primers and crRNAs would have to specifically bind the SARS-CoV-2 target DNA but not the DNA of other related coronaviruses. We analyzed the binding sites of the primers and crRNAs that were originally designed based on the consensus sequence of the genomes of 264 SARS-CoV-2 strains, available on the GISAID database [Ding, X. et al. Nature communications 11: 4711 (2020); Shu, Y. and McCauley, J. Eurosurveillance 22: 30494 (2017)]. These consensus sequences were aligned with the corresponding regions of SARS-CoV-2-related beta coronaviruses, such as SARS-CoV, MERS-CoV, and human coronaviruses Human-CoV 229E/HKU1/NL63/OC43. No cross-binding regions were observed with the SARS-CoV-2-related coronaviruses analyzed (
Previous studies have reported that RPA reactions could be inhibited by high concentrations of background human DNA 33, 34. We therefore first tested the RPA-Cas12a bulk reaction under varying concentrations of background human DNA.
The RADICA reaction mix for SARS-CoV-2 DNA was prepared by mixing 300 nM forward primer (N-AIOD-F), 300 nM reverse primer (N-AIOD-R), 500 nM FQ reporter, 1×RPA rehydration buffer containing 1×RPA Pellet (TwistDx), 200 nM EnGen® Lba Cas12a (New England Biolabs), 200 nM crRNA (synthesized by N-AIOD-crRNA-IVT-F and T7-3G IVT primer), 1× Clarity™ JN solution (JN Medsys), followed by adding various amounts of DNA input with or without human genomic DNA, and 14 mM magnesium acetate. All reactions were prepared on ice and samples were loaded within one minute to prevent premature target amplification. 15 μL of the mixture was loaded on the chip by a Clarity™ autoloader for sample partitioning. The reaction partitions were sealed with the Clarity™ Sealing Enhancer and 230 μL Clarity™ Sealing Fluid. The partitioned reactions were incubated in water baths or heat blocks at 42° C. for 1 hour, unless otherwise indicated. After incubation, a Clarity™ Reader was used to read the fluorescent signal in the partitions, and Clarity™ software was used to calculate input DNA copy numbers.
RPA reactions can be inhibited by high concentrations of background human DNA [Rohrman, B. and Richards-Kortum, R. Analytical chemistry 87: 1963-1967 (2015)]. We tested for possible inhibitory effects of background DNA on reactions carried out in small partitions. In an RPA-Cas12a reaction with 400 copies/μL of target DNA, 1 ng/μL of background human DNA (4350 human cells per reaction) did not affect the RADICA reaction (
We tested the effect of 1 ng/μL of background human DNA on RADICA reactions with various concentrations of target DNA (
As SARS-CoV-2 is an RNA virus, we tested whether RADICA could be combined with reverse transcription (RT) in a one-pot reaction for the absolute quantification of RNA. The method was optimized with the primers and crRNA shown in Table 3 for SARS-CoV-2 RNA detection.
The RADICA reaction mix for SARS-CoV-2 DNA was prepared by mixing 300 nM forward primer (N-AIOD-F for N1 or NF-CoV-F for N0 region), 300 nM reverse primer (N-AIOD-R for N1 or NF-CoV-R for N0 region), 300 nM reverse primer2 (N-RPA-RR for N1 or NF-CoV-RR for N0 region), 500 nM FQ reporter, 1×RPA rehydration buffer containing 1× RPA Pellet (TwistDx), 200 nM EnGen® Lba Cas12a (New England Biolabs), 200 nM crRNA (synthesized by N-AIOD-crRNA-IVT-F and T7-3G IVT for N1; synthesized by NF-crRNA-1F and T7-Cas12scaffold-F for N0), 10 U/μL PhotoScript Reverse transcriptase (New England Biolabs) or 10 U/μL SuperScript™ IV Reverse Transcriptase (Invitrogen) and 0.5 U/μL RNase H (Invitrogen or New England Biolabs), 1× Clarity™ JN solution (JN Medsys), followed by adding various amounts of RNA target, and 14 mM magnesium acetate. All reactions were prepared on ice and samples were loaded within one minute to prevent premature target amplification. 15 μL of the mixture was loaded on the chip by a Clarity™ autoloader for sample partitioning. The reaction partitions were sealed with the Clarity™ Sealing Enhancer and 230 μL Clarity™ Sealing Fluid. The partitioned reactions were incubated in water baths or heat blocks at 42° C. for 1 hour, unless otherwise indicated. After incubation, a Clarity™ Reader was used to read the fluorescent signal in the partitions, and Clarity™ software was used to calculate input DNA copy numbers.
The sensitivity of the one-pot RT-RPA-Cas12a bulk reaction was lower-than-expected, with an LoD at 244 copies/μL, with an increased sensitivity (61 copies/μL) when two reverse primers were used (
Absolute Quantification of Epstein-Barr Virus from Infected B Cells by RADICA (Digital RPA-Cas12a)
We tested the ability of RADICA to perform absolute quantification on Epstein-Barr virus (EBV), a member of the human herpesvirus (HHV4) reported as viral contamination in the biologic and cell manufacturing process [Barone, P. W. et al. Nature biotechnology 38: 563-572 (2020)]. The primers and crRNA shown in Table 4 were used for Epstein-Barr virus detection in infected B cells.
∧Vo, J. H. et al., Scientific Reports 6:13-13 (2016).
Primer and crRNA Design for RADICA Targeting EBV:
Epstein-Barr virus primers and crRNA were designed based on consensus sequences of 16 virus genomes, including both type I and type II EBV (NCBI: AP015016.1, AY961628.3, HQ020558.1, JQ009376.2, KC207813.1, KC207814.1, KC440851.1, KC440852.1, KC617875.1, KF373730.1, KF717093.1, KP735248.1, LN827800.1, NC_007605.1, NC_009334.1, V01555.2).
Serial dilutions of EBV DNA were used for dPCR quantification by Clarity™ Epstein-Barr Virus Quantification Kit (JN Medsys) or primers and probes from published papers [Tay, J. K. et al. International Journal of Cancer 146: 2923-2931 (2020); Vo, J. H. et al. Scientific reports 6: 13-13 (2016)] with TaqMan™ Fast Advanced Master Mix (Applied Biosystems), 1× Clarity™ JN solution (JN Medsys) according to the manufacturer's protocol.
Growing EBV-2 from Jijoye Cells:
Jijoye cells were treated with 4 mM sodium butyrate and 24 ng/ml tetradecanoyl phorbol acetate (TPA). Supernatants were harvested 4-5 days post-treatment by centrifugation at 4,000 g for 20 min and passing over a 0.45 μm filter to remove cellular debris. Viral particles were pelleted by ultracentrifugation at 20,000 rpm for 90 min and resuspended in 1/100 the initial volume using complete RPMI or PBS if viruses were to be further purified. Concentrated viruses were further purified using OptiPrep gradient density ultracentrifugation at 20,000 rpm for 120 min, and the virus interface band was collected and stored at −80° C. for downstream analysis.
Epstein-Barr Virus DNA Extraction from Jijoye Cells:
EBV DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's protocol.
Each 15 μL RADICA reaction consisted of 300 nM forward primer (EBV-EBNA1-F2 or EBV-BamHIW-F3), 300 nM reverse primer (EBV-EBNA1-R2 or EBV-BamHIW-R3), 500 nM FQ reporter, 1×RPA rehydration buffer containing 1×RPA Pellet (TwistDx), 200 nM EnGen® Lba Cas12a (New England Biolabs), 200 nM EBNA-2R1-crRNA or BamHIW-3F-crRNA, 0.01 mg/mL BSA, 1× Clarity JN Solution (JN Medsys), different concentrations of DNA or controls, and 14 mM magnesium acetate. Each reaction mix was partitioned in the Clarity Digital PCR tube-strip (JN Medsys) followed by 42° C. incubation for 1 h in a water bath. After incubation, a Clarity™ Reader was used to read the fluorescent signal in the partitions, and Clarity™ software was used to calculate input DNA copy numbers.
To design primers and crRNA that were universal to both type I and type II EBV, we analyzed the genomes of 16 EBV strains and identified the conserved regions across all 16 strains. A conserved DNA region within the Epstein-Barr nuclear antigen 1 (EBNA1) and repetitive BamHI-W sequences were used as the target sequences (
Clinical Validation of RADICA (Digital RPA-Cas12a) and Comparison with qPCR and dPCR
To validate RADICA in clinical samples, we compared RADICA with qPCR- and dPCR-based quantification methods to analyze the EBV load in 79 serum samples obtained from 39 nasopharyngeal cancer (NPC) patients and 40 healthy controls. NPC is an EBV-associated malignancy and the circulating EBV cell-free DNA is elevated in 53-96% of NPC patients [Tay, J. K. et al. International Journal of Cancer 146: 2923-2931 (2020)].
Two sets of clinical samples from a serum bank of nasopharyngeal cancer patients and healthy controls were used in this study. The first set comprised 79 serum samples of which 39 were from nasopharyngeal cancer (NPC) patients and 40 were from healthy controls. The second set comprised of 66 serum samples taken from 22 NPC patients at three time points: at the time of initial diagnosis, one year after treatment and at the time of recurrence. All participants were recruited with informed consent, and the study was approved by the Institutional Review Board of the National Healthcare Group, Singapore (Approval numbers: 2006/00149, 2006/00409).
Epstein-Barr Virus DNA Extraction from Serum:
EBV cell-free DNA was extracted from 200 μL of serum using the ReliaPrep™ Blood gDNA Miniprep System (Promega) according to the manufacturer's protocol. The DNA was eluted using 50 μL of ddH2O and further diluted using another 50 μL of ddH2O, resulting in 100 μL of DNA solution.
qPCR Quantification of Epstein-Barr Virus in Serum Cell-Free DNA:
qPCR was performed using 400 nM EBNA-PCR-F, 400 nM EBNA-PCR-R, 200 nM EBNA-P-FAM, 200 nM BamHIW-PCR-44F, 200 nM BamHIW-PCR-119R, 100 nM BamHIW-P-HEX, 3 μL of serum DNA (after 1:1 dilution) or controls in 1× TaqMann™ Fast Advanced Master Mix (Applied Biosystems). Each reaction mix was incubated at 50° C. 2 min to allow UNG to degrade carry-over PCR products, followed by 1 cycle at 95° C. for 2 min, 40 cycles at 95° C. for 3 s and 59° C. for 30 s. A standard curve using DNA extracted from the Jijoye EBV positive cell line was run in parallel with each reaction to quantify the input DNA concentration.
dPCR Quantification of Epstein-Barr Virus in Serum Cell-Free DNA:
dPCR was performed using the Clarity Digital PCR System (JN Medsys). Each 15 μL dPCR reaction consisted of 400 nM EBNA-PCR-F, 400 nM EBNA-PCR-R, 200 nM EBNA-P-FAM, 200 nM BamHIW-PCR-44F, 200 nM BamHIW-PCR-119R, 100 nM BamHIW-P-HEX, 1× Clarity JN Solution (JN Medsys), 1× TaqMann™ Fast Advanced Master Mix (Applied Biosystems), and 3 μL of serum DNA or controls. Each reaction mix was partitioned in the Clarity Digital PCR tube-strip (JN Medsys) followed by 1 cycle at 95° C. for 10 min, 40 cycles at 95° C. for 50 s and 57° C. for 90 s and 1 cycle at 70° C. for 5 min. After thermal cycling, a Clarity™ Reader was used to read the fluorescent signal in the partitions, and Clarity™ software was used to calculate input DNA copy numbers.
Each 15 μL RADICA reaction consisted of 300 nM EBV-BamHIW-F3, 300 nM EBV-BamHIW-R3, 500 nM FQ reporter, 1×RPA rehydration buffer containing 1×RPA Pellet (TwistDx), 200 nM EnGen® Lba Cas12a (New England Biolabs), 200 nM BamHIW-3F-crRNA, 0.01 mg/mL BSA, 1× Clarity JN Solution (JN Medsys), 3 μL of serum DNA or controls, and 14 mM magnesium acetate. Each reaction mix was partitioned in the Clarity Digital PCR tube-strip (JN Medsys) followed by 42° C. incubation for 1 h in a water bath. After incubation, a Clarity™ Reader was used to read the fluorescent signal in the partitions, and Clarity™ software was used to calculate input DNA copy numbers.
Cell-free DNA from 79 frozen serum samples were blinded and the viral load was quantified using the EBV BamHI-W target. First, to confirm the integrity of the frozen serum samples after long-term storage, qPCR-based quantification of the EBV load in each frozen serum sample was performed and found to be comparable with that obtained previously using the fresh serum. We next compared dPCR- and RADICA-based EBV quantification with that obtained from qPCR on the same serum samples, and found a high correlation between both methods and qPCR, with RADICA demonstrating a higher correlation with qPCR (r=0.872, p=1.28e-25) compared to dPCR (r=0.831, p=2.51e-21) (
As EBV cell-free DNA is routinely used to monitor the virus load in NPC patients after treatment, frozen serum samples obtained from 22 NPC patients at their initial diagnoses, one year after treatment and the point of recurrence were blinded, and EBV loads were quantified by qPCR, dPCR and RADICA (
Human genomic DNA is usually used as a control target to indicate the success of the reaction. Thus, we designed primers and crRNAs targeting human RNAse P gene and primer, and screened these primers and crRNAs (Table 5) using bulk RPA-Cas12a assay.
Screen the primer and crRNA using RPA-Cas12a bulk assay: The one-pot reaction combining RPA-DNA amplification and Cas12a detection was performed as follows: 300 nM forward primer, 300 nM reverse primer, 500 nM FQ reporter, 1×RPA rehydration buffer containing 1×RPA Pellet (TwistDx), 200 nM EnGen® Lba Cas12a (New England Biolabs), 200 nM crRNA, were prepared followed by adding various amounts of DNA input, and 14 mM magnesium acetate. The reaction mixture was incubated at 42° C. unless otherwise indicated and fluorescence kinetics were monitored every 1 min.
RADICA quantification of human DNA: Each 15 μL RADICA reaction consisted of 300 nM forward primer (RNAseP-RPA-6L), 300 nM reverse primer (RNAseP-RPA-6R), 500 nM FQ reporter, 1×RPA rehydration buffer containing 1×RPA Pellet (TwistDx), 200 nM EnGen® Lba Cas12a (New England Biolabs), 200 nM P-CR6-F-crRNA, 0.01 mg/mL BSA, lx Clarity JN Solution (JN Medsys), different concentrations of DNA or controls, and 14 mM magnesium acetate. Each reaction mix was partitioned in the Clarity Digital PCR tube-strip (JN Medsys) followed by 42° C. incubation for 1 h in a water bath. After incubation, a Clarity™ Reader was used to read the fluorescent signal in the partitions, and Clarity™ software was used to calculate input DNA copy numbers.
We designed primers and crRNAs targeting human RNAse P gene and primer, screened these primers and crRNAs using bulk RPA-Cas12a assay (
Another format of RADICA combines RT-LAMP and Cas12b in a one-pot reaction and digitalizes the reaction into thousands of nanoliter or sub-nanoliter reactions for quantitative results. The design principle of RADICA is illustrated in
As the RT-LAMP reaction is warm-start, the reaction will be inhibited at temperatures below 45° C. and will start only after the samples are partitioned and incubated at 60° C., which enables room-temperature reaction setup and increases the accuracy and consistency of the results. The Cas enzyme we used was the thermostable Cas12b from Alicyclobacillus acidiphilus (AapCas12b), which has been shown to be compatible with the one-pot RT-LAMP reaction with high sensitivity [Joung, J. et al. New England Journal of Medicine (2020)]. Compared to a bulk reaction (like the conventional SHERLOCK reaction) [Joung, J. et al. New England Journal of Medicine (2020)] (
Systematic studies of one-pot RT-LAMP-Cas12b reaction were conducted to optimize the assay performance. The primers and crRNAs used are shown in Table 6.
The DNA/RNA target samples were mixed with 1.6 μM FIP primers, 1.6 μM BIP primers, 0.2 μM F3 primers, 0.2 μM B3 primers, 0.4 μM LoopF primers, 0.4 μM LoopB primers, 1.4 mM dNTPs, 8 mM MgSO4, 2 μM FQ-5T reporters, 0.96 U/μL Bst 2.0 WarmStart polymerase, 0.3 U/μL WarmStart RTx Reverse Transcriptase (for RNA), 1 U/μL RNase Inhibitor, 50 mM Taurine, 50 nM Cas12b, and 50 nM crRNA in 1× isothermal amplification buffer, unless otherwise indicated. To detect the LAMP signal, 250 mM SYTO-82 fluorescent nucleic acid stain was added to the reaction. The reaction mixture was incubated at 60° C. and fluorescence kinetics were monitored for 1-2 h using Roche Light Cycler 96. The fluorescence values displayed in the results were the fluorescence levels determined by Roche Light Cycler 96 software. To mimic the complexity of real samples, 1 ng/μL human genomic DNA was also added to the reactions.
The RADICA reaction was prepared by adding 250 nM Cyanine 680 succinimidyl ester (biotium) as reference dye to the RT-LAMP-Cas12b bulk reactions stated above and partitioned on QIAcuity digital nanoplates (˜0.91 nL partition volume, 26,000 partitions per reaction). 40 μL reaction mixtures were loaded onto the QIAcuity digital nanoplate and into the QIAcuity Digital PCR System. In the QIAcuity machine, the reactions were automatically partitioned into 26,000 microwells followed by 60° C. incubation for 1 hour and end-point fluorescence detection. To get the best signal to noise ratio, exposure duration (200 ms to 800 ms) and gain (2 to 8) were adjusted for each experiment. Usually, a 600 ms/6 will be used for the sample reading. Positive partition percentages, as well as nucleic acid concentration, were calculated by QIAcuity software.
To increase the signal-to-noise ratio (S/N) and reduce the reaction time, the FQ reporter with 5 thymine (T) was chosen because it showed the highest S/N and reaction rate compared to those of other FQ reporters with different base compositions (poly-A, poly-T, poly C, poly G and poly AT,
Using the optimized parameters above, we subsequently tested the real-time performance of bulk reactions at concentrations of RNA ranging from 1 to 18391 copies/μL. In the one-pot assay, RT-LAMP reactions were monitored using a SYTO-82 orange fluorescent nucleic acid stain (
We then investigated the reaction time of the reaction on a digital device by measuring the percentages of positive partitions at different reaction times. From the end-point fluorescence results at different time (
To investigate the performance of RADICA, the specificity, quantification capability, and applicability of this method were evaluated on different devices, using the primers and crRNA shown in Table 7.
The DNA/RNA target samples were mixed with 1.6 μM FIP primers, 1.6 μM BIP primers, 0.2 μM F3 primers, 0.2 μM B3 primers, 0.4 μM LoopF primers, 0.4 μM LoopB primers, 1.4 mM dNTPs, 8 mM MgSO4, 2 μM FQ-5T reporters, 0.96 U/μL Bst 2.0 WarmStart polymerase, 0.3 U/μL WarmStart RTx Reverse Transcriptase (for RNA), 1 U/μL RNase Inhibitor, 50 mM Taurine, 50 nM Cas12b, and 50 nM crRNA in 1× isothermal amplification buffer, unless otherwise indicated. To detect the LAMP signal, 250 mM SYTO-82 fluorescent nucleic acid stain was added to the reaction. The reaction mixture was incubated at 60° C. and fluorescence kinetics were monitored for 1-2 h using Roche Light Cycler 96. The fluorescence values displayed in the results were the fluorescence levels determined by Roche Light Cycler 96 software. To mimic the complexity of real samples, 1 ng/μL human genomic DNA was also added to the reactions.
RADICA reaction was prepared by adding 1× Clarity JN solution to the RT-LAMP-Cas12b bulk reactions described above and partitioned on Clarity digital chip (˜1.336 nL partition volume, ˜10,000 partitions per reaction). 15 μL reaction mixtures were loaded onto the digital chip followed by treatment with the Clarity sealing enhancer and sealing with 230 μL Clarity sealing fluid. The tube containing the digital chip was warmed in a water bath at for 1 hour, unless otherwise indicated. After incubation, the end-point fluorescence in the 10,000 partitions was detected by Clarity Reader. Using Clarity software, the threshold was determined based on the florescence distribution of the partitions and positive partition percentages and input nucleic acids concentration were then calculated based on the threshold.
The RADICA reaction was prepared by adding 250 nM Cyanine 680 succinimidyl ester (biotium) as reference dye to the RT-LAMP-Cas12b bulk reactions stated above and partitioned on QIAcuity digital nanoplates (˜0.91 nL partition volume, ˜26,000 partitions per reaction). 40 μL reaction mixtures were loaded onto the QIAcuity digital nanoplate and into the QIAcuity Digital PCR System. In the QIAcuity machine, the reactions were automatically partitioned into 26,000 microwells followed by 60° C. incubation for 1 hour and end-point fluorescence detection. To get the best signal to noise ratio, exposure duration (200 ms to 800 ms) and gain (2 to 8) were adjusted for each experiment. Usually, a 600 ms/6 will be used for the sample reading. Positive partition percentages, as well as nucleic acid concentration, were calculated by QIAcuity software.
DNA or RNA containing the SARS-CoV-2 sequence was added to the reaction as the target and LAMP primers and crRNA targeting N gene of SARS-CoV-2 were used to evaluate the performance of the digital reaction on commercially available Clarity digital chips (1.336 nL partition volume, ˜10,000 partitions per reaction) [Joung, J. et al. New England Journal of Medicine (2020)]. First, the specificity of RADICA was tested by comparing results of SARS-CoV-2 DNA with SARS-CoV DNA, MERS-CoV DNA, and a human genomic DNA control. From the result (
As it would be useful to quantify nucleic acids with this method as well as to detect their presence, RADICA was evaluated for its ability to distinguish different concentrations of SARS-CoV-2 DNA (12777 to 0.8 copies/μL) or RNA (18391 to 1.2 copies/μL). From the results of about 10,000 partitions (
Having shown the specificity and quantification capability on the Clarity digital chip, we determined whether RADICA could be easily used with other digital devices which had been previously designed for digital PCR and which are in common use. A plate-based QIAcuity Digital System was tested for this purpose (˜0.91 nL partition volume, 26,000 partitions per reaction). The same one-pot reactions were digitalized using the sample partition system on QIAcuity to divide the samples into 26,000 individual wells in the plate followed by 60° C. incubation for 60 min. For both DNA (0.8-12777 copies/μL) and RNA (1.2-18391 copies/μL), the number of positive wells in the plate increased when more targets were present (
Comparison of RADICA (Digital RT-LAMP-Cas12b) with Other Detection Methods
To determine whether RADICA is competitive with other nucleic acid detection methods, such as RT-qPCR, RT-dPCR, and RT-LAMP-Cas12b bulk assay, we performed these methods and compared their detection of the same concentrations of SARS-CoV-2 RNA in the presence of a human genomic DNA background.
Serial dilutions of RNA targets were mixed with 1×USCDC N2 assay, 1× TaqMan™ Fast Virus 1-Step Master Mix and loaded on Roche Light Cycler. The reactions were incubated at 55° C. for 5 min followed by 95° C. for 20 s (one cycle), 95° C. 10 s and 60° C. 30 s (45 cycles). Fluorescent signals were monitored and Cq (Ct) values were calculated automatically using Roche Light Cycler 96 software.
DNA Quantification by dPCR:
Plasmids containing the N genes of SARS-CoV-2, SARS-CoV, and MERS-CoV were linearized with FastDigest Scal (Thermo Scientific) then mixed with 1×USCDC N2 assay, lx TaqMan™ Fast Advanced Master Mix (Applied Biosystems) and 1× Clarity™ JN solution (JN Medsys). The 15 μL reactions were loaded onto the Clarity digital chip using the method mentioned above, and the tubes containing the digital chip were transferred to a PCR machine using the following parameters (ramp rate=1° C./s): 95° C. for 15 min (one cycle), 95° C. for 50 s and 58° C. for 90 s (40 cycles), and 70° C. for 5 min. The endpoint fluorescence of the partitions was detected with a Clarity™ Reader, and the input DNA copy numbers were calculated by Clarity™ software.
Serial dilutions of RNA targets were mixed with 1×USCDC N2 assay, 1× TaqMan™ Fast Virus 1-Step Master Mix (Applied Biosystems) and 1× Clarity™ JN solution (JN Medsys) followed by incubation at 55° C. 5 min. After incubation, the reactions were partitioned on the Clarity digital chip and transferred to a PCR thermocycler with the following parameters (ramp rate=1° C./s): 95° C. for 15 min (one cycle), 95° C. for 50 s and 58° C. for 90 s (40 cycles), and 70° C. for 5 min. The endpoint fluorescence of the partitions was detected using a Clarity™ Reader and the input RNA copy numbers were calculated by Clarity™ software.
To test the inhibitor tolerance, 2.5 U/mL heparin, 0.01% sodium dodecyl sulphate (SDS), 1 mM ethylenediaminetetraacetic acid (EDTA), or 5% ethanol was added to the reactions mentioned above. For bulk reactions (RT-qPCR and bulk RT-LAMP-Cas12b), the 1 h end-point fluorescent signals of reactions with inhibitors were divided by the reactions without inhibitors. For digital reactions (RT-dPCR and RADICA), the positive partition percentage of the reactions with inhibitors was divided by that of the reactions without inhibitors, and the corresponding ratio was taken as the percentage of efficiency.
To determine whether RADICA is competitive with other nucleic acid detection methods, such as RT-qPCR, RT-dPCR, and RT-LAMP-Cas12b bulk assay, we performed the above four methods and compared their detection of the same concentrations of SARS-CoV-2 RNA (18391 to 1 copies/μL) in the presence of a human genomic DNA (1 ng/A) background (
Compared to PCR-based methods, RT-LAMP-Cas12b-based method is relatively simple, as isothermal amplification does not require thermal cyclers. As to sensitivity, the bulk reaction was slightly weaker in detecting low copy number samples, resulting in a detection sensitivity of 6 copies/μL RNA (
As inhibitors in the samples are likely to alter the reaction and might affect the accuracy of the result, we tested the effect of various inhibitors on the above four detection methods (
After the validation of RADICA on SARS-CoV-2 sequence, we asked whether the method can be adapted to detect and quantify other targets. Biomanufacture for protein therapeutics, vaccines and cell therapy usually need cell culture, which is susceptible to contamination with viruses. Thus, a rapid quantification method is needed to monitor the virus contaminations in biomanufacturing processes. To make use of the speed and quantification ability of RADICA, we have designed RADICA assays to detect and quantify the most common viral contaminants in biomanufacture: human adenovirus and herpes simplex virus [Barone, P. W. et al. Nature biotechnology 38: 563-572 (2020)]. Different LAMP primer sets and crRNA were designed (Table 8) and screened by bulk LAMP-Cas12b reaction, and the combination of LAMP primer set and crRNA with the highest speed were selected for the following RADICA experiment.
Human adenovirus 1 (ATCC VR-1) was propagated using A549 human lung epithelial cell line (ATCC CCL-185) grown in Ham's F-12K (Kaighn's) Medium (Thermo Fisher Scientific). Viruses were concentrated and purified using a chromatography-based Adeno-X Maxi Purification system (Takara Bio) according to the manufacturer's instructions. Herpes simplex virus 1 (ATCC VR-260) was propagated using Vero cell line (ATCC CCL-81) grown in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific). Viruses were concentrated by ultracentrifugation and purified using iodixanol density gradient ultracentrifugation.
DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's protocol.
The DNA/RNA target samples were mixed with 1.6 μM FIP primers, 1.6 μM BIP primers, 0.2 μM F3 primers, 0.2 μM B3 primers, 0.4 μM LoopF primers, 0.4 μM LoopB primers, 1.4 mM dNTPs, 8 mM MgSO4, 2 μM FQ-5T reporters, 0.96 U/μL Bst 2.0 WarmStart polymerase, 0.3 U/μL WarmStart RTx Reverse Transcriptase (for RNA), 1 U/μL RNase Inhibitor, 50 mM Taurine, 50 nM Cas12b, and 50 nM crRNA in 1× isothermal amplification buffer, unless otherwise indicated. To detect the LAMP signal, 250 mM SYTO-82 fluorescent nucleic acid stain was added to the reaction. The reaction mixture was incubated at 60° C. and fluorescence kinetics were monitored for 1-2 h using Roche Light Cycler 96. The fluorescence values displayed in the results were the fluorescence levels determined by Roche Light Cycler 96 software. To mimic the complexity of real samples, 1 ng/μL human genomic DNA was also added to the reactions.
The RADICA reaction was prepared by adding 250 nM Cyanine 680 succinimidyl ester (biotium) as reference dye to the RT-LAMP-Cas12b bulk reactions stated above and partitioned on QIAcuity digital nanoplates (˜0.91 nL partition volume, 26,000 partitions per reaction). 40 μL reaction mixtures were loaded onto the QIAcuity digital nanoplate and into the QIAcuity Digital PCR System. In the QIAcuity machine, the reactions were automatically partitioned into 26,000 microwells followed by 60° C. incubation for 1 hour and end-point fluorescence detection. To get the best signal to noise ratio, exposure duration (200 ms to 800 ms) and gain (2 to 8) were adjusted for each experiment. Usually, a 600 ms/6 will be used for the sample reading. Positive partition percentages, as well as nucleic acid concentration, were calculated by QIAcuity software.
Results:
To make use of the speed and quantification ability of RADICA, we have designed RADICA assays to detect and quantify the most common viral contaminants in biomanufacture: human adenovirus and herpes simplex virus [Barone, P. W. et al. Nature biotechnology 38: 563-572 (2020)]. Different LAMP primer sets and crRNA were designed and screened by bulk LAMP-Cas12b reaction, and the combination of LAMP primer set and crRNA with the highest speed were selected for the following RADICA experiment (
In clinical settings, to confirm the success of sampling as well as the reaction, there is a need to test the presence of human control as well as the intended target in a same reaction. Thus, a multiplex assay is needed to test both the target and the human control. One strategy of multiplex RADICA is use different Cas effectors with different specificity on the reporter [Gootenberg, J. S. et al. Science 360: 439-444 (2018)], another strategy is couple RADICA with other probe-based isothermal amplification methods. Here we validated the multiplex RADICA method using the second strategy, with primers and crRNA shown in Table 9.
∧Zhang, Y. et al. Biotechniques 69: 178-185 (2020)
ACTB-probe were prepared by mixing equal molar of ACTB-FIP-ROX/ACTB-F1-RQ, followed by 95° C. 5 min followed by slowly cooling down (ramp rate=1° C./s) to room temperature. The DNA/RNA target samples were mixed with 1.6 μM FIP primers (N2-WSLAMP-FIP and ACTB-FIP), 1.6 μM BIP primers (N2-WSLAMP-BIP and ACTB-BIP), 0.2 μM F3 primers (N2-WSLAMP-F3 and ACTB-F3), 0.2 μM B3 primers (N2-WSLAMP-B3 and ACTB-B3), 0.4 μM LoopF primers (N2-WSLAMP-LoopF and ACTB-LF), 0.4 μM LoopB primers (N2-WSLAMP-LoopB and ACTB-LB), 0.4 μM ACTB-FIP-ROX/ACTB-F1-RQ duplex, 1.4 mM dNTPs, 8 mM MgSO4, 2 μM FQ-5T reporters, 0.96 U/μL Bst 2.0 WarmStart polymerase, 0.3 U/μL WarmStart RTx Reverse Transcriptase (for RNA), 1 U/μL RNase Inhibitor, 50 mM Taurine, 50 nM Cas12b, 50 nM crRNA and 250 nM Cyanine 680 succinimidyl ester (biotium) as reference dye in 1× isothermal amplification buffer, unless otherwise indicated. The reactions were partitioned on QIAcuity digital nanoplates (˜0.91 nL partition volume, ˜26,000 partitions per reaction). 40 μL reaction mixtures were loaded onto the QIAcuity digital nanoplate and into the QIAcuity Digital PCR System. In the QIAcuity machine, the reactions were automatically partitioned into 26,000 microwells followed by 60° C. incubation for 1 hour and end-point fluorescence detection. To get the best signal to noise ratio, exposure duration (200 ms to 800 ms) and gain (2 to 8) were adjusted for each experiment. Usually, a 600 ms/6 will be used for the sample reading. Positive partition percentages, as well as nucleic acid concentration, were calculated by QIAcuity software.
Here we validated a multiplex RADICA method. First, a multiplex method targeting two targets that combine RADICA (FAM fluorescent colour signal, targeting SARS-CoV-2 N gene) with probe-based LAMP reaction (ROX fluorescent colour signal, targeting human ACTB gene) were designed and tested [Zhang, Y. et al. Biotechniques 69: 178-185 (2020)]. SARS-CoV-2 N gene were detected by N gene-specific LAMP primer and crRNA, human ACTB gene were detected by ACTB gene-specific LAMP primer and probe. Different concentrations of SARS-CoV-2 RNA in a constant amount of human background were tested using this method. From the result, the two detection targets are compatible and do not interfere with each other in the same multiplex reaction. With the decrease of SARS-CoV-2 RNA (from 25000 to 0 copies/μL) in a constant human DNA (1 ng/μL), the result showed decreasing FAM-positive partitions and constant ROX-positive partitions in both the position plot and scatter plot (
As there is an emerging number of SARS-CoV-2 mutants with high transmission and infection activity, we tested whether multiplex RADICA could be used to detect both the wildtype and mutant using primers and crRNA shown in Table 10.
S gene-probe were prepared by mixing equal molar of S1-F1P-alpha-CY5-4 bp/S1-F1C-RQ or S1-F1P-beta-TEX615-1mismatch/S1-F1C-RQ, followed by 95° C. 5 min followed by slowly cooling down (ramp rate=1° C./s) to room temperature. The RNA target samples were mixed with 1.6 μM FIP primers (N2-WSLAMP-FIP and S1-FIP-WT), 1.6 μM BIP primers (N2-WSLAMP-BIP and S1-BIP-WT), 0.2 μM F3 primers (N2-WSLAMP-F3 and S1-F3), 0.2 μM B3 primers (N2-WSLAMP-B3 and S1-133), 0.4 μM LoopF primers (N2-WSLAMP-LoopF and S1-LF), 0.4 μM LoopB primers (N2-WSLAMP-LoopB and S1-LB), 0.1 μM S1-F1P-alpha-CY5-4 bp/S1-F1C-RQ and 0.1 μM S1-F1P-beta-TEX615-1mismatch/S1-F1C-RQ, 1.4 mM dNTPs, 8 mM MgSO4, 2 μM FQ-5T reporters, 0.96 U/μL Bst 2.0 WarmStart polymerase, 0.3 U/μL WarmStart RTx Reverse Transcriptase (for RNA), 1 U/μL RNase Inhibitor, 50 mM Taurine, 50 nM Cas12b, 50 nM crRNA and 250 nM Cyanine 680 succinimidyl ester (biotium) as reference dye in 1× isothermal amplification buffer, unless otherwise indicated. The reactions were partitioned on QIAcuity digital nanoplates (˜0.91 nL partition volume, 26,000 partitions per reaction). 40 μL reaction mixtures were loaded onto the QIAcuity digital nanoplate and into the QIAcuity Digital PCR System. In the QIAcuity machine, the reactions were automatically partitioned into 26,000 microwells followed by 60° C. incubation for 1 hour and end-point fluorescence detection. To get the best signal to noise ratio, exposure duration (200 ms to 800 ms) and gain (2 to 8) were adjusted for each experiment. Usually, a 600 ms/6 will be used for the sample reading. Positive partition percentages, as well as nucleic acid concentration, were calculated by QIAcuity software.
For multiplex detection of wildtype and mutant in one reaction, RADICA targeting N gene of SARS-CoV-2 (FAM fluorescent colour signal, could detect both wildtype, alpha and beta mutant) and probe-based digital RT-LAMP targeting mutant (CY5 fluorescent colour signal specific for alpha mutant detection and ROX fluorescent colour signal specific for beta mutant detection) were tested on different SARS-CoV-2 wildtype and mutant. N gene LAMP primer/crRNA were used to detect SARS-CoV-2 both wildtype and mutant. For the mutant-specific primer design, LAMP primers and probes specifically targeting SARS-CoV-2 mutant but not wildtype were designed by the principle of allele-specific LAMP [Gill, P. and Hadian Amree, A. Avicenna J Med Biotechnol 12: 2-8 (2020)]. The multiplexed assays were validated using a mixture of synthetic RNA of SARS-CoV-2 wildtype, alpha and beta mutant. From the result (
As there is a need to detect more than 2 targets in many applications, we tested the performance of multiplex RADICA in detecting 4 targets simultaneously. Primers, probes and crRNA (Table 11) were designed to detect SARS-CoV-2 N gene (FAM channel), SARS-CoV-2 E gene (HEX channel), SARS-CoV-2 ORF1ab gene (CY5 channel) and human ACTB gene (ROX channel) in one reaction.
∧Zhang, Y. et al. Biotechniques 69: 178-185 (2020)
LAMP probe set were prepared by mixing equal molar of E1-HP-HEX/E1-F1-1-PQ, or ACTB-FIP-ROX/ACTB-F1-RQ or Orf1a_FIP-CY5/001a_F1C-RQ, followed by 95° C. 5 min followed by slowly cooling down (ramp rate=1° C./s) to room temperature. 10×LAMP primer sets were prepared as follows: 1.6 μM FIP primers, 1.6 μM BIP primers, 0.2 μM F3 primers, μM B3 primers, 0.4 μM LoopF primers, 0.4 μM LoopB primers were premixed for each gene. The RNA target samples were mixed with 1×N LAMP primer set, 0.5×E LAMP primer set, 0.5×ACTB LAMP primer set, 0.5×ORF1ab LAMP primer set, 0.3 μM E1-FIP-HEX/E1-F1-FQ duplex, 0.1 μM ACTB-FIP-ROX/ACTB-F1-RQ duplex, 0.2 μMOrf1a_FIP-CY5/Orf1a_F1C-RQ duplex, 1.4 mM dNTPs, 8 mM MgSO4, 2 μM FQ-5T reporters, 0.96 U/μL Bst 2.0 WarmStart polymerase, 0.3 U/μL WarmStart RTx Reverse Transcriptase (for RNA), 1 U/μL RNase Inhibitor, 50 mM Taurine, 50 nM Cas12b, 50 nM crRNA and 250 nM Cyanine 680 succinimidyl ester (biotium) as reference dye in 1× isothermal amplification buffer, unless otherwise indicated. The reactions were partitioned on QIAcuity digital nanoplates (˜0.91 nL partition volume, ˜26,000 partitions per reaction). 40 μL reaction mixtures were loaded onto the QIAcuity digital nanoplate and into the QIAcuity Digital PCR System. In the QIAcuity machine, the reactions were automatically partitioned into 26,000 microwells followed by 60° C. incubation for 1 hour and end-point fluorescence detection. To get the best signal-to-noise ratio, exposure duration (200 ms to 800 ms) and gain (2 to 8) were adjusted for each experiment. Usually, a 600 ms/2 will be used for the sample reading. Positive partition percentages, as well as nucleic acid concentration, were calculated by QIAcuity software.
Serial dilutions of SARS-CoV-2 RNA in a constant human genomic DNA background were used to validate this multiplex reaction. From the scatter plot (
RADICA can be easily extended to a variety of clinical, research, environmental, and biomanufacturing applications, such as liquid biopsy, rare mutation detection, gene expression analysis, gene editing detection, sequencing library quantification, environmental monitoring, cancer research, and cell therapies. RADICA offers a customizable solution that is amenable to many DNA isothermal amplification platforms such as recombinase polymerase amplification (RPA) [Piepenburg, O. et al. PLOS Biology 4: e204 (2006)], loop-mediated isothermal amplification (LAMP) [Notomi, T. et al. Nucleic acids research 28: E63-E63 (2000)], rolling circle amplification (RCA) [Lizardi, P. M. et al. Nature Genetics 19: 225-232 (1998)], strand displacement amplification (SDA) [Walker, G. T. et al. Nucleic acids research 20: 1691-1696 (1992)], or other isothermal amplifications, as well as the use of other Cas proteins, such as Cas13a, Cas12b, Cas14 for multiplex detection [Gootenberg, J. S. et al. Science 360: 439-444 (2018)]. Also, RADICA could be used on different microfluidic or droplet-based partition devices such as Clarity digital PCR system (JNMedsys), QuantStudio 3D Digital PCR System (Thermo Fisher), QIAcuity Digital PCR System (QIAGEN), Droplet Digital PCR System (Bio-Rad), Naica Crystal Digital PCR System (Stilla Technologies), RainDrop digital PCR system (RainDance Technologies, Bio-rad), BioMark digital PCR system (Fluidigm) and so on. Crude samples, without initial step of nucleic acid extraction, could also be used for single-cell detection. As RADICA only requires one temperature for the reaction, it can be integrated with a portable heater and smartphone-based fluorescence detection for point-of-care quantification. Based on the superior performance in sensitivity, speed, inhibitor resistance, and quantitative detection and the great potential for improvement and applicability, we anticipate that RADICA will be a promising quantitative molecular tool applicable to the clinical setting, as well as to research, biomanufacturing, and environmental and food industries.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2021/050661 | 10/28/2021 | WO |
