WO2021262422A1 - Procédé et système de détection d'adn et d'arn - Google Patents

Procédé et système de détection d'adn et d'arn Download PDF

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WO2021262422A1
WO2021262422A1 PCT/US2021/036043 US2021036043W WO2021262422A1 WO 2021262422 A1 WO2021262422 A1 WO 2021262422A1 US 2021036043 W US2021036043 W US 2021036043W WO 2021262422 A1 WO2021262422 A1 WO 2021262422A1
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sgid
primer
gene
sample
tag
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WO2021262422A9 (fr
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Chung-Ying Huang
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Huang Chung Ying
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

Definitions

  • the current gold standard detection assay of SARS-Cov-2 is Realtime RT-qPCR.
  • Each sample needs to run three RT-qPCR reactions for N1 gene, N2 gene (virus) and RP gene (human) according to the FDA approved CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel.
  • Real-Time qRT-PCR (Real-Time Quantitative Reverse Transcription PCR) is a major development of PCR technology that enables reliable detection and measurement of products generated during each cycle of PCR process. This technique became possible after introduction of an oligonucleotide probe which was designed to hybridize within the target sequence. Cleavage of the probe during PCR because of the 5' nuclease activity of Taq polymerase can be used to detect amplification of the target-specific product. Information about Real-Time qRT PCR is readily available in many publications. For example, Thermo Fisher Scientific described basic principles of RT-qPCR on its website as follow:
  • RNA is first transcribed into complementary DNA (cDNA) by reverse transcriptase from total RNA or messenger RNA (mRNA).
  • cDNA complementary DNA
  • mRNA messenger RNA
  • RT-qPCR is used in a variety of applications including gene expression analysis, RNAi validation, microarray validation, pathogen detection, genetic testing, and disease research.
  • RT-qPCR can be performed in a one-step or a two-step assay ( Figure 1, Table 1).
  • One-step assays combine reverse transcription and PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase.
  • One-step RT-qPCR only utilizes sequence-specific primers.
  • the reverse transcription and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming strategies.
  • RNA may provide slightly more sensitivity, but total RNA is often used because it has important advantages over mRNA as a starting material.
  • fewer purification steps are required, which ensures a more quantitative recovery of the template and a better ability to normalize the results to the starting number of cells.
  • oligo(dT) primers Three different approaches can be used for priming cDNA reactions in twostep assays: oligo(dT) primers, random primers, or sequence specific primers. Often, a mixture of oligo(dT)s and random primers is used. These primers anneal to the template mRNA strand and provide reverse transcriptase enzymes a starting point for synthesis.
  • Primer considerations for the cDNA synthesis step of RT-qPCR Combining random primers and anchored oligo(dT) primers improves the reverse transcription efficiency and qPCR sensitivity.
  • Oligo(dT)s Stretch of thymine residues Generation of Only amplify
  • anchored oligo(dT)s from poly(A)- poly(A) tail oligo(dT)s contain one G, tailed mRNA Truncated cDNA
  • RNA RNA transcript rRNA, and which is not mRNA
  • Reverse Transcriptase is the enzyme that makes DNA from RNA. Some enzymes have RNase activity to degrade the RNA strand in the RNA-DNA hybrid after transcription. If an enzyme does not possess RNase activity, an RNaseH may be added for better qPCR efficiency. Commonly used enzymes include Moloney murine leukemia virus reverse transcriptase and Avian myeloblastosis virus reverse transcriptase. For RT-qPCR, it is ideal to choose a reverse transcriptase with high thermal stability, because this allows cDNA synthesis to be performed at higher temperatures, ensuring successful transcription of RNA with high levels of secondary structure, while maintaining their full activity throughout the reaction producing higher cDNA yields.
  • RNase H activity degrades RNA from RNA-DNA duplexes to allow efficient synthesis of double- stranded DNA.
  • RNA may be degraded prematurely resulting in truncated cDNA.
  • reverse transcriptases with intrinsic RNase H activity are often favored in qPCR applications because they enhance the melting of RNA-DNA duplex during the first cycles of PCR.
  • PCR primers for the qPCR step of RT-qPCR should ideally be designed to span an exon-exon junction, with one of the amplification primers potentially spanning the actual exon-intron boundary ( Figure 4). This design reduces the risk of false positives from amplification of any contaminating genomic DNA, since the intron-containing genomic DNA sequence would not be amplified.
  • RNA sample If primers cannot be designed to separate exons or exon-exon boundaries, it is necessary to treat the RNA sample with RNase-free DNase I or dsDNase in order to remove contaminating genomic DNA.
  • a minus Reverse Transcription control should be included in all RT-qPCR experiments to test for contaminating DNA (such as genomic DNA or PCR product from a previous run).
  • a control contains all the reaction components except for the reverse transcriptase. Reverse transcription should not occur in this control, so if PCR amplification is seen, it is most likely derived from contaminating DNA.
  • Primer Design for the qPCR 1) If one primer is designed to span an exon-intron boundary, the possible contaminating genomic DNA is not amplified, because the primer cannot anneal to the template. In contrast, cDNA does not contain any introns, and is efficiently primed and amplified. 2) When primers flank a long (e.g. 1 kb) intron, the amplification cannot occur because the short extension time is sufficient for the short cDNA sequence but not for the longer genomic target.
  • sequence Nl (110) includes forward primer 1101, probe 1102, and reverse primer 1103.
  • Sequence N2 120 includes forward primer 1201, probe 1202, and reverse primer 1203.
  • RNA sample isolated and purified from upper and lower respiratory specimen is reverse transcribed to cDNA and subsequently amplified in the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with SDS version 1.4 software. In this process, the probe anneals to a specific target sequence located between the forward and reverse primers.
  • the 5' nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal.
  • additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored at each PCR cycle by Applied Biosystems 7500 Fast Dx Real-Time PCR System with SDS version 1.4 software. The following shows CDC's N1 and N2 primer and probe design for SARS-CoV-2 virus, plus RP as a control.
  • Hybridization between the cDNA reverse transcribed from a biological sample to a pre designed complementary DNA probe arranged on a slide, or array is the basis of DNA microarrays.
  • a microarray therefore consists of a pre-designed library of synthetic nucleic acid probes that are immobilized and spatially arrayed on a solid matrix.
  • Microarrays evolved from a technique known as Southern blotting, where DNA fragments are attached to a substrate and then probed with a known gene sequence.
  • the first DNA arrays were constructed by immobilizing cDNAs onto filter paper. However, it was not until 1995 that the first DNA microarrays capable of analyzing thousands of sequences were constructed by "spotting", or attaching short synthetic probes to designated locations on the solid surface, usually glass or silicon chip.
  • spotted arrays can be produced. Some methods basically use a robot to "print" pre-designed probes that have been attached to fine needles onto a chemical matrix surface using surface engineering (examples include fine-pointed pins, needles and ink-jet printing). Other methods employ photo-activated chemistry and masking to synthesize probes one nucleotide at a time on a solid surface in repeated steps to build up probes of specific sequence in designated locations.
  • a basic protocol for a DNA microarray is as follows:
  • Isolate and purify mRNA from samples of interest When comparing gene expression, one sample usually serves as control, and another sample would be the experiment (healthy vs. disease, etc)
  • Reverse transcribe and label the mRNA In order to detect the transcripts by hybridization, they need to be labeled with florescence, and because starting material maybe limited, an amplification step is also used. Labeling usually involves performing a reverse transcription (RT) reaction to produce a complementary DNA strand (cDNA) and incorporating a florescent dye that has been linked to a DNA nucleotide, producing a fluorescent cDNA strand. Disease and healthy samples can be labeled with different dyes and cohybridized onto the same microarray in the following step. Some protocols do not label the cDNA but use a second step of amplification, where the cDNA from RT step serves as a template to produce a labeled cRNA strand.
  • RT reverse transcription
  • Hybridize the labeled target to the microarray involves placing labeled cDNAs onto a DNA microarray where it will hybridize to their synthetic complementary DNA probes attached on the microarray. A series of washes are used to remove non-bound sequences.
  • the fluorescent labels on bound cDNA are excited by a laser and the fluorescently labeled target sequences that bind to a probe generate a signal.
  • the total strength of the signal depends upon the amount of target sample binding to the probes present on that spot.
  • the amount of target sequence bound to each probe correlates to the expression level of various genes expressed in the sample.
  • the signals are detected, quantified, and used to create a digital image of the array.
  • each sample is labeled with a different dye, the resulting image is analyzed by calculating the ratio of the two dyes. If a gene is over-expressed in the experimental sample, then more of that sample cDNA than control cDNA will hybridize to the spot representing that expressed gene. In turn, the spot will fluoresce red with greater intensity than it will fluoresce green. The red-to-green fluorescence ratio thus indicates which gene is up or downregulated in the appropriate sample.
  • NGS (Next-Generation Sequencing) Technology lllumina is one of those companies who provide NGS technologies. Here is a brief description on NGS technology from lllumina's "An introduction to Next-Generation Sequencing Technology", available online. b. The Basics of NGS Chemistry
  • NGS nucleotide triphosphates
  • the sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followed by 5'and 3'adapter ligation. Alternatively, “tagmentation” combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process.9 Adapter-ligated fragments are then PCR amplified and gel purified.
  • Cluster Generation For cluster generation, the library is loaded into a flow cell where fragments are captured on a lawn of surface-bound oligos complementary to the library adapters. Each fragment is then amplified into distinct, clonal clusters through bridge amplification. When cluster generation is complete, the templates are ready for sequencing.
  • Sequencing lllumina SBS technology uses a proprietary reversible terminator-based method that detects single bases as they are incorporated into DNA template strands. As all four reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias and greatly reduces raw error rates compared to other technologies.6, 7 The result is highly accurate base-by-base sequencing that virtually eliminates sequence context-specific errors, even within repetitive sequence regions and homopolymers.
  • PE sequencing involves sequencing both ends of the DNA fragments in a library and aligning the forward and reverse reads as read pairs. In addition to producing twice the number of reads for the same time and effort in library preparation, sequences aligned as read pairs enable more accurate read alignment and the ability to detect indels, which is not possible with singleread data.8 Analysis of differential read-pair spacing also allows removal of PCR duplicates, a common artifact resulting from PCR amplification during library preparation. Furthermore, PE sequencing produces a higher number of SNV calls following read-pair alignment.8, 9 While some methods are best served by single-read sequencing, such as small RNA sequencing, most researchers currently use the paired-end approach. nCounter technology by Nanostring
  • nCounter technology detect the mRNA directly without using enzyme.
  • mRNA sample is mixed with a capture probe and a target-specific reporter probe. If the target mRNA exists, the capture probe and the reporter probe will form a target-probe complex.
  • the tripartite molecule is affinity-purified first by the 3C-repeat sequence and then by the 5C-repeat sequence to remove excess reporter and capture probes, respectively.
  • the purified complexes are attached to a streptavidin-coated slide via biotinylated capture probes. Voltage is applied to elongate and align the molecules.
  • Biotinylated anti-5C oligonucleotides that hybridize to the 5C-repeat sequence are added.
  • the stretched reporters are immobilized by the binding of the anti-5C oligonucleotides to the slide surface via the biotin. Voltage is turned off and the immobilized reporters are prepared for imaging and counting.
  • All probes are mixed together with total RNA in a single hybridization reaction that proceeds in solution.
  • the capture probe and reporter probe hybridize to a complementary target mRNA in solution via the gene-specific sequences. Hybridization results in the formation of tripartite structures, each comprised of a target mRNA bound to its specific reporter and capture probes.
  • Unhybridized reporter and capture probes are removed by affinity purification, and the remaining complexes are washed across a surface that is coated with the appropriate capture reagent (e.g., streptavidin). After capture on the cartridge surface, an applied electric field extends and orients each complex in the solution in the same direction. The complexes are then immobilized in an elongated state (Fig. lc).
  • the appropriate capture reagent e.g., streptavidin
  • the immobilized reporters are prepared for imaging and counting.
  • Fig. Id Each target molecule of interest is identified by the color code generated by the ordered fluorescent segments present on the reporter probe.
  • the level of expression is measured by counting the number of codes for each mRNA.
  • US Patent Publication Number US 2014/0371088 A1 describes the nCounter technology, which is incorporated by reference.
  • the above technologies can process/detect large number of genes, but the samples from different specimens have to be tested separately.
  • the identity of the sample/specimen is labeled on the carrier/tube/plate/container so that the test data can be correctly filed/categorized. It is very difficult to achieve detection of large number of patients (for general screening purpose, for example) using existing technologies.
  • the present invention provides a method and system to insert an identity indicator (tag) about the identity of each specimen (from which patient or origin it was collected) into the cDNA of mRNA or duplicated DNA.
  • the identity indicator can also be gene-specific.
  • multiple samples can be pooled together to be amplified (for example, using PCR).
  • the amplified sample pool is then detected using mass-detection methods. Data containing test results of hundreds, thousands, or tens of thousands of specimens are sorted or categorized according to the tag information, and analysis of the data for each specimen can be performed accordingly.
  • Fig. 1. shows the CDC N1 and N2 primer and probe design for SARS-CoV-2 virus.
  • Fig. 2a shows the SGID-tagging process of a mRNA sample
  • Fig. 2b shows the Gene(x) cDNA with SGID-Tag (n-x) and URP attached
  • Fig. 3 shows pooled PCR amplification process.
  • Fig. 4a shows the microarray before hybridization.
  • Fig. 4b shows the microarray after hybridization with pooled PCR products.
  • Fig. 5 explains briefly the nCounter probe hybridization.
  • Fig. 6a shows an example of scanning data of microarray technology.
  • Fig. 6b shows an example of NGS read count data.
  • Fig. 6c shows an example of nCounter data.
  • Fig. 7 illustrates the 1st SGID-Tag PCR process of another embodiment.
  • Fig. 8. Shows the 2nd Pool PCR process of the embodiment.
  • sample-and-gene ID tag sequences SGID-Tags, preferably 8 to 60 nucleotides
  • each SGID-Tag corresponds to a unique gene from a specific sample.
  • the SGID-Tag will enable tracking individual target DNA/RNA molecule from a specific sample.
  • a SGID-Tag Reverse Primer 220 includes a gene-specific RT primer 221, a SGID-Tag 222, and universal reverse primer (URP) 223.
  • nCounter and microarray technologies require longer nucleotide tags for the purpose of identifications because both are not sequencing-based technologies. They depend on the hybridization of complementary nucleotide sequences of the targets to the probes and cannot differentiate single or a few nucleotide differences (2-10 nucleotides). Hybridization complexes will become unstable and separate if the surrounding temperature goes above the melting temperature (Tm).
  • Tm melting temperature
  • the Tm can be decided by the sequence length, the composition of the complementary DNA/RNA nucleotide sequences, and salt conditions of the hybridization solution.
  • Microarray and nCounter technologies perform hybridization of DNA or RNA under stringent temperature control for a fixed period (0.5 to 24 hours) to detect RNA or DNA whose sequences are complementary to the oligonucleotide probes on the microarray or in the nCounter assay. Both technologies usually require longer than 30 nucleotides for better results.
  • the tag sequence should avoid those sequences that may exist in the samples.
  • the sequence of SGID-Tag 222 should be selected to avoid cross-hybridization to any known virus, pathogen, or human genes.
  • SGID-Tag for example, 6 sets of 10-nucloetide sequences, 4 sets of 15-nucleotide sequences...etc.
  • SRP(n,x) means the sample ID is n and the gene ID is x.
  • the composition of SRP(n,x) includes a gene x-specific RT primer, a SGID-Tag(n-x), and a universal reverse primer.
  • SRP Pool (n) means a mixture of multiple SRPs with SGID-Tags assigned to sample n. Taking SARS-CoV-2 virus as an example, SRP Pool (0001) is a mixture of SRP(0001, Nl), SRP(0001, N2), and SRP(0001, RP).
  • the proposed parallel sample detection method comprises the following steps:
  • RNA sample 0001 runs with SRP Pool (0001)
  • RNA sample 0002 runs with SRP Pool (0002)
  • RNA sample 0003 runs with SRP Pool (0003)
  • RNA sample 0099 runs with SRP Pool (0099), ..., and so on.
  • SRP Pool (1) which is prepared for sample 1, will have three equal molar gene-specific reverse primers in the pool:
  • SRP Pool (2) which is prepared for sample 2, will have three equal molar gene-specific reverse primers in the pool:
  • SRP Pool (3) which is prepared for sample 3, will have three equal molar gene-specific reverse primers in the pool:
  • SRP 220 is mixed with sample RNA 200 for reverse transcription.
  • Gene (x) RT primer 221 will attach to Gene (x) 211 and the RT reaction begins.
  • a cDNA solution is formed for each sample.
  • the cDNA 300 of Human RP genes should have been generated with SGID-Tag(n-RP) 222 and URP 223 attached. If SARS-CoV-2 RNA is present in sample n, the cDNA of N1 gene and N2 gene shall also be generated - the cDNA of N1 gene will include SGID-Tag(n-Nl) and URP, while the cDNA of N2 gene will include SGID-Tag(n-N2) and URP.
  • the forward primers 320 for amplifying those cDNAs can be gene-specific forward primer 321, or a universal forward primer (UFP) 323 can also be added. URP 223 can be used as the reverse primer.
  • the Forward Primer Pool comprising all Gene (x) forward-primers.
  • the Forward Primer Pool contains a N1 forward primer, a N2 forward primer, and a Human RP forward primer. If NGS is the detecting method, add an optional universal forward primer (UFP) sequence (UFP 323) to all forward primers.
  • UFP universal forward primer
  • the pooled PCR product is ready for SGID-Tag counting.
  • Microarray technology may require longer SGID-Tag oligonucleotides for the hybridization-based assay, as mentioned above.
  • Fig. 4a shows a few detection spots 410-460 of microarray 400. Each detection spot has its unique complementary-sequence oligonucleotide probes attached. For example, probe 4101 is attached to spot 410, probe 4201 is attached to spot 420, ... and so on.
  • Probe 4101 is specifically designed to detect SGID-Tag (1-1)
  • probe 4201 is specifically designed to detect SGID-Tag (1-2)
  • probe 4301 is specifically designed to detect SGID-Tag (1-3).
  • SGID-Tags (1-1), (1-2), and (1-3) correspond to Nl, N2, RP genes from Sample 1, respectively.
  • Probe 4301 is specifically designed to detect SGID-Tag (2-1)
  • probe 4401 is specifically designed to detect SGID-Tag (2-2)
  • probe 4601 is specifically designed to detect SGID-Tag (2-3).
  • SGID-Tags (2-1), (2-2), and (2-3) correspond to Nl, N2, RP genes from Sample 2, respectively.
  • Fig. 4b shows an example after the microarray hybridized with the PCR product.
  • Probes 4401 and 4501 do not bound to any complementary SGID-TAGs.
  • NGS sequencing technology can sequence all SGID-Tags in a massively parallel way in a flow cell. It can differentiate the SGID-Tags down to single nucleotide differences; hence, the SGID-Tags for NGS sequencing technology may only require as low as 8 nucleotides to have 65,536 combinations of the SGID-Tag.
  • the procedures of NGS SGID-Tag counting by the lllumina sequencing technology as an example is discussed below.
  • nCounter technology is a molecule counting assay by NanoString Technologies. It is also a probe hybridization-based assay. However, it does not spot the probe on a fixed array. Instead it requires two adjacent probes to hybridize to the targets.
  • One probe called capture probe has the biotin label, which will help deposit the hybridization complex including target and reporter probe on a streptavidin-coated slide.
  • nCounter utilizes 800 probes of these 4,096 combinations.
  • the nCounter system uses a 12-lane cartridge for each run.
  • pooled cDNA 300 can be tested without the pool PCR amplification.
  • Nl and N2 positive, RP positive or negative - possible positive, need repeat Nl and N2 negative, RP negative - undetermined Nl and N2 negative, RP positive - negative
  • spot n-1 detects Nl gene
  • spot n-2 detects N2 gene
  • spot n-3 detects RP gene from Sample n.
  • Nl and N2 of sample 1 are all negative with fluorescent signals below threshold values.
  • Tag n-1 is Nl gene
  • Tag n-2 is N2 gene
  • Tag n-3 is RP gene from Sample n.
  • Nl, N2 of sample 1 (Tag 1-1, 1-2) and Nl, N2, RP of sample 2 (Tag 2-1, 2-2, 2-3) are all negative with counts below the threshold read count (assuming 10 is the threshold read count).
  • RP of sample 1 (Tag 1-3) and Nl, N2, RP of sample 3 (Tag 3-1, 3-2, 3-3) are all positive, with counts significantly above the threshold read count.
  • the results of sample 1 is negative, sample 2 is undetermined, and sample 3 is positive.
  • Digital scanner will automatically convert scanning data to nCounter counts of all SGID-Tags for all samples and genes.
  • Tag n-1 is N1 gene
  • Tag n-2 is N2 gene
  • Tag n-3 is RP gene from Sample n.
  • Nl, N2 of sample 1 (Tag 1-1, 1-2) and Nl, N2, RP of sample 2 (Tag 2-1, 2-2, 2-3) are all negative with counts below the threshold count (assuming 50 is the threshold count).
  • RP of sample 1 (Tag 1-3) and Nl, N2, RP of sample 3 (Tag 3-1, 3-2, 3-3) are all positive, with counts significantly above the threshold count. Hence, the results of sample 1 is negative, sample 2 is undetermined, and sample 3 is positive.
  • the unique SGID-Tag for identification of specific samples and genes can be attached in the 1st PCR cycle, as described below.
  • each sample runs the 1st PCR with a pool of Gene (x) reverse primers (RP) 720 and a pool of SGID-Tag Forward Primers (SFP) 730 for 5 - 20 cycles to attach the unique SGID-Tag (n-x) to individual gene and sample.
  • RP 720 includes gene-specific reverse primer sequence 721 and universal reverse primer (URP) 723.
  • SFP 730 includes a gene-specific forward primer (Gene (x) forward primer) sequence 731 for each target gene, a unique SGID-Tag sequence (8 to 60 nucleotides) 732 for sample and gene identification, and a universal forward primer (UFP) 733, each individual sample will use a pool of SFP 730.
  • Gene (x) forward primer Gene-specific forward primer
  • unique SGID-Tag sequence 8 to 60 nucleotides
  • UFP universal forward primer
  • SFP(n,x) means the sample ID is n, and the gene ID is x.
  • the composition of SFP(n,x) includes a universal forward primer, a SGID-Tag(n-x), and a gene x-specific forward primer.
  • SFP Pool (n) means a mixture of multiple SFPs with SGID-Tags assigned to sample n.
  • SFP Pool (0001) is a mixture of SFP(0001, Gene 1), SFP(0001, Gene 2), and SFP(0001, Gene 3).
  • Each DNA or cDNA sample runs a sample-specific 1 st PCR with the corresponding SFP Pool: DNA/cDNA sample 0001 runs with SFP Pool (0001), DNA/cDNA sample 0002 runs with SFP Pool (0002), DNA/cDNA sample 0003 runs with SFP Pool (0003), ..., DNA/cDNA sample 0099 runs with SFP Pool (0099), ..., and so on.
  • Reverse Primer Pool comprises all gene-specific reverse primers and a universal reverse primer (URP). All samples will use the same Reverse Primer Pool.
  • This SGID-Tag 1st PCR process will generate DNAs 800 to be used as a template in the next amplification process.
  • universal forward primer (UFP) 833, universal reverse primer (URP) 723, and PCR enzyme mix are added to the pooled PCR product, and then run 10- to 40- cycle PCR.
  • the 2nd PCR product is ready for SGID-Tag counting and data analysis in the same workflow described above.
  • SARS-CoV-2 Nl, N2, and Fluman RP as examples, application of this invention is not limited to those genes, or limited to this virus, or limited to RNA detection.
  • the detection is also not limited to three genes per sample.
  • the long stretch of SGID-Tag will tolerate one or a few point mutations, which makes the detection of mutated genes possible.
  • the application of this invention can also be used to detect multiple samples from the same patient, or multiple samples from multiple patients. For example, spacemen can be collected on day 1 ⁇ 20 from patients 1 ⁇ 100; 2000 RNA samples are prepared accordingly; and 3 genes from a first RNA and 2 genes from a second RNA are subject to detection (total 10,000 spots if using microarray technology). It is also possible that different genes are detected using different microarrays.

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Abstract

La présente invention concerne un procédé et un système permettant d'insérer un indicateur d'identité (marqueur) concernant l'identité de chaque échantillon (patient ou origine de l'échantillon) dans l'ADNc de l'ARNm ou l'ADN dupliqué. L'indicateur d'identité peut également être spécifique à un gène. À savoir, le marqueur est un indicateur d'identité spécifique à l'échantillon et au gène (marqueur SGID). Après avoir fixé le marqueur SDIG sur l'ADNc ou l'ADN, plusieurs échantillons peuvent être regroupés pour être amplifiés (par exemple, en utilisant la PCR). L'ensemble des échantillons amplifiés est ensuite détecté à l'aide de procédés de détection de masse. Les données contenant les résultats de tests de centaines, de milliers ou de dizaines de milliers d'échantillons sont triées ou catégorisées en fonction des informations de marquage, et l'analyse des données de chaque échantillon peut ainsi être réalisée.
PCT/US2021/036043 2020-06-25 2021-06-04 Procédé et système de détection d'adn et d'arn WO2021262422A1 (fr)

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US20110092387A1 (en) * 2003-09-10 2011-04-21 Althea DX Expression profiling using microarrays
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US20180010176A1 (en) * 2015-02-13 2018-01-11 Abhijit Ajit Patel Methods for highly parallel and accurate measurement of nucleic acids

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