KRAS G12C gene mutation detection method based on programmable enzyme system and application

Abstract

The invention provides a KRAS G12C gene mutation detection method based on a programmable enzyme system and application of the KRAS G12C gene mutation detection method. Specifically, the invention provides a KRAS G12C gene mutation detection reagent which is rapid, efficient and high in detection sensitivity, and the detection reagent comprises a guide ssDNA (gDNA) for guiding a gene editing enzyme Ago protein to specifically cut a KRAS wild type gene, and a primer and a detection probe which are used for PCR (Polymerase Chain Reaction) amplification. The invention also discloses a detection kit and a detection method adopting the gDNA, the primer and the probe.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a KRAS G12C gene mutation detection method based on a programmable enzyme system and its application. Background Art

[0002] The KRAS gene is an important upstream gene in the RAS (Rat sarcoma) family. The KRAS protein is a small membrane-bound GTPase that plays an important role in a variety of cell signaling functions. KRAS is the most frequently mutated oncogene in human cancer. The number of cancer patients carrying KRAS mutations is astonishingly large, including about 90% of pancreatic cancer (pancreatic cancer), about 50% of colorectal cancer (CRC) and about 20% of non-small cell lung cancer (NSCLC), some of the most lethal tumor types. Therefore, KRAS gene mutations indicate early cancer or adenoma. Among the KRAS gene mutations, more than 90% of the mutations occur at the 12th glycine. Among them, the KRAS G12C gene mutation has a worse prognosis than mutations at other KRAS sites, making it a hot target for the development of new diagnostic technologies and the development of new anticancer drugs.

[0003] At present, KRAS gene mutations can be detected in DNA samples from tissues, blood, feces or urine through Sanger sequencing, second-generation sequencing, or qPCR, dPCR and other methods. Although the mutant gene abundance in tissue samples is high and relatively easy to detect, it is suitable for various detection technologies, but invasive sampling is required and patients are not adaptable. Therefore, the collection of samples such as blood, feces or urine for free DNA detection is more widely used. Although free DNA comes from the presence of somatic mutations, the ratio of mutant DNA to wild-type DNA may be as low as 1 / 10000, which is usually beyond the capabilities of qPCR or dPCR methods, and traditional mutation detection methods through sequencing cannot accurately detect it. As a result, the current clinical detection and application of KRAS gene mutations is still not ideal, and it is impossible to take into account both the detection cost and sensitivity issues. Summary of the invention

[0004] The purpose of the present invention is to provide a KRAS G12C gene mutation detection method and application based on a programmable enzyme system.

[0005] In a first aspect of the present invention, a KRAS G12C gene mutation detection reaction system is provided, the reaction system comprising:

[0006] (a) a pre-amplification and enrichment primer pair for amplifying the KRAS G12C mutant gene in the sample to be tested; and

[0007] (b) Ago enzyme and guide ssDNA (gDNA) that specifically cleave the KRAS wild-type gene;

[0008] Wherein, the guide ssDNA (gDNA) is selected from the following group: SEQ ID NO. 8, 13, 12, 5, 6, 7, 9, 10, 11, 14, 15, 16, or a combination thereof.

[0009] In another preferred example, the gDNA comprises a combination of SEQ ID NOs. 8 and 13.

[0010] In another preferred embodiment, the length of the gDNA is 10-60 nt, preferably, 10-40 nt, more preferably, 13-20 nt, and most preferably, 16 nt.

[0011] In another preferred embodiment, the 5' and / or 3' end of the gDNA contains phosphorylation modification.

[0012] In another preferred embodiment, the first base at the 5' end of the gDNA is T.

[0013] In another preferred example, the size of the cleavage product formed by the gDNA-guided Ago enzyme includes cleavage products of 20-40 nt.

[0014] In another preferred example, the size of the cleavage product is 20-30 nt, preferably 22-28 nt, more preferably 25 nt.

[0015] In another preferred example, the size of the cleavage product is 30-40 nt, preferably 33-37 nt, more preferably 35 nt.

[0016] In another preferred example, the gDNA targets the KRAS wild-type gene.

[0017] In another preferred example, the gDNA-targeted sequence is selected from SEQ ID NO. 1, 2 or a combination thereof.

[0018] In another preferred example, the gDNA targets the KRAS wild-type positive strand, including the sequence shown in SEQ ID NO.1.

[0019] In another preferred example, the gDNA targets the wild-type negative strand of KRAS, including the sequence shown in SEQ ID NO.2.

[0020] In another preferred embodiment, the working concentration of the gDNA in the reaction system is 400-4800 nM.

[0021] In another preferred embodiment, the Ago enzyme is selected from the following group: PfAgo (Pyrococcus furiosus Ago), MfAgo (Methanocaldococcus fervens Ago), TcAgo (Thermogladius calderae Ago), TfAgo (Thermus filiformis Ago), AaAgo (Aquifex aeolicus Ago), TpAgo (Thermusparvatiensis Ago), FpAgo (Ferroglobus placidus Ago) or a combination thereof, more preferably, PfAgo is selected.

[0022] In another preferred embodiment, the working concentration of the PfAgo enzyme in the reaction system is 20-240 nM.

[0023] In another preferred example, the pre-amplification and enrichment primer pair includes an upstream primer as shown in SEQ ID NO.17 and a downstream primer as shown in SEQ ID NO.18.

[0024] In another preferred embodiment, the working concentration of the pre-amplification and enrichment primers in the reaction system is 100-300 nM, preferably 150-250 nM, more preferably 180-220 nM, most preferably 200 nM.

[0025] In another preferred embodiment, the reaction system further comprises: (c) a qPCR primer pair and probe for detecting the KRAS G12C mutant gene;

[0026] In another preferred example, the qPCR primer pair includes an upstream primer as shown in SEQ ID NO.19 and a downstream primer as shown in SEQ ID NO.20.

[0027] In another preferred example, the working concentration of the qPCR primer is 100-300 nM, preferably 150-250 nM, more preferably 180-220 nM, most preferably 200 nM.

[0028] In another preferred embodiment, the probe comprises a TaqMan probe combination.

[0029] In another preferred embodiment, the probe includes a probe targeting the KRAS wild-type gene, a probe targeting the KRAS G12C mutant gene, or a combination thereof.

[0030] In another preferred embodiment, the probe targeting the wild-type KRAS gene includes the probe shown in SEQ ID NO.21.

[0031] In another preferred embodiment, the probe targeting the KRAS G12C mutant gene includes the probe shown in SEQ ID NO.22.

[0032] In another preferred embodiment, the probe comprises a fluorescent modification.

[0033] In another preferred embodiment, the 5' end of the probe is modified with a fluorescent reporter group, and the 3' end is modified with a fluorescent quencher group.

[0034] In another preferred embodiment, the fluorescent reporter groups include: FAM, ROX, VIC, HEX, Cy5, Cy3, TAMRA, NED, JOE, TET, Texas Red, LC RED640, and LC RED705.

[0035] In another preferred embodiment, the fluorescent reporter group of the probe is modified to FAM or VIC.

[0036] In another preferred embodiment, the fluorescence quenching group includes: BHQ1, BHQ2, TAMRA, and MGB.

[0037] In another preferred embodiment, the fluorescence quenching group of the probe is modified to BHQ1.

[0038] In another preferred embodiment, the working concentration of the probe is 100-300 nM, preferably 150-250 nM, more preferably 180-220 nM, most preferably 200 nM.

[0039] In another preferred embodiment, the reaction system includes: a pre-amplification reaction system, an enrichment reaction system and a qPCR detection system.

[0040] In another preferred example, the pre-amplification reaction system includes: 2×PCR high-fidelity Taq enzyme Mix 25μL, 10μM pre-amplification and enrichment upstream and downstream primers 1μL each, 100nM standard template or test sample 5μL, supplemented with ddH2O to 50μL; the reaction conditions of the pre-amplification reaction system are 94℃ pre-denaturation for 3min; 94℃ denaturation for 10sec, 55℃ annealing for 30sec, 72℃ extension for 20sec, 30 cycles; 72℃ supplementary extension for 5min.

[0041] In another preferred example, the enrichment reaction system includes: 2×PCR ordinary Taq enzyme Mix 25μL, 2mM MnCl21.25μL, 10μM pre-amplification and enrichment upstream and downstream primers 0.5μL each, pre-amplification product 2μL, 100μM positive and negative chain gDNA 0.8μL each, 5μM PfAgo 0.8μL, supplemented with ddH2O to 25μL; the reaction conditions of the enrichment reaction system are 94℃ pre-denaturation for 3min; 94℃ denaturation for 30sec, 55℃ annealing for 30sec, 72℃ extension for 20sec, 25 cycles; 72℃ supplementary extension for 5min.

[0042] In another preferred embodiment, the qPCR detection system includes: 2×qPCR Mix 10 μL, 10 μM qPCR upstream and downstream primers 0.5 μL each, wild-type and mutant TaqMan probes 0.4 μL each, -3 5 μL of the diluted enriched product was supplemented with ddH2O to 20 μL; the reaction conditions were 95°C pre-denaturation for 8 min; 95°C denaturation for 15 s, 61.5°C annealing, extension and signal collection for 40 sec, and 40 cycles.

[0043] In a second aspect of the present invention, a KRAS G12C gene mutation detection reagent is provided, the detection reagent comprising guide ssDNA (gDNA), and the gDNA is selected from the following group: SEQ ID NO.8, 13, 12, 5, 6, 7, 9, 10, 11, 14, 15, 16, or a combination thereof.

[0044] In another preferred example, the gDNA comprises a combination of SEQ ID NOs. 8 and 13.

[0045] In another preferred embodiment, the length of the gDNA is 10-60 nt, preferably, 10-40 nt, more preferably, 13-20 nt, and most preferably, 16 nt.

[0046] In another preferred embodiment, the 5' and / or 3' end of the gDNA contains phosphorylation modification.

[0047] In another preferred embodiment, the first base at the 5' end of the gDNA is T.

[0048] In another preferred example, the size of the cleavage product formed by the gDNA-guided Ago enzyme includes cleavage products of 20-40 nt.

[0049] In another preferred example, the size of the cleavage product is 20-30 nt, preferably 22-28 nt, more preferably 25 nt.

[0050] In another preferred example, the size of the cleavage product is 30-40 nt, preferably 33-37 nt, more preferably 35 nt.

[0051] In another preferred example, the gDNA-targeted sequence is selected from SEQ ID NO. 1, 2 or a combination thereof.

[0052] In another preferred example, the gDNA targets the KRAS wild-type positive strand, including the sequence shown in SEQ ID NO.1.

[0053] In another preferred example, the gDNA targets the wild-type negative strand of KRAS, including the sequence shown in SEQ ID NO.2.

[0054] In another preferred embodiment, the working concentration of the gDNA is 400-4800 nM.

[0055] In another preferred embodiment, the detection reagent also includes a pre-amplification and enrichment primer pair for amplifying the KRAS G12C mutant gene in the sample to be tested.

[0056] In another preferred example, the length of the DNA product that can be amplified by the pre-amplification and enrichment primer pairs includes 60-200 bp, preferably 80-180 bp, more preferably 100-160 bp, and most preferably 158 bp.

[0057] In another preferred example, the pre-amplification and enrichment primer pair includes an upstream primer as shown in SEQ ID NO.17 and a downstream primer as shown in SEQ ID NO.18.

[0058] In another preferred embodiment, the working concentration of the pre-amplification and enrichment primers is 100-300 nM, preferably 150-250 nM, more preferably 180-220 nM, most preferably 200 nM.

[0059] In another preferred embodiment, the detection reagent further comprises a qPCR primer pair for detecting the KRAS G12C mutant gene in the pre-amplification and enrichment products.

[0060] In another preferred example, the length of the DNA product that can be amplified by the qPCR primer pair includes 50-110 bp, preferably 70-90 bp, more preferably 75-85 bp, and most preferably 80 bp.

[0061] In another preferred example, the qPCR primer pair includes an upstream primer as shown in SEQ ID NO.19 and a downstream primer as shown in SEQ ID NO.20.

[0062] In another preferred example, the working concentration of the qPCR primer is 100-300 nM, preferably 150-250 nM, more preferably 180-220 nM, most preferably 200 nM.

[0063] In another preferred embodiment, the detection reagent further comprises a probe.

[0064] In another preferred embodiment, the probe comprises a TaqMan probe combination.

[0065] In another preferred embodiment, the probe includes a probe targeting the KRAS wild-type gene, a probe targeting the KRAS G12C mutant gene, or a combination thereof.

[0066] In another preferred embodiment, the probe targeting the wild-type KRAS gene includes the probe shown in SEQ ID NO.21.

[0067] In another preferred embodiment, the probe targeting the KRAS G12C mutant gene includes the probe shown in SEQ ID NO.22.

[0068] In another preferred embodiment, the probe comprises a fluorescent modification.

[0069] In another preferred embodiment, the 5' end of the probe is modified with a fluorescent reporter group, and the 3' end is modified with a fluorescent quencher group.

[0070] In another preferred embodiment, the fluorescent reporter groups include: FAM, ROX, VIC, HEX, Cy5, Cy3, TAMRA, NED, JOE, TET, Texas Red, LC RED640, and LC RED705.

[0071] In another preferred embodiment, the fluorescent reporter group of the probe is modified to FAM or VIC.

[0072] In another preferred embodiment, the fluorescence quenching group includes: BHQ1, BHQ2, TAMRA, and MGB.

[0073] In another preferred embodiment, the fluorescence quenching group of the probe is modified to BHQ1.

[0074] In another preferred embodiment, the working concentration of the probe is 100-300 nM, preferably 150-250 nM, more preferably 180-220 nM, most preferably 200 nM.

[0075] In another preferred embodiment, the detection reagent also includes: gene editing enzyme Ago enzyme.

[0076] In another preferred embodiment, the Ago is selected from the following group: PfAgo (Pyrococcus furiosus Ago), MfAgo (Methanocaldococcus fervens Ago), TcAgo (Thermogladius calderae Ago), TfAgo (Thermus filiformis Ago), AaAgo (Aquifex aeolicus Ago), TpAgo (Thermusparvatiensis Ago), FpAgo (Ferroglobus placidus Ago) or a combination thereof, more preferably, PfAgo is selected.

[0077] In another preferred embodiment, the working concentration of the PfAgo enzyme is 20-240 nM.

[0078] In another preferred embodiment, the detection reagent further comprises a DNA extraction reagent.

[0079] In another preferred embodiment, the DNA extraction reagent includes a DNA extraction reagent for extracting genomic or free DNA from tissues, body fluids or excrement.

[0080] In another preferred example, the detection reagent further comprises a PCR reagent.

[0081] In another preferred embodiment, the PCR kit comprises dNTPs, DNA polymerase, a cofactor of DNA polymerase (e.g. Mg 2+ ), PCR buffer, or a combination thereof.

[0082] In another preferred embodiment, the DNA polymerase is Taq enzyme, including high-fidelity Taq enzyme and ordinary Taq enzyme.

[0083] In another preferred embodiment, the components exist alone or in a mixture.

[0084] In another preferred example, the PCR reagent includes a fluorescent quantitative PCR reagent.

[0085] In the third aspect of the present invention, a KRAS G12C gene mutation detection kit is provided, wherein the kit comprises the detection reagent as described in the second aspect of the present invention.

[0086] In another preferred embodiment, the kit further comprises an instruction manual, which indicates that the kit is used to detect the KRAS G12C mutant gene in a sample to be tested.

[0087] In another preferred embodiment, the detection includes qualitative detection and quantitative detection.

[0088] In another preferred example, the kit comprises the components of 2×PCR high-fidelity Taq enzyme Mix, 10 μM pre-amplification and enrichment upstream and downstream primers, 100 nM 1%, 1‰, 1 / 10000 KRAS G12C standards, 2×PCR ordinary Taq enzyme Mix, 2 mMMnCl2, 100 μM positive and negative chain gDNA, 5 μM PfAgo enzyme, 2×qPCR Mix, 10 μM qPCR upstream and downstream primers, and 10 μM wild-type and mutant probes.

[0089] In a fourth aspect of the present invention, a method for detecting KRAS G12C gene mutation is provided, the method comprising the steps of:

[0090] (S1) providing a sample to be tested;

[0091] (S2) Detecting the sample using the reaction system described in the first aspect of the present invention, the detection reagent described in the second aspect of the present invention, the kit described in the third aspect of the present invention, or a combination thereof.

[0092] Specifically, the method comprises the following steps:

[0093] (i) providing samples to be tested;

[0094] (ii) performing PCR pre-amplification on the sample using a pre-amplification and enrichment primer pair to obtain a pre-amplification product;

[0095] (iii) enriching the KRASG12C mutant gene in the pre-amplification product using a pre-amplification and enrichment primer pair, a guide ssDNA, and an Ago enzyme to obtain an enriched product;

[0096] (iv) detecting the enriched product.

[0097] In another preferred embodiment, step (iv) of the method comprises collecting the enriched product and performing a detection selected from the group consisting of: Sanger sequencing, qPCR, dPCR, or a combination thereof.

[0098] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0099] In another preferred embodiment, the method is an in vitro method.

[0100] In another preferred embodiment, the sample to be tested comes from a subject.

[0101] In another preferred embodiment, the subject includes an animal model.

[0102] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0103] In another preferred embodiment, the subject includes primates and rodents.

[0104] In another preferred embodiment, the subject includes Carnivora.

[0105] In another preferred embodiment, the subject includes humans, mice, rats, rabbits, monkeys, dogs, cats, or a combination thereof.

[0106] In another preferred embodiment, the subject includes a tumor patient.

[0107] As used herein, "tumor" and "cancer" are used interchangeably.

[0108] In another preferred embodiment, the tumor is a tumor associated with KRAS G12C gene mutation.

[0109] In another preferred embodiment, the tumor includes lung cancer, pancreatic cancer, and colorectal cancer.

[0110] In another preferred embodiment, the lung cancer includes non-small cell lung cancer.

[0111] In another preferred embodiment, the sample includes tissue, body fluid, excrement, or a combination thereof.

[0112] In another preferred embodiment, nucleic acid is extracted from the sample using a nucleic acid extraction kit, and nucleic acid quantification is performed.

[0113] In another preferred embodiment, the inlet amount of the quantified nucleic acid during pre-amplification is higher than 15 ng.

[0114] In the fifth aspect of the present invention, provided is the use of the reaction system as described in the first aspect of the present invention and the detection reagent as described in the second aspect of the present invention in detecting KRAS G12C gene mutation, and / or preparing a reagent for detecting KRAS G12C gene mutation.

[0115] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 1 Shows the electrophoresis of positive-strand oligonucleotide fragments guided by gDNA to cleave KRAS wild-type and KRAS G12C mutant genes.

[0117] Figure 2 Shows the electrophoresis of gDNA-guided PfAgo cleavage of negative-strand oligonucleotide fragments of KRAS wild-type and KRAS G12C mutant genes.

[0118] Figure 3 Shows the electrophoresis of gDNA-guided PfAgo cleavage of double-stranded fragments of KRAS wild-type and KRAS G12C mutant genes.

[0119] Figure 4 The figure shows the effect of the pre-amplification (enrichment) primer annealing temperature on the amplification effect. The annealing temperatures corresponding to lanes 1-12 are 52°C, 52.5°C, 53.7°C, 55.6°C, 58.2°C, 60.8°C, 63.2°C, 65.8°C, 68.4°C, 70.3°C, 71.5°C, and 72°C, respectively.

[0120] Figure 5 It shows the effect of qPCR primer annealing temperature on amplification effect. The annealing temperatures corresponding to lanes 1-12 are 52°C, 52.5°C, 53.7°C, 55.6°C, 58.2°C, 60.8°C, 63.2°C, 65.8°C, 68.4°C, 70.3°C, 71.5°C, and 72°C, respectively.

[0121] Figure 6 Shows the screening and specificity validation of qPCR TaqMan probes.

[0122] Figure 7 Shows the test results of human serum clinical samples containing KRAS G12C gene mutation. DETAILED DESCRIPTION

[0123] After in-depth and extensive research and extensive screening, the inventors first coupled nucleic acid amplification technology with PfAgo protease cleavage reaction to construct a KRAS G12C gene mutation detection method, and screened gDNA, primers, and detection probes for KRAS G12C gene mutation detection that are particularly suitable for this method. The inventors unexpectedly found that the detection reagent of the present invention can highly sensitively detect KRAS G12C gene mutations, and has excellent effects such as simplicity, speed, high specificity and sensitivity. On this basis, the present invention was completed.

[0124] the term

[0125] In order to better understand the present invention, the following terms are defined.

[0126] Unless otherwise specified, all singular terms also include plural, active and past tenses of the terms. Thus, for example, reference to "a protein" includes more than one protein, and reference to "a compound" refers to more than one compound.

[0127] Unless the context clearly dictates otherwise, the term "about" includes values ​​within a standard deviation of the stated value.

[0128] Where a numerical range is provided, it is to be understood that each intermediate integer of the value and each tenth of each intermediate integer of the value (unless the context clearly indicates otherwise), between the upper and lower limits of the range and any other specified or intermediate values ​​in the specified range are included in the present invention, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed in the present invention, subject to any explicitly excluded limitations within the specified ranges. When the specified range includes one or two of these limits, the range excluding (i) any one or (ii) two of those included limits is also included in the present invention. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0129] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0130] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0131] As used herein, the term "nucleic acid" means single-stranded and double-stranded polymers of nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bonds or internucleotide analogs, and associated counterions, such as H + NH4 + , trialkylammonium, tetraalkylammonium, Mg 2+ 、Na + Etc. Nucleic acid can be a polynucleotide or an oligonucleotide. Nucleic acid can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or can be a chimeric mixture thereof. Nucleotide monomer units can include any nucleotide described herein, including but not limited to naturally occurring nucleotides and nucleotide analogs. The size range of nucleic acids is typically from several monomer units such as 5-40 to several thousand monomer nucleotide units. Nucleic acids include but are not limited to genomic DNA, cDNA, hnRNA, mRNA, rRNA, tRNA, fragmented nucleic acids, nucleic acids obtained from subcellular organelles such as mitochondria or chloroplasts, and nucleic acids obtained from microorganisms or DNA or RNA viruses that may be present on or in biological samples.

[0132] As used herein, the term "target nucleic acid" is intended to mean a nucleic acid that is a target of analysis or action. Analysis or action includes subjecting the nucleic acid to copying, amplification, sequencing and / or other procedures for nucleic acid interrogation. The target nucleic acid may include a nucleotide sequence other than the target sequence to be analyzed. For example, the target nucleic acid may include one or more adapters, including adapters that function as primer binding sites on the flanks of the target nucleic acid sequence to be analyzed. The target nucleic acid hybridized with a capture oligonucleotide or a capture primer may include nucleotides that extend beyond the 5' or 3' ends of the capture oligonucleotide, in such a way that not all target nucleic acids are suitable for extension.

[0133] As used herein, when referring to guide RNA or DNA, crRNA or its derivatives, 5' phosphorylated single-stranded nucleic acid or other nucleotides, the term "target specificity" is intended to mean such a polynucleotide, which includes a nucleotide sequence specific to the target polynucleotide sequence, i.e., a nucleotide sequence that can selectively anneal to the recognition region of the target polynucleotide (e.g., target DNA). The target-specific nucleotide can have a single type of oligonucleotide, or the target-specific nucleotide can include two or more types of oligonucleotides with different sequences. Therefore, the target-specific nucleotide can be two or more sequences, including 3, 4, 5, 6, 7, 8, 9 or 10 or more different sequences. In one embodiment, crRNA or its derivatives include a target-specific nucleotide region complementary to a region of the target DNA sequence. In one embodiment, crRNA or its derivatives may include other nucleotide sequences except the target-specific nucleotide region. In one embodiment, other nucleotide sequences may be from tracrRNA sequences. In one embodiment, 5' phosphorylated single-stranded nucleic acid or its derivatives include a target-specific nucleotide region complementary to a region of the target DNA sequence. In one embodiment, the 5'-phosphorylated single-stranded nucleic acid or its derivative may contain other nucleotide sequences in addition to the target-specific nucleotide region.

[0134] As used herein, when referring to polynucleotide use, the term "complementary" is intended to mean a polynucleotide comprising a nucleotide sequence that can selectively anneal to the identification region of a target polynucleotide under certain conditions. As used herein, the term "substantially complementary" and grammatical equivalents are intended to mean a polynucleotide comprising a nucleotide sequence that can specifically anneal to the identification region of a target polynucleotide under certain conditions. Annealing refers to the nucleotide base pairing interaction of a nucleic acid and another nucleic acid, which interaction results in the formation of a duplex, triplex or other higher-order structures. By Watson-Crick (Watson-Crick) and Hoogsteen type hydrogen bonding, the main interaction is typically nucleotide base specific, for example, A:T, A:U and G:C. In certain embodiments, base stacking and hydrophobic interactions can also contribute to duplex stability. Conditions for annealing a polynucleotide to a complementary or substantially complementary region of a target nucleic acid are well known in the art, for example, as described in Nucleic Acid Hybridization, A Practical Approach, Hames and Higgins, eds., IRL Press, Washington, DC (1985) and Wetmur and Davidson, Mol. Biol. 31:349 (1968). Annealing conditions will depend on the specific application and can be routinely determined by one skilled in the art without undue experimentation.

[0135] As used herein, the term "polymerase chain reaction" or "PCR" refers to a procedure in which a small amount of nucleic acid, e.g., RNA and / or DNA, is amplified, as described, for example, in U.S. Pat. No. 4,683,195 to Mullis. Typically, sequence information from the ends of or beyond the region of interest needs to be available so that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to the opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers can be consistent with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, phage or plasmid sequences, and the like. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).

[0136] As used herein, the term "Argonaute system" refers to an enzyme system comprising a guide nucleic acid and an Argonaute family protein or a variant thereof, wherein the guide nucleic acid comprises a nucleotide sequence that is complementary or substantially complementary to a region of a target polynucleotide, such as a guide DNA. The Argonaute system includes an engineered and / or programmed nuclease system derived from a naturally occurring Argonaute system. The Argonaute system may comprise an engineered and / or mutated Argonaute protein. The Argonaute system may comprise an engineered and / or programmed guide DNA.

[0137] As used herein, the terms "guide DNA," "gDNA," and "guide DNA" are used interchangeably to refer to a gDNA comprising a sequence that is complementary or substantially complementary to a region of a target DNA sequence. The gDNA may comprise a nucleotide sequence other than a region that is complementary or substantially complementary to a region of a target DNA sequence.

[0138] As used herein, the term "nuclease" refers to an enzyme that is capable of cleaving phosphodiester bonds between nucleotide subunits of a nucleic acid.

[0139] As used herein, the term "detecting" a nucleic acid molecule or a fragment thereof refers to determining the presence of a nucleic acid molecule, typically when the nucleic acid molecule or a fragment thereof has been completely or partially separated from other components of a sample or composition, and may also include determining the charge-to-mass ratio, mass, amount, absorbance, fluorescence or other properties of a nucleic acid molecule or a fragment thereof.

[0140] As used herein, the term "primer" refers to a natural or synthetic oligonucleotide primer that can serve as the starting point of nucleic acid synthesis when placed in a primer extension (not limited to the number of extended bases) that is initiated. Primer can be a single-stranded oligodeoxyribonucleotide. The length of primer can be in the range of from about 10 to about 50 nucleotides, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or about 50 nucleotides. The primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template for primer extension to occur. If desired, the primer can be labeled by incorporating a detectable marker, for example, by spectroscopy, photochemistry, biochemistry, immunochemistry, or chemical means. Exemplary markers include, but are not limited to, biotin, amines, radioactive labels (e.g., 32P), fluorescent dyes, electron-dense reagents, enzymes (commonly used in ELISA), or biotin.

[0141] As used herein, the term "linear amplification" refers to an amplification process that uses multiple cycles of primer extension reactions to amplify target nucleic acids. With linear amplification, the abundance of transcripts increases proportionally with the number of cycles and is linearly amplified. Examples of linear amplification programs are LCR, the aRNA method of Phillips and Eberwine above, and the linear amplification method described herein. Unlike exponential amplification, the amount of amplified product does not increase exponentially. For example, in an ideal 4-hour linear amplification reaction with a copy rate of 2000 copies / minute, 2000 copies of template DNA will produce 960,000,000 copies.

[0142] As used herein, the term "exponential amplification" refers to an amplification procedure in which the product (i.e., amplicon) doubles with each reaction cycle. "Exponential amplification" is a nonlinear amplification that results in an exponential increase in the number of nucleic acid copies present. For example, exponential amplification can occur when primer extensions are initiated at both ends of an amplicon in an amplification cycle. For example, PCR is an exponential amplification procedure. For example, in an ideal PCR reaction with 30 cycles, 2 copies of template DNA will produce 2^30 or 1,073,741,824 copies.

[0143] As used herein, the terms "enrich," "enriching," or "enrichment" in the context of enriching a target polynucleotide refer to a process that produces a higher percentage of a target polynucleotide in a polynucleotide population. In one embodiment, the percentage is increased by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In one embodiment, the percentage is increased by about 2-fold, 5-fold, 10-fold, 50-fold, or 100-fold. In one embodiment, the target polynucleotide is substantially isolated from the polynucleotide population.

[0144] As used herein, the term "polymerase" refers to a protein that can catalyze the specific incorporation of nucleotides to extend the 3' hydroxyl terminus of a primer molecule (such as, for example, a template oligonucleotide) for a nucleic acid target sequence. The polymerase can be, for example, thermophilic, such that it is active at elevated reaction temperatures. For example, it can also have strand displacement capability.

[0145] As used herein, the term "single nucleotide polymorphism (SNP)" refers to a DNA sequence variation that occurs when a single nucleotide - A, T, C or G - in a genome (or other consensus sequence) differs between members of a species (or between paired chromosomes in an individual).

[0146] As used herein, the term "single nucleotide variant (SNV)" refers to a genotype or polynucleotide that includes a single nucleotide polymorphism (SNP) or a point mutation site.

[0147] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the subject is preferably a mammal, such as a non-primate (e.g., a cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., a monkey and a human). In a specific embodiment, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) having cancer.

[0148] KRAS G12C gene mutation

[0149] RAS represents a group of closely related monomeric globular proteins (21kDa molecular weight) with 189 amino acids, which are associated with the plasma membrane and bind GDP or GTP. RAS acts as a molecular switch. When RAS contains bound GDP, it is in a static or closed position and "inactive". When cells are exposed to certain growth-promoting stimuli in response, RAS is induced to exchange its bound GDP for GTP. In the case of binding GTP, RAS is "turned on" and is able to interact with other proteins (its "downstream targets") and activate the proteins. The RAS protein itself has an extremely low intrinsic ability to hydrolyze GTP back to GDP, thereby turning itself into a closed state. Closing RAS requires an exogenous protein called GTPase activating protein (GAP), which interacts with RAS and greatly accelerates the conversion of GTP to GDP. Any mutation in RAS that affects its ability to interact with GAP or convert GTP back to GDP will result in prolonged protein activation, and therefore prolonged signals to cells telling them to continue growing and dividing. Because these signals cause cell growth and division, overactivated RAS signaling can ultimately lead to cancer.

[0150] Structurally, the RAS protein contains a G domain responsible for the enzymatic activity of RAS, guanine nucleotide binding and hydrolysis (GTPase reaction). It also contains a C-terminal extension region called the CAAX box, which can be post-translationally modified and is responsible for targeting the protein to the membrane. The G domain is approximately 21-25 kDa in size and contains a phosphate binding loop (P-loop). The P-loop represents a pocket in the protein that binds nucleotides, and this is a rigid part of the domain with conserved amino acid residues that are necessary for nucleotide binding and hydrolysis (glycine 12, threonine 26, and lysine 16). The G domain also contains the so-called switch I region (residues 30-40) and switch II region (residues 60-76), which are both dynamic parts of the protein, and are often represented as a "spring-loaded" mechanism due to the ability of the dynamic part to switch between static and loaded states. The main interaction is a hydrogen bond formed by threonine-35 and glycine-60 with the γ-phosphate of GTP, which maintains their active conformations in switch 1 region and switch 2 region, respectively. Following hydrolysis of GTP and release of phosphate, both relax into the inactive GDP conformation.

[0151] The most notable members of the RAS subfamily are HRAS, KRAS, and NRAS, which are primarily involved in many types of cancer. However, there are many other members, including DIRAS1; DIRAS2; DIRAS3; ERAS; GEM; MRAS; NKIRAS1; NKIRAS2; NRAS; RALA; RALB; RAP1A; RAP1B; RAP2A; RAP2B; RAP2C; RASD1; RASD2; RASL10A; RASL10B; RASL11A; RASL11B; RASL12; REM1; REM2; RERG; RERGL; RRAD; RRAS, and RRAS2.

[0152] Mutation of any of the three major isoforms of the RAS gene (HRAS, NRAS or KRAS) is one of the most common events in human tumor formation. It was found that about 30% of all human tumors carry some mutations in the RAS gene. It is worth noting that KRAS mutations are detected in 25%-30% of tumors. In contrast, the rate of oncogenic mutations in NRAS and HRAS family members is much lower (8% and 3%, respectively). The most common KRAS mutations are found at residues G12 and G13 in the P-loop and at residue Q61.

[0153] G12C is a frequently occurring KRAS gene mutation (glycine-12 to cysteine). This mutation has been found in about 13% of cancers, about 43% of lung cancers, and almost 100% of MYH-associated polyposis (familial colon cancer syndrome).

[0154] Ago enzyme

[0155] In the present invention and detection method, a core component is a gene editing enzyme, such as Ago enzyme.

[0156] In some embodiments, the Ago is derived from the Argonaute system. The Argonaute protein family was first discovered genetically and identified for the first time in plants. Members are defined by the presence of PAZ (Piwi-Argonaute-Zwille) and PIWI domains. Argonaute proteins are highly conserved between species, and many organisms encode multiple members of the family. The number of Argonaute genes ranges from 1 in the fission yeast Schizosaccharomyces pombe to 27 in the nematode worm Caenorhabditis elegans. In mammals, there are eight Argonaute genes. The Argonaute protein family can be divided into the Ago subfamily and the Piwi subfamily. In most organisms investigated so far, including fruit flies, zebrafish and mice, the expression of Piwi proteins is limited to the germline, in which Piwi proteins bind to Piwi interacting proteins (piRNAs). In contrast, Ago proteins are ubiquitously expressed in many organisms. The human Ago1, Ago3, and Ago4 genes are clustered on chromosome 1, while the Ago2 gene is located on chromosome 8. The human Piwi subfamily includes HIWI1, HIWI2, HIWI3, and HILI; they are encoded by genes on chromosomes 12, 11, 22, and 8, respectively. Members of the Argonaute protein family have been shown to be key players in gene silencing pathways directed by small RNAs. Small RNAs, such as short interfering RNAs (siRNAs), microRNAs (miRNAs), or Piwi interacting RNAs (piRNAs), are anchored in specific binding pockets and direct Argonaute proteins to target mRNA molecules for silencing or destruction. See, e.g., Hock and Meister, Genome Biol, .2008, 9(2):210. Recently, it has also been shown that certain Argonaute proteins are directed by small DNAs for DNA cleavage. See, e.g., Gao et al., nature biotechnology, 2016, doi:10.1038 / nbt.3547. Thus, the Argonaute system provides a mechanism for targeting specific sequences and certain enzymatic activities to that sequence.

[0157] Thus, in some embodiments, the Ago is an Ago subfamily system or a derivative thereof. In other embodiments, the system is a Piwi subfamily system or a derivative thereof. In some specific embodiments, the Argonaute protein is Ng Argonaute or a variant thereof.

[0158] The key elements of the Argonaute system include guide nucleic acids (gDNA or gRNA), such as 5' phosphorylated single-stranded nucleic acids. 5' phosphorylated single-stranded nucleic acids or their derivatives include target-specific nucleotide regions that are complementary or substantially complementary to a region of the target nucleic acid. In some embodiments, 5' phosphorylated single-stranded nucleic acids or their derivatives include user-selectable DNA sequences that allow enzyme-specific targeting of complementary double-stranded DNA. In some embodiments, the user-selectable DNA sequence includes 20-50 nucleotides that are complementary or substantially complementary to a region of the target DNA sequence. In some embodiments, 5' phosphorylated single-stranded nucleic acids are user-selectable single-stranded DNA sequences that are less than 100 nucleotides. In some embodiments, 5' phosphorylated single-stranded nucleic acids are single-stranded DNAs that are less than 50 nucleotides. In some embodiments, 5' phosphorylated single-stranded nucleic acids are single-stranded DNAs that are less than 25 nucleotides. Exemplary lengths of single-stranded DNA include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 nucleotides. In some embodiments, the user-selectable DNA sequence comprises less than 20 nucleotides that are complementary or substantially complementary to a region of the target DNA sequence. Exemplary user-selectable DNA sequences comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides that are complementary or substantially complementary to a region of a target DNA sequence.

[0159] In some embodiments, the target-specific nucleotide region of the 5' phosphorylated single-stranded nucleic acid has a 100% base pair match with a region of the target nucleic acid. In some embodiments, the target-specific nucleotide region of the 5' phosphorylated single-stranded nucleic acid has a 90%-100%, 80%-100%, or 70%-100% base pair match with a region of the target nucleic acid. In some embodiments, there is a base pair mismatch between the target-specific nucleotide region of the crRNA and a region of the target nucleic acid. In some embodiments, there are two base pair mismatches between the target-specific nucleotide region of the 5' phosphorylated single-stranded nucleic acid and a region of the target nucleic acid. In some embodiments, there are three base pair mismatches between the target-specific nucleotide region of the 5' phosphorylated single-stranded nucleic acid and a region of the target nucleic acid. In some embodiments, there are four base pair mismatches between the target-specific nucleotide region of the 5' phosphorylated single-stranded nucleic acid and a region of the target nucleic acid. In some embodiments, there are five base pair mismatches between the target-specific nucleotide region of the 5' phosphorylated single-stranded nucleic acid and a region of the target nucleic acid.

[0160] Argonaute used in the present application includes engineered and / or programmed nuclease systems derived from naturally occurring Argonaute systems. The Argonaute system may comprise engineered and / or mutated Argonaute proteins. The Argonaute system may also comprise engineered and / or programmed guide nucleic acids. In some embodiments, the Argonaute protein or its variant is engineered to remove endonuclease activity so that the Argonaute system does not produce double-stranded DNA breaks or single-stranded DNA breaks. In other embodiments, the Argonaute protein or its variant is engineered to be able to produce single-stranded DNA breaks.

[0161] It will be appreciated that any Argonaute system capable of binding nucleic acid or disrupting double stranded nucleic acid and creating a loop structure by directing nucleic acid may be used in the present methods.

[0162] In the present invention, the preferred Ago enzyme is PfAgo enzyme, which is derived from the archaeon Pyrococcus furiosus, has a gene length of 2313 bp, and an amino acid sequence consisting of 770 amino acids.

[0163] The enzymatic cleavage characteristics of PfAgo enzyme are as follows: the enzyme can use 5' phosphorylated oligonucleotide as guide ssDNA to guide the enzyme to accurately cleave the target nucleic acid sequence; the cleavage site is located at the phosphodiester bond between the target nucleic acid (ssDNA) corresponding to the 10th and 11th nucleotides of the guide ssDNA.

[0164] Generally, the preferred working temperature of PfAgo enzyme is 95±2 degrees.

[0165] Guide ssDNA

[0166] In the detection method of the present invention, a core component is the guide ssDNA, especially two ssDNAs, which are adjacent to each other and have no intervening bases or intervening sequences between them.

[0167] In the present invention, the preferred guide ssDNAs are oligonucleotides with a length of 10-60 nt, preferably 10-40 nt, and more preferably 13-20 nt, and the first 5' nucleotide is thymine (T) and can be phosphorylated.

[0168] In another preferred embodiment, the two guide ssDNAs of each target are 16 nt in length, and the enzyme cleavage products guided therefrom are reverse complementary to the probe.

[0169] In another preferred embodiment, the length of each guide ssDNA is independently 10-30 nt, more preferably, 13-20 nt.

[0170] In another preferred embodiment, each guide ssDNA is a single-stranded DNA molecule with a chemically modified 5' end.

[0171] In some embodiments of the present invention, the guide ssDNA is selected from the group consisting of SEQ ID NO. 8, 13, 12, 5, 6, 7, 9, 10, 11, 14, 15, 16, or a combination thereof. In a preferred embodiment of the present invention, the guide ssDNA comprises a combination of SEQ ID NO. 8 and 13.

[0172] Primers of the present invention

[0173] In some embodiments, the primer used in the application is a single-stranded oligodeoxyribonucleotide. In some embodiments, the primer length is from about 10 to about 50 nucleotides. The exemplary length of the primer provided herein is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.

[0174] In some embodiments, the primer used in the present application has a 100% base pair match with a region of the target nucleic acid. In some embodiments, the primer used in the present application has a 90%-100%, 80%-100% or 70%-100% base pair match with a region of the target nucleic acid. In some embodiments, there is a base pair mismatch between a region of the primer and the target nucleic acid. In some embodiments, there are two base pair mismatches between a region of the primer and the target nucleic acid. In some embodiments, there are three base pair mismatches between a region of the primer and the target nucleic acid. In some embodiments, there are four base pair mismatches between a region of the primer and the target nucleic acid. In some embodiments, there are five base pair mismatches between a region of the primer and the target nucleic acid.

[0175] In some embodiments, the primer used in the present application is labeled, for example, for enrichment or detection of amplified products. In some embodiments, the primer used in the present application is labeled for detection by spectroscopy, photochemistry, biochemistry, immunochemistry or chemical means. In some embodiments, primers are labeled with biotin, amine, radioactive markers (for example, 32P), fluorescent dyes, electron-dense reagents, enzymes (generally used in ELISA) or biotin.

[0176] The primers of the present invention include pre-amplification and enrichment primers for amplifying the KRAS G12C mutant gene in the sample to be tested. In a preferred embodiment, the pre-amplification and enrichment primer pair includes an upstream primer as shown in SEQ ID NO.17 and a downstream primer as shown in SEQ ID NO.18.

[0177] In some embodiments, the primers of the present invention further include qPCR primers for detecting KRAS G12C mutant gene. In a preferred embodiment, the qPCR primer pair includes an upstream primer as shown in SEQ ID NO.19 and a downstream primer as shown in SEQ ID NO.20.

[0178] Probe

[0179] The term "primer" used herein has the meaning commonly understood by those skilled in the art, that is, a short single-stranded DNA or RNA fragment used to detect a nucleic acid sequence complementary thereto.

[0180] In view of the teachings of the present invention and the common knowledge in the art, it should be understood by those skilled in the art that, knowing the primer pair, those skilled in the art can independently design the probe according to the template sequence between the forward primer and the reverse primer binding site, and detect the technical effect of the probe and the primer pair. In a specific embodiment, a person of ordinary skill in the art can specifically design the probe as needed, and the probe can be in a liquid phase or fixed on a solid phase; it can be combined before amplification or after amplification. Therefore, the probe of the present invention is not limited to the probe specifically disclosed in the embodiment. The primer pair of the present invention is also not limited to the use of paired with the probe specifically disclosed in the embodiment.

[0181] In a specific embodiment, the probe of the present invention is shown as SEQ ID NO:21,22.

[0182] As used herein, "probe," "reporter molecule," and "reporter nucleic acid molecule" are used interchangeably.

[0183] In the detection method of the present invention, a core component is a reporter nucleic acid carrying a reporter molecule.

[0184] In some embodiments, the reporter nucleic acid molecule of the present invention is a nucleic acid molecule carrying a fluorescent group and a quencher group, respectively. For example, the 5' end is labeled with a fluorescent group (F) and the 3' end is labeled with a quencher group (Q).

[0185] Detection principle of the present invention

[0186] The present invention also provides a nucleic acid detection method based on the gene editing enzyme Ago, such as Pyrococcus furiosus Argonaute (PfAgo).

[0187] In the method of the present invention, based on the cleavage activity of the PfAgo enzyme, a guide ssDNA can be designed according to the target nucleic acid molecule (such as single-stranded DNA, preferably the amplified target nucleic acid molecule), and the guide ssDNA targets the target nucleic acid molecule and mediates the cleavage of the target nucleic acid molecule by the PfAgo enzyme.

[0188] According to the design requirements of the guide ssDNA, the PfAgo enzyme can be specially designed to selectively cleave nucleic acid sequences with differences in some sites, so it is very suitable for typing detection. In the present invention, the designed guide ssDNA allows the PfAgo enzyme to only cleave the KRAS wild-type gene, but not the KRAS G12C mutant gene, thereby enriching the KRASG12C mutant gene and realizing the typing detection of the KRAS gene.

[0189] Detection reagent and kit of the present invention

[0190] The present invention also provides a KRAS G12C gene mutation detection reagent, which comprises a guide ssDNA (gDNA) for guiding the Ago enzyme to cleave the target sequence (i.e., the KRAS wild-type gene), and other components required for PCR such as the primer pair and probe of the present invention, such as Taq enzyme, dNTP, Mg 2+ etc.

[0191] In a specific embodiment, the detection reagent of the present invention comprises a gDNA selected from SEQ ID NO. 8, 13, 12, 5, 6, 7, 9, 10, 11, 14, 15, 16, or a combination thereof, preferably, a gDNA comprising a combination of SEQ ID NO. 8 and 13;

[0192] The detection reagent of the present invention also includes an upstream primer shown in SEQ ID NO.17 and a downstream primer shown in SEQ ID NO.18 used for PCR amplification; an upstream primer shown in SEQ ID NO.19 and a downstream primer shown in SEQ ID NO.20 used for qPCR detection; and TaqMan probes shown in SEQ ID NOs:21 and 22.

[0193] In a specific embodiment, the detection sensitivity of the detection reagent of the present invention reaches single copy / reaction.

[0194] On the basis of the detection reagent of the present invention, the present invention further provides a method for detecting KRAS G12C gene mutation, the method comprising: using the detection reagent of the present invention, performing PCR on the sample to be tested, and detecting the PCR amplification product.

[0195] Based on the detection reagent of the present invention, the present invention further provides a PCR kit, which comprises a container and the detection reagent of the present invention located in the container.

[0196] In a specific embodiment, the PCR kit of the present invention is also equipped with other components required for implementing PCR and instructions for using the kit for PCR detection. In a preferred embodiment, the kit is also equipped with a standard control.

[0197] Technical solution of the present invention

[0198] One of the preferred embodiments of the present invention is as follows.

[0199] At present, the present invention has developed a new low-abundance gene mutation detection technology based on the emerging multifunctional programmable nuclease, PfAgo protein (Argonaute protein from Pyrococcus furiosus), which has the characteristics of endonuclease. This technology couples nucleic acid amplification technology with PfAgo protease cleavage reaction, and through the designed guide DNA (gDNA) sequence, shears wild-type DNA under specific reaction conditions (temperature, ionic strength) to enrich mutant DNA, which is conducive to the subsequent detection of mutant genes, and the detection sensitivity can reach less than 1 / 10000. Its specific application method is to use the gDNA design strategy to make Ago protein selectively shear wild-type targets and retain mutant targets, so as to achieve the effect of enriching mutant genes.

[0200] The technical problem to be solved by the present invention is to provide a rapid, efficient and highly sensitive KRAS G12C gene mutation detection primer, gDNA and TaqMan probe, a detection kit and a detection method using the primer, gDNA and TaqMan probe.

[0201] In a first aspect of the present invention, a primer, gDNA, and TaqMan probe set for detecting KRAS G12C gene mutation are provided. Specifically, the primer, gDNA, and TaqMan probe set include:

[0202] Pre-amplification and enrichment primer pairs for detecting KRAS G12C:

[0203] Upstream primer: 5'-GTGACATGTTCTAATATAGTC-3' (SEQ ID NO. 17)

[0204] Downstream primer: 5'-GGATCATATTCGTCCACAAA-3' (SEQ ID NO.18)

[0205] gDNA for KRAS G12C detection:

[0206] Positive strand gDNA: 5'-TTTGGAGCTGATGGCG-3' (SEQ ID NO.8)

[0207] Negative strand gDNA: 5'-TCTACGCCAGCAGCTC-3'(SEQ ID NO.13)

[0208] qPCR primer pairs for detecting KRAS G12C:

[0209] Upstream primer: 5'-AGGCCTGCTGAAAATGACTG-3' (SEQ ID NO.19)

[0210] Downstream primer: 5'-GCTGTATCGTCAAGGCACTCT-3' (SEQ ID NO.20)

[0211] TaqMan Probe Panel for KRAS G12C Detection:

[0212] Wild-type probe: 5'-TTGGAGCTGGTGGCGTA-3' (SEQ ID NO.21)

[0213] Mutation probe: 5'-TTGGAGCTTGTGGCGTA-3' (SEQ ID NO.22)

[0214] The pre-amplification and enrichment primer pairs for detecting KRAS G12C can amplify a DNA product with a length of 158 bp, which covers the general size of free DNA (about 166 bp).

[0215] During the enrichment process, the gDNA pair that guides Ago protein to cleave the wild-type gene is a 16nt oligonucleotide chain with phosphorylated 5' and 3' ends. On the one hand, it can increase the cleavage activity of Ago protein, and on the other hand, it can avoid non-specific amplification of gDNA as a primer. At the same time, the first base of the 5' end of gDNA is unified as T, which can increase the cleavage activity and cleavage specificity of Ago protein.

[0216] The qPCR primer pair for detecting KRAS G12C can amplify a DNA product with a length of 80 bp, and the amplified fragment must be within the fragment of the pre-amplified and enriched product.

[0217] The TaqMan probe for detecting KRAS G12C was fluorescently modified. The fluorescent reporter groups for the mutant and wild-type probes of the KRAS gene were FAM and VIC, respectively, and the fluorescent quenching group was BHQ1.

[0218] In a second aspect of the present invention, a kit for detecting KRAS G12C gene mutation is provided, which comprises the above-mentioned primers, gDNA and TaqMan probe set.

[0219] In some embodiments of the present invention, the concentration of each primer and TaqMan probe is 200 nM.

[0220] In some embodiments of the present invention, gDNA is used at a concentration of 800-4800 nM.

[0221] In some embodiments of the present invention, the PfAgo protein is used at a concentration of 40-240 nM.

[0222] In some embodiments of the present invention, the kit further comprises a DNA extraction reagent, specifically a DNA extraction kit for extracting genomic or free DNA from tissues, body fluids or excrement.

[0223] In some embodiments of the present invention, the kit further comprises PCR reagents, including Mg 2+ , dNTPs, DNA polymerase, and PCR buffer components exist alone or in a mixture. 2+ It is a cofactor of DNA polymerase and has an impact on amplification efficiency and specificity. 2+ The concentrations of , dNTPs, DNA polymerase, and PCR buffer can be adjusted according to conventional methods in the art.

[0224] In some embodiments of the present invention, the kit further comprises a fluorescent quantitative PCR kit.

[0225] The third aspect of the present invention provides methods and applications of the above reagents (primers, gDNA, TaqMan probe set) or kits in related tumor screening, companion diagnosis or medication guidance.

[0226] In some embodiments of the present invention, the test sample is selected from tissue, body fluid or excrement of a tumor patient who may have KRAS G12C gene mutation.

[0227] In some embodiments of the present invention, the sample is extracted using a nucleic acid extraction kit and nucleic acid quantification is performed.

[0228] In some embodiments of the present invention, the quantified nucleic acid is pre-amplified according to an input amount of 33.3 ng.

[0229] In some embodiments of the present invention, the pre-amplification product is subjected to gDNA-guided PfAgo-specific shearing to enrich the KRAS G12C mutation.

[0230] In some embodiments of the present invention, the enriched products are detected or quantified by Sanger sequencing, qPCR or dPCR.

[0231] Studies related to KRAS gene mutations have shown that KRAS gene mutations can serve as an important adverse prognostic factor for progression-free survival in non-small cell lung cancer, and that there is a certain relationship between its mutation and the progression-free survival of pancreatic cancer patients. On the other hand, KRAS gene mutations are closely related to the efficacy of immunotherapy for colorectal cancer and non-small cell lung cancer. Colorectal cancer patients carrying KRAS gene mutations have a poor response to panitumumab or cetuximab, and KRAS gene mutations are also related to the efficacy of EGFR-TKI in the treatment of non-small cell lung cancer. Therefore, the detection of KRAS gene mutation sites in patients can, to a certain extent, achieve an auxiliary role in the prognosis of the above cancers.

[0232] application

[0233] The present invention is particularly suitable for detecting target nucleic acid molecules and has wide applicability.

[0234] In the present invention, the target nucleic acid molecule may be DNA.

[0235] In terms of disease monitoring, the present invention can achieve active management such as prediction and prevention of diseases, and achieve early detection and early treatment, or early prediction and early prevention. Since the detection sensitivity and accuracy of the present invention are very high and have obvious advantages, it is suitable for typing diagnosis and disease differentiation and identification, prescribing the right medicine, saving patients' treatment time, and improving the success rate of treatment. The present invention reduces the waste of high medical costs and strives for the golden opportunity for treatment.

[0236] The applicability of the present invention is that it can assist in the prediction and diagnosis of a variety of diseases, including but not limited to: acute myeloid leukemia, juvenile cancer, childhood adrenocortical carcinoma, AIDS-related cancers (such as lymphoma and Kaposi's sarcoma) Sarcoma), anal cancer, appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumor, burkitt lymphoma, carcinoid tumor, atypical teratoid, embryonal tumor, germ cell tumor, primary lymphoma, cervical cancer, childhood cancer, chordoma, heart tumor, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, extrahepatic ductal carcinoma in situ (DCIS), embryonal tumor, CNS cancer, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, Ewing's sarcoma sarcoma), extracranial germ cell tumors, extragonadal germ cell tumors, eye cancer, fibrous histiocytoma of bone, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational trophoblastic tumors, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney cancer, laryngeal cancer, lip and oral cancer, liver cancer, lobular carcinoma in situ (LCIS), lung cancer, lymphoma, metastatic squamous neck cancer with occult primary, midline tract cancer, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, multiple myeloma, merkel cell carcinoma cellcarcinoma), malignant mesothelioma, malignant fibrous histiocytoma and osteosarcoma of the bone, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer (NSCLC), oral cancer, lip and oral cavity cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumors, unusual cancers of children, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, virus-induced cancers, or a combination thereof.

[0237] Advantages of the present invention include:

[0238] 1. The primer pair or detection reagent of the present invention can detect KRAS G12C gene mutation with high sensitivity.

[0239] 2. The detection method of the present invention is simple and quick to operate, and has high specificity and sensitivity.

[0240] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001), or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0241] Example 1

[0242] This embodiment provides a primer, gDNA, and TaqMan probe set for detecting KRAS G12C gene mutation, and a design method of the primer, gDNA, and TaqMan probe set.

[0243] Around the KRAS G12C gene mutation site region, 60nt wild-type and mutant ssDNA targets (including sense and antisense strands) were designed and synthesized, and then gDNA was designed for the target. The gDNA length is generally 16nt, with 5' end phosphorylation modification, and a mismatch is introduced at the 10th / 11th gDNA, for a total of 12 gDNAs. The shear product sizes are generally 25nt and 35nt, respectively. The specific sequence information is as follows:

[0244] Table 1 ssDNA targets and gDNA sequences

[0245]

[0246]

[0247] Design A-Star enrichment primers based on the synthesized 500bp gene sequence: You can use the online NCBI Primer-Blast tool for design. The amplified product size is generally 130-160bp, and the primer parameters are selected by default. The mutation site is generally designed in the middle region of the amplified product. Primers are usually designed and synthesized in 2-3 groups, and then PCR amplification tests are performed, and a group with good specificity is screened by agarose gel electrophoresis.

[0248] Taqman probes and corresponding primers for qPCR detection were designed for the sequence of KRAS G12C enriched products. The design steps are as follows: first, in Beacon Designer 8 software, click File>New>Sequence, paste the sequence, click Tools>SNP>Add, fill in the mutated base (G>T) in Mutant Base, fill in the position of the mutated base in SNP Position in this sequence, then click Add SNP, click Analyze>Taqman Search>Standard, set the default parameters, and click Search. If there is no result, you need to modify the parameters yourself, usually the probe length and Tm value, and then go to Search. MGB modification is performed on the 3' end of the designed sequence during synthesis to increase the probe Tm value. Generally, the Tm value of the probe is required to be 5 to 10°C higher than that of the primer.

[0249] Example 2

[0250] This embodiment provides a method for screening primers, gDNA, and TaqMan probe groups for detecting KRAS G12C gene mutations, as well as optimization of reaction conditions.

[0251] The single-stranded target was a synthetic KRAS wild-type and KRAS G12C mutant gene positive and negative strand oligonucleotide fragment with a length of 60 nt. The shearing reaction system was 2.5 μL of 10× reaction buffer, 1.25 μL of 10 mM MnCl2, 2 μL of 10 μM single-stranded target, 0.5 μL of 100 μM gDNA, 1 μL of 5 μM PfAgo, and 25 μL of ddH2O. The reaction conditions were 95°C for 15 min; slowly cooled to 10°C at 0.1°C / s. 6 μL of the sheared product was subjected to 16% urea-TBE denaturing electrophoresis ( Figure 1 , Figure 2 ) to screen out gDNA that only cuts the wild-type positive and negative chains but not the mutant positive and negative chains.

[0252] Shearing of double-stranded targets, the double-stranded targets are synthetic KRAS wild-type and G12C mutant gene fragments, with a length of 500bp. The shearing reaction system is 2.5μL of 10× reaction buffer, 1.25μL of 10mM MnCl2, 200ng of wild-type or mutant double-stranded targets, 0.5μL of 100μM gDNA and gDNA complementary to the positive and negative strand targets, 1μL of 5μM PfAgo, and ddH2O is added to 25μL. The reaction conditions are 95℃15min; slowly cool to 10℃ at 0.1℃ / s. Take 10μL of the reaction product for 1.5% agarose gel electrophoresis ( Figure 3). The gDNA combination that only cuts the wild-type double-stranded target but not the mutant double-stranded target was screened out. It can be seen that the combination of FW-11A and RV-10G can only cut the wild-type double-stranded target but not the mutant double-stranded target.

[0253] The screening of pre-amplification, enrichment and qPCR primers and the optimization of amplification conditions were performed. The reaction system was 2×PCR Taq MasterMix 12.5μL, 10μM upstream and downstream primers (pre-amplification and enrichment primer amplification products were 158bp, qPCR primer amplification products were 80bp) 0.5μL each, 500bp double-stranded target final concentration was 10nM, and ddH2O was added to 25μL. The reaction conditions were 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, annealing at 52-72℃ temperature range for 30s, 72℃ extension for 20s for 35 cycles, and 72℃ extension for 5min. 5μL of the reaction product was subjected to 1% agarose gel electrophoresis ( Figure 4 , Figure 5 ).

[0254] Screening and specificity verification of qPCR TaqMan probes, the reaction system is 2×qPCR Probe Mix 10μL, 10μM qPCR upstream and downstream primers 0.5μL each, wild-type and mutant TaqMan probes 0.4μL each, 500bp wild-type or mutant double-stranded target final concentration 10nM, supplemented with ddH2O to 20μL. The reaction conditions are 95℃ pre-denaturation for 8min; 95℃ denaturation for 15sec annealing, extension and collection of fluorescence signals in the temperature range of 57.5-67.5℃ for 40 cycles for 40s. Read the fluorescence signals under wild-type and mutant double-stranded targets respectively, and select the reaction conditions where the wild-type double-stranded target has only the wild-type TaqMan probe signal and the mutant double-stranded target has only the mutant TaqMan probe signal ( Figure 6 ).

[0255] In this example, the pre-amplification and enrichment primer pairs obtained by screening are shown in SEQ ID NO.17 and SEQ ID NO.18; the qPCR primer pairs are shown in SEQ ID NO.19 and SEQ ID NO.20; and the Taqman probes are shown in SEQ ID NO.21 and SEQ ID NO.22.

[0256] Example 3

[0257] This embodiment provides a KRAS G12C gene mutation detection kit and the components of the kit.

[0258] The kit comprises the following components: 2×PCR high-fidelity Taq enzyme Mix, 10 μM PCR upstream and downstream primers (SEQ ID NO.17 and SEQ ID NO.18), 100 nM 1%, 1‰, 1 / 10000 KRAS G12C standards, 2×PCR ordinary Taq enzyme Mix, 2 mM MnCl2, 100 μM positive and negative chain gDNA (SEQ ID NO.8 and SEQ ID NO.13), 5 μM PfAgo protein, 2×qPCR Mix, 10 μM qPCR upstream and downstream primers (SEQ ID NO.19 and SEQ ID NO.20), 10 μM qPCR wild-type and mutant probe combinations (SEQ ID NO.21 and SEQ ID NO.22), and dd H2O.

[0259] Example 4

[0260] This example provides the kit in Example 3 for detecting standard samples containing 1 / 100, 1 / 1000 and 1 / 10000 KRAS G12C gene mutations and human serum clinical samples containing KRAS G12C gene mutations.

[0261] The kit consisting of the above-screened primers, gDNA, and TaqMan probe group and the optimized reaction conditions were used to detect 1 / 100, 1 / 1000, and 1 / 10000 KRAS G12C gene mutation standard samples.

[0262] The pre-amplification reaction system was 25 μL of 2×PCR high-fidelity Taq enzyme Mix, 1 μL of 10 μM PCR upstream and downstream primers, 5 μL of 100 nM standard template, and ddH2O added to 50 μL. The reaction conditions were 94°C pre-denaturation for 3 min; 94°C denaturation for 10 sec, 55°C annealing for 30 sec, 72°C extension for 20 sec, 30 cycles; 72°C extension for 5 min.

[0263] The enrichment reaction system was 2×PCR ordinary Taq enzyme Mix 25μL, 2mM MnCl2 1.25μL, 10μM PCR upstream and downstream primers 0.5μL each, pre-amplification product 2μL, 100μM positive and negative chain gDNA 0.8μL each, 5μM PfAgo 0.8μL, supplemented with ddH2O to 25μL. The reaction conditions were 94℃ pre-denaturation for 3min; 94℃ denaturation for 30sec, 55℃ annealing for 30sec, 72℃ extension for 20sec, 25 cycles; 72℃ additional extension for 5min.

[0264] The qPCR detection system consisted of 10 μL of 2×qPCR Probe Mix, 0.5 μL of 10 μM qPCR upstream and downstream primers, and 0.4 μL of wild-type and mutant TaqMan probes. -3 5 μL of the diluted enriched product was supplemented with ddH2O to 20 μL. The reaction conditions were 95°C pre-denaturation for 8 min, 95°C denaturation for 15 s, 61.5°C annealing, extension and signal collection for 40 sec, and 40 cycles.

[0265] Example 5

[0266] The kit consisting of the above-screened primers, gDNA, and TaqMan probe group and the optimized reaction conditions were used to detect human serum clinical samples containing KRAS G12C gene mutations.

[0267] The free DNA of serum samples was extracted and quantitatively tested according to the instructions of the free DNA extraction kit (magnetic bead method) and the free DNA of serum samples was extracted and quantitatively tested using a dsDNA quantification kit.

[0268] The free DNA of clinical samples was detected according to the method described in Example 4 and substituted into the standard curve to calculate the copy number of wild-type and mutant genes and the enrichment efficiency of mutants.

[0269] The results are as follows Figure 7 As shown. The results showed that the abundance of KRAS G12C gene mutations was significantly increased after enrichment, and the mutation abundance increased from undetected to 21.08%. At the same time, by comparing with the amplification signals of 0.1% and 0.01% standard samples, the original KRAS G12C gene mutation abundance in the sample should be less than 0.01%, that is, the detection method can detect KRAS G12C gene mutations less than 1 / 10000.

[0270] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A KRAS G12C gene mutation detection reaction system, characterized in that: The reaction system comprises: (a) a pre-amplification and enrichment primer pair for amplifying the KRAS G12C mutant gene in the sample to be tested; and (b) Ago enzyme and guide ssDNA (gDNA) that specifically cleave the KRAS wild-type gene; Wherein, the guide ssDNA (gDNA) is selected from the following group: SEQ ID NO. 8, 13, 12, 5, 6, 7, 9, 10, 11, 14, 15, 16, or a combination thereof.

2. The reaction system according to claim 1, characterized in that The gDNA includes a combination of SEQ ID NOs. 8 and 13.

3. The reaction system according to claim 1, characterized in that The Ago enzyme is selected from the following group: PfAgo (Pyrococcusfuriosus Ago), MfAgo (Methanocaldococcus fervens Ago), TcAgo (Thermogladiuscalderae Ago), TfAgo (Thermus filiformis Ago), AaAgo (Aquifex aeolicus Ago), TpAgo (Thermus parvatiensis Ago), FpAgo (Ferroglobus placidus Ago) or a combination thereof, more preferably, PfAgo is selected.

4. The reaction system according to claim 1, characterized in that The reaction system also includes: (c) a qPCR primer pair and a probe for detecting the KRASG12C mutant gene.

5. A KRAS G12C mutation detection reagent, characterized in that: The mutation detection reagent includes a guide ssDNA (gDNA), and the gDNA is selected from the following group: SEQ ID NO. 8, 13, 12, 5, 6, 7, 9, 10, 11, 14, 15, 16, or a combination thereof.

6. The detection reagent according to claim 5, characterized in that The gDNA includes a combination of SEQ ID NOs. 8 and 13.

7. A KRAS G12C mutation detection kit, characterized in that: The kit comprises the detection reagent as claimed in claim 5 or 6.

8. A method for detecting a KRAS G12C mutation, the method comprising the steps of: (S1) providing a sample to be tested; (S2) Detecting the sample using the reaction system described in the first aspect of the present invention, the detection reagent described in the second aspect of the present invention, the kit described in the third aspect of the present invention, or a combination thereof.

9. The method according to claim 8, characterized in that The method comprises the following steps: (i) providing samples to be tested; (ii) performing PCR pre-amplification on the sample using a pre-amplification and enrichment primer pair to obtain a pre-amplification product; (iii) enriching the KRAS G12C mutant gene in the pre-amplification product using a pre-amplification and enrichment primer pair, a guide ssDNA, and an Ago enzyme to obtain an enriched product; (iv) detecting the enriched product.

10. Use of the reaction system according to claim 1 and the detection reagent according to claim 5 in detecting KRAS G12C gene mutation and / or preparing a reagent for detecting KRAS G12C gene mutation.

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