WO2020215652A1 - 基于核酸酶偶联pcr原理富集低丰度dna突变的检测技术体系及应用 - Google Patents

基于核酸酶偶联pcr原理富集低丰度dna突变的检测技术体系及应用 Download PDF

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WO2020215652A1
WO2020215652A1 PCT/CN2019/115151 CN2019115151W WO2020215652A1 WO 2020215652 A1 WO2020215652 A1 WO 2020215652A1 CN 2019115151 W CN2019115151 W CN 2019115151W WO 2020215652 A1 WO2020215652 A1 WO 2020215652A1
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nucleic acid
amplification
pcr
target
target nucleic
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French (fr)
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冯雁
刘倩
荀冠华
郭翔
李忠磊
崇曰盛
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上海交通大学
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Priority to EP19926362.5A priority Critical patent/EP3998352A4/en
Priority to JP2021563419A priority patent/JP7384459B2/ja
Priority to US17/605,669 priority patent/US20220119877A1/en
Publication of WO2020215652A1 publication Critical patent/WO2020215652A1/zh

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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
    • C12Q1/6858—Allele-specific amplification
    • 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

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a detection system based on the principle of nuclease-coupled PCR for enriching low-abundance single-nucleotide mutant genes (Single Nucleotide Variant, SNV).
  • liquid biopsy In recent years, the concept of "liquid biopsy” is emerging. The basic idea is to use blood and other body fluid samples to replace tumor tissue samples for pathological and molecular biological testing, and to detect circulating tumor DNA in patient body fluid samples (mainly blood). Obtaining tumor gene mutation information has become a trend. Compared with the current standard tissue biopsy, the revolutionary liquid biopsy has the following irreplaceable advantages: less trauma, reproducibility, homogenization of heterogeneity, real-time judgment of curative effect, and dynamic adjustment of treatment decisions as the tumor develops. Therefore, in 2015, MIT Technology Review released the top ten breakthrough technologies of the year (Breakthrough Technologies 2015), ASCO's annual progress (Clinical cancer advance 2015), the expectations for the next ten years, liquid biopsy are all on the list.
  • ctDNA detection By detecting ctDNA to track the specific genetic changes of the tumor throughout the course of the disease, it is of great value for tumor screening, diagnosis, efficacy monitoring, and prognosis judgment. At the same time, it can explore the molecular mechanism of tumor metastasis, recurrence and drug resistance, and identify new targets. Therefore, ctDNA detection has become one of the three popular directions for tumor liquid biopsy applications.
  • cfDNA cell-free DNA
  • cfDNA cell-free DNA
  • 1mL of plasma of a healthy person contains 25ng cfDNA.
  • the content of cfDNA in cancer patients is several times higher than normal, part of which is ctDNA (circulating tumor DNA).
  • the relative content of ctDNA is related to tumor load and response to treatment, and can be used to identify driver genes, guide clinical treatment, monitor clinical treatment effects and cancer recurrence, reveal treatment resistance, and detect disease progression. In some aspects, the sensitivity of the ctDNA method is even higher than that of traditional methods.
  • cfDNA KRAS mutation in lung cancer and bowel cancer also has important diagnostic value for lung cancer.
  • ctDNA can be detected in the early stages of cancer. Because cfDNA is easy to collect and has been shown to be highly consistent with the variation in tissues in lung cancer, liquid biopsy of ctDNA has attracted more and more attention.
  • BEAMing amplification method greatly improves the sensitivity of DNA detection technology.
  • Bert Vogelstein and Kenneth Kinzler of Johns Hopkins University in the United States tracked the circulating tumor DNA of 18 patients with colorectal cancer.
  • the sensitivity of BEAMing technology is high, which can reach 0.1% to 0.01%. It is an ideal technique for detecting circulating tumor DNA. However, due to its complicated operation and expensive equipment, it is not suitable for large-scale clinical promotion.
  • the current rare mutation detection methods mainly include gene sequencing as the "gold standard".
  • the sensitivity of sequencing is limited. Under the background of a large number of wild-type genes, sequencing can only detect 20% of mutations, which will lead to false negative results and take a long time.
  • the sensitivity of denaturing high performance liquid chromatography has been improved, but it requires PCR post-processing, which is likely to cause laboratory contamination, easily lead to false positive results, specificity is also limited, and the operation steps are complicated and the cycle is long.
  • Detection methods based on the principle of nucleic acid hybridization, such as TaqMan probes have a selective detection level equivalent to sequencing methods.
  • Amplification hindered mutation system is a commonly used method for detecting rare mutations.
  • mutant templates are specifically selected for amplification, but due to limited distinguishing ability, the selectivity is general. And different types of mutations are quite different.
  • Lifetechnology developed a highly selective mutation detection technology—cast PCR technology, which is based on ARMS technology and uses the high specificity of MGB probes to further improve the selectivity of the reaction.
  • MGB probes are difficult and expensive, which is not conducive to wide application.
  • Digital PCR is another high-sensitivity detection technology that has emerged in recent years. This technology can reach a sensitivity of 0.01%, but this technology is very prone to false positive results.
  • high equipment and reagent prices, and extremely high experimental operation requirements also restrict its large-scale promotion.
  • the purpose of the present invention is to provide a method for enriching and detecting mutant DNA with high specificity, high sensitivity and low abundance.
  • a method for increasing the relative abundance of target nucleic acid which includes the steps:
  • nucleic acid sample (a) providing a nucleic acid sample, the nucleic acid sample containing a first nucleic acid and a second nucleic acid, wherein the first nucleic acid is the target nucleic acid and the second nucleic acid is a non-target nucleic acid,
  • the abundance of the target nucleic acid in the nucleic acid sample is F1a;
  • the nucleic acid cleavage reaction is used to specifically cut the non-target nucleic acid, but not the target nucleic acid;
  • the amplification-cleavage reaction system contains (i) reagents required for PCR reaction and (ii) reagents required for nucleic acid cleavage reaction;
  • the abundance of the target nucleic acid in the amplification-cleavage reaction product is F1b
  • the ratio of F1b/F1a is ⁇ 10.
  • the target nucleic acid and the non-target nucleic acid differ by only one base.
  • the ratio of F1b/F1a ⁇ 10 when 1% ⁇ F1a ⁇ 10%, the ratio of F1b/F1a ⁇ 10, when 0.1% ⁇ F1a ⁇ 0.5%, the ratio of F1b/F1a ⁇ 100, when F1a ⁇ 0.1%, The ratio of F1b/F1a is ⁇ 200.
  • the nucleic acid sample includes a nucleic acid sample that is directly heated and lysed, a nucleic acid sample that is treated with a direct lytic enzyme protease, an extracted nucleic acid sample, a nucleic acid sample pre-amplified by PCR, or any sample containing nucleic acid .
  • the nucleic acid samples pre-amplified by PCR are PCR amplified products of 1-30 cycles, preferably 10-20, more preferably 15-30 cycles.
  • the target nucleic acid is a nucleotide sequence containing mutations.
  • the mutation is selected from the following group: nucleotide insertion, deletion, substitution, or a combination thereof.
  • the mutation includes SNV.
  • the non-target nucleic acid (or the second nucleic acid) is a wild-type nucleotide sequence, a highly abundant nucleotide sequence, or a combination thereof.
  • the abundance of the non-target nucleic acid in the nucleic acid sample is F2a.
  • F1a+F2a 100%.
  • the ratio of F2a/F1a is ⁇ 20, preferably ⁇ 50, more preferably ⁇ 100, and most preferably ⁇ 1000 or ⁇ 5000.
  • the abundance of the non-target nucleic acid in the amplification-cleavage reaction product is F2b.
  • F1b+F2b 100%.
  • said F1b/F2b ⁇ 0.5, preferably ⁇ 1, more preferably ⁇ 2, most preferably ⁇ 3 or ⁇ 5.
  • the ratio of F1b/F1a is ⁇ 200, preferably ⁇ 500, more preferably ⁇ 1000, and most preferably ⁇ 2000 or ⁇ 5000 or higher.
  • F1a ⁇ 0.5%, preferably ⁇ 0.2%, more preferably ⁇ 0.1%, most preferably ⁇ 0.01%.
  • F1b ⁇ 10%, preferably ⁇ 30%, more preferably ⁇ 50%, and most preferably ⁇ 70%.
  • the "reagents required for PCR reaction” include: DNA polymerase.
  • the "reagents required for performing PCR reactions" further include: dNTP, 1-5 Mm Mg 2+ , PCR buffer.
  • the "reagents required for nucleic acid cleavage reaction” include: nucleic acid cleavage tool enzyme and guide DNA (gDNA).
  • the nucleic acid cutting tool enzyme is a high-temperature stable double-stranded DNA cutting tool enzyme.
  • the nucleic acid cleavage tool enzyme is selected from but not limited to the following Argonaute proteins from thermophilic microorganisms ( ⁇ 60°C) and their mutants: PfAgo (Pyrococcus furiosus Ago), MfAgo (Methanocaldococcus fervens Ago), TcAgo (Thermogladius calderae Ago), TfAgo (Thermus filiformis Ago), AaAgo (Aquifex aeolicus Ago), etc.
  • PfAgo Polyrococcus furiosus Ago
  • MfAgo Metalhanocaldococcus fervens Ago
  • TcAgo Thermogladius calderae Ago
  • TfAgo Thermus filiformis Ago
  • AaAgo Aquifex aeolicus Ago
  • the nucleic acid cutting tool enzyme is PfAgo.
  • the gDNA forms a complex with the nucleic acid cutting tool enzyme, and the complex specifically cleaves non-target nucleic acids.
  • the gDNA and the nucleic acid sequence of the target region of the target nucleic acid form a first complementary binding region; and the gDNA also interacts with the non-target nucleic acid (ie, the second nucleic acid).
  • the nucleic acid sequence of the targeted region of the forms the second complementary binding region.
  • the first complementary binding region contains at least 2 unmatched base pairs.
  • the second complementary binding region contains 0 or 1 unmatched base pair.
  • the second complementary binding region contains 1 base pair that does not match.
  • the first complementary binding region contains at least 2 unmatched base pairs, so that the complex does not cleave the target nucleic acid; and the second complementary binding region contains 1 unmatched base pair. Matched base pairs, thereby causing the complex to cleave the non-target nucleic acid.
  • the targeted region of the target nucleic acid corresponds to the targeted region of the non-target nucleic acid (i.e., the second nucleic acid).
  • the length of the gDNA is 15-30 nt.
  • the 7th and/or 10th position of the gDNA is a mismatched base, and the mismatched base is used to form a mismatch in both the first complementary binding region and the second complementary binding region. Matched base pairs.
  • positions 2-8 of the gDNA are "seed region" regions, and positions 10 and 11 are key sites for PfAgo cutting.
  • the ratio (molar ratio) of the nucleic acid cutting tool enzyme and gDNAs is 1:2 to 1:20.
  • the nucleic acid cleavage tool enzyme is 30 nM
  • the DNA polymerase is a high temperature polymerase, preferably Taq DNA polymerase, LA Taq DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Phusion DNA polymerase, KOD DNA polymerase, etc., better 2X PCR Precision TM Master Mix.
  • the amount of nucleic acid used as a template is 0.1-100 nM.
  • the method further includes:
  • the detection in step (c) includes quantitative detection, qualitative detection, or a combination thereof.
  • the quantitative detection is selected from the following group: q-PCR, ddPCR, chemiluminescence method, high-resolution melting curve method, Sanger sequencing, NGS and the like.
  • the first nucleic acid includes n different nucleic acid sequences, wherein n is a positive integer ⁇ 1.
  • n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 , 97, 98, 99, 100 or greater.
  • n is 2-1000, preferably 3-100, more preferably 3-50.
  • step (b) C "high temperature denaturation-extension" cycles are performed, where C is ⁇ 5.
  • the high-temperature denaturation temperature corresponds to the melting temperature of the DNA double strand in the PCR reaction and the cutting temperature of the nucleic acid cutting tool enzyme.
  • the high temperature denaturation temperature is 85-95°C.
  • the C is 5-35.
  • the method is non-diagnostic and non-therapeutic.
  • the nucleic acid sample includes nucleic acid from a sample, wherein the sample is selected from the group consisting of blood, cells, serum, saliva, body fluid, plasma, urine, prostatic fluid, bronchial lavage Fluid, cerebrospinal fluid, gastric fluid, bile, lymph fluid, abdominal cavity fluid, feces, etc. or a combination thereof.
  • an amplification-cleavage reaction system is provided, the reaction system is used to simultaneously perform polymerase chain reaction (PCR) and nucleic acid cleavage reactions on a nucleic acid sample, thereby obtaining an amplification-cleavage reaction product;
  • PCR polymerase chain reaction
  • the nucleic acid sample contains a first nucleic acid and a second nucleic acid, wherein the first nucleic acid is the target nucleic acid, and the second nucleic acid is a non-target nucleic acid;
  • the nucleic acid cleavage reaction is used to specifically cut the non-target nucleic acid, but not the target nucleic acid;
  • the amplification-cleavage reaction system contains (i) reagents required for PCR reaction and (ii) reagents required for nucleic acid cleavage reaction.
  • the amplification-cleavage reaction system does not contain or contains the nucleic acid sample.
  • the concentration of Mn ions in the amplification-cleavage reaction system is 0.1-1 mM.
  • the concentration of Mg ions in the amplification-cleavage reaction system is 1-3 mM.
  • FIG 1 shows the principle diagram of the technical scheme of the present invention.
  • Figure 2 shows the recognition of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) substrates by gDNA and the shearing mechanism of the PfAgo-gDNA complex.
  • Figure 3 shows the differential shearing of ssDNA, dsDNA and SNV of dsDNA under the PCR working system by the PfAgo-gDNA complex.
  • Figure 3C-PfAgo-gDNA complex under the PCR system preferably under the combination of forward and reverse gDNA, distinguishes shearing of wild-type and mutant dsDNA substrates and the mutant type Enrichment of dsDNA.
  • Figure 4 shows the optimization of the enrichment conditions for the KRAS-G12D low-abundance mutant dsDNA substrate by the PfAgo-gDNA complex, and the optimal working concentration of PfAgo protein at a mutation ratio of 10 nM 1.0%.
  • Figure 5 shows the standard curve of the double TaqMan probe method for the detection of KRAS-G12D low-abundance mutant DNA substrates.
  • Figure 6 shows the high sensitivity detection of KRAS-G12D low-abundance mutant DNA (0.1%, 0.01%) substrate by PfAgo-gDNA complex.
  • Figure 7 shows the highly sensitive detection and optimal enrichment results of the PfAgo-gDNA complex on EGFR-delE746-A750 low-abundance mutant DNA (0.1%, 0.01%) substrates.
  • Figure 8 shows the high sensitivity detection and optimal enrichment results of PfAgo-gDNA complex on KRAS-G12D, PIK3CA-E545K and EGFR-delE746-A750 triple low abundance mutant DNA (0.01%) substrates.
  • PCR pre-amplification pre-process samples from different sources, obtain nucleic acid samples containing low-abundance target genes, and perform PCR pre-amplification to increase the molar concentration of target genes to meet the initial sample volume required for Ago-PCR enrichment.
  • the target products enriched in the above system can be combined with multi-terminal detection equipment and methods, such as q-PCR, NGS, chemiluminescence, high-resolution melting curve method, Sanger sequencing, ddPCR, etc., to quantitatively detect target genes Mutation situation.
  • the invention has the advantages of non-invasiveness, easy operation, fast speed, etc., the sensitivity can reach 0.01%, the DNA amount of the sample can be as low as aM level, and it can better detect low-abundance mutant genes in human liquid biopsy.
  • the technology of the invention It can be widely used in various fields of molecular diagnosis involving nucleic acid detection, such as tumor liquid biopsy, infectious diseases such as major infectious diseases and pathogenic infectious diseases (viruses, pathogens) detection fields. The present invention has been completed on this basis.
  • the core of the present invention is to develop a novel nucleic acid cleavage tool enzyme PfAgo with single-point nucleic acid recognition specificity and high temperature stability, and to couple the PCR reaction to realize the cutting-while-amplification process, and establish "A-STAR( A go- mediated S pecific Tar get detection)" technology, the principle details are as follows: in the high temperature denaturation step of each cycle of PCR, dsDNA is denatured and melted into ssDNA.
  • PfAgo melts a pair of gDNA under the guidance of a specifically designed pair Wild-type gene ssDNA cutting, that is, this process can specifically cut wild-type genes while retaining mutant genes; in the subsequent PCR annealing step, the designed primers are located at least 20 nt upstream and downstream of the SNV site of the target nucleic acid. Selectively combine wild-type genes and mutant genes; in the subsequent PCR extension step, since the wild-type gene has been cut at the mutation site, it cannot be used as a template for extension, while the mutant gene retains its original length and can be used as a template. Amplification.
  • this PfAgo high-temperature specific cleavage and PCR amplification coupling reaction can be performed in each cycle of conventional PCR (20-35 cycles), it can realize cutting-while amplification to efficiently enrich low-abundance mutant genes .
  • the technical advantages are: 1) Differential shearing at high temperature, easy to operate; 2) gDNA sequence matches the target sequence, with high specificity; 3) Can be designed for any target sequence without sequence preference; 4) Single enzyme for multiple nucleic acid targets Realize multiple detection; 5) Can be combined with multiple terminal detection technology.
  • the reaction when the PfAgo-gDNA complex is used for the "PCR while cutting" coupling reaction, the reaction can be carried out under suitable conditions of the corresponding cleaving enzyme and the corresponding amplification enzyme, as long as the conditions are The cleavage and amplification enzymes described above can perform their corresponding functions.
  • the research of the present invention shows that, for the enrichment of mutant dsDNA signal through the coupling reaction, some key factors mainly include the following aspects:
  • the initial template concentration in the enrichment reaction system wild type (wt) and mutant (mutant type, mut) total concentration (nM ⁇ fM)): preferably 0.1-100 nM.
  • the initial PfAgo protein concentration in the enrichment reaction system preferably 20-100 nM;
  • Pre-processing time preferably 3-10 minutes;
  • the initial gDNAs concentration in the enrichment reaction system preferably 200-2000nM;
  • KRAS-G12D wild and mutant fragments were used as substrates, and experiments were conducted on the factors.
  • the parameters can be seen in Table A.
  • the method of the present invention requires only a small amount of test samples, and has high detection sensitivity and accuracy;
  • the rapid detection technology for low-abundance mutant genes of the present invention can be used in the fields of early detection of trace nucleic acid markers of disease, dynamic monitoring of disease driver genes, and prognostic evaluation of certain diseases;
  • the method of the present invention can also be applied to the detection of infectious diseases, such as major infectious diseases and pathogen infectious diseases, so as to achieve proactive management such as prediction and prevention.
  • the method of the present invention can also screen for genetic metabolic diseases related to mutant genes, and achieve proactive management such as prediction and early treatment.
  • the method of the present invention can also be used for screening of obstetrics and gynecology diseases, obstetrics and genetics and metabolic diseases of newborns, so as to achieve effective measures such as prediction and early treatment.
  • the method of the present invention can also expand the detection of susceptibility genes, predict a small amount of disease risk in advance, and take effective preventive measures before the occurrence of the disease to minimize the possibility of disease.
  • Primer design follows the principles: Primer requirements: 1The primer sequence should avoid a series of bases, especially a series of G; 2Tm is generally required to be 50 ⁇ 60°C; 3The ratio of (G+C)% should be controlled within 28% ⁇ 80%; 4 The last 5 bases of the 3'end of the primer cannot have more than 2 (G+C); 5 The closer the downstream primer is to the probe, the better, and the fragments can overlap.
  • the amplified fragments are preferably 75-150bp.
  • the detection probe specifically binds to the target gene, and its binding site is in any region of the target gene.
  • the 5'end of the probe is labeled with a fluorescent reporter (Reporter, R), such as FAM, VIC, etc., and the 3'end is labeled with a quencher (Quencher, Q).
  • Probe design requirements 1The 5′ end of the probe cannot be G; 2The length of the probe should not be less than 13bp; 3Avoid a series of repeated base sequences; 4Tm is 65 ⁇ 70°C, the theoretical annealing temperature difference between the primer and the corresponding probe The best temperature is 5 ⁇ 10°C; 5The SNV site to be detected is best located in the middle of the probe and as close as possible to the 3'end. If there is no suitable probe in this segment of SNV that can achieve the required Tm value, the quenching group BHQ can be introduced into the 3'end.
  • target genes include KRAS-G12D, PIK3CA-E545K, Multiple tumor mutant genes such as EGFR-delE746-A750, NRAS-A59T and their corresponding wild-type genes correspond to the primer pairs of group numbers in Table 7.
  • the present invention provides forward and reverse gDNAs and primers required for amplification of target genes designed for different target genes for SNV mutation or fragment deletion mutation enrichment, as well as specific detection probes, which include four groups of targets similar to those in Table 1. Oligonucleotide sequences for gene enrichment and amplification.
  • the core principle of this method for the enrichment of low-abundance mutant target genes is: on the one hand, the phosphorylation modification of the 5'end of gDNAs significantly improves the affinity of the PfAgo-gDNA complex to nucleic acid substrates. At the same time, this method found that there is a seed region on gDNA at the beginning of its establishment. The specificity of the interaction between the PfAgo-gDNA complex and the substrate is determined by the seed sequence in gDNAs. This method explores the different positions of the gDNAs seed region (the second to the fifteenth nucleotides), and the different nucleotides (bases) can improve the specific target binding of the PfAgo-gDNA complex to the target DNA substrate.
  • the rules, analysis and summary of the design rules of gDNAs seed regions used to identify single nucleotide variations are as follows:
  • the principle of gDNAs design forward and reverse oligonucleotide gDNAs sequence must be absolutely conservative.
  • the gDNAs seed region spans the second to fifteenth nucleotides of gDNA when targeting ss DNA. Its characteristics are similar to the seed regions reported by other Agos, and the specific effects of the 3rd, 6th, 7th, 9th, 10th and 11th nucleotides on the PfAgo-gDNA complex binding to the target ssDNA substrate maximum. Therefore, when gDNA is designed, the key nucleic acids and their positions that affect the substrate specificity of the gDNAs seed region are first optimized to improve the specificity of the PfAgo-gDNA complex to single-stranded DNA substrates.
  • a programming method of introducing several (more than 2) base substitutions (permutations) at positions 2-15 of gDNA can distinguish only a single nucleoside Acid-differentiated ssDNA substrate.
  • the appropriate forward and reverse gDNAs can be selected according to the specific distribution law of PfAgo-gDNA complex to single-stranded DNA substrates, and the PfAgo-gDNA under mixed conditions of double gDNA can be tested.
  • the ability of the complex to distinguish between only a single nucleotide difference substrate. Screen the highly specific PfAgo-gDNA complex against specific nucleic acid substrates-for subsequent enrichment of low-abundance DNA mutations.
  • the specific oligonucleotide gDNA its characteristics also include: 5'end, 3'end are modified with phosphate groups; gDNA length ( ⁇ 15nt), mismatch position, mismatch sites introduced on gDNA and The effect of the number on the specific recognition of nucleic acid substrates by the PfAgo-gDNA complex.
  • the enzyme has a good distinction.
  • the ability of only a single nucleotide as a different substrate can display highly specific shearing of wild-type DNA, that is, it can distinguish wild-type and mutant target genes well.
  • specific oligonucleotide gDNAs designed for different circulating tumor DNA (ctDNA) for SNV mutation or fragment deletion mutation enrichment including KRAS-G12D, PIK3CA-E545K
  • the wild-type genes corresponding to multiple tumor mutant genes such as EGFR-delE746-A750, NRAS-A59T, etc., respectively correspond to different gDNAs pairs in Table 6.
  • the main test is to test whether the PfAgo-gDNA complex still has a good distinguishing and shearing ability for ssDNA, dsDNA and dsDNA under the PCR working system under ordinary PCR reaction buffer and other components.
  • the components and working conditions involved in this example mainly include: 2 ⁇ PCR Taq Master Mix, forward and reverse primers, forward and reverse gDNAs, PfAgo, MnCl 2 , templates (pure wild, pure mutation, and wild and Half mutations), etc., as shown in Table 8.
  • Table 8 takes the 25 ⁇ L system as an example, the PfAgo enrichment reaction system components and preparation sequence.
  • 2 ⁇ PCR Taq Master Mix reaction solution is prepared by 2 ⁇ PCR buffer, dNTPs and hot-start enzyme.
  • 2 ⁇ PCR buffer KCl, (NH 4 ) 2 SO 4 , 3mM MgCl 2 , Tris-HCl, pH 8.3 (25°C).
  • dNTPs include dATP, dGTP, dCTP and dTTP, and the final concentration in the reaction system is 0.4mM.
  • the hot-start enzyme uses Taq DNA polymerase with a concentration of 5U/ ⁇ L, and the final concentration in the reaction system is 0.1-0.5U/ ⁇ L.
  • the 2 ⁇ PCR Taq Master Mix reaction solution was from abm biotechnology company (product number: G013).
  • the primers corresponding to each target gene in Table 6 were used.
  • the forward and reverse gDNAs in Table 5 correspond to each target gene, preferably the gDNAs pair.
  • the concentration of the PfAgo mother solution stored after the pre-purification is 5 ⁇ M. In actual use, it needs to be diluted with the prepared Reaction Buffer under ice bath conditions in advance: first dilute the 5 ⁇ MPfAgo mother solution to 1 ⁇ M, and then dilute it to 0.3 ⁇ M with Reaction Buffer.
  • Reaction Buffer components 15mM Tris-Cl, 250mM NaCl, pH 8.0.
  • the ssDNA and dsDNA in the template are 60nt KRAS-G12D ssDNA wild-type and mutant fragments, and 620bp KRAS-G12D dsDNA wild-type and mutant fragments, respectively.
  • reaction conditions for the shearing of 60nt KRAS-G12D ssDNA wild-type and mutant fragments by PfAgo-gDNA complex 95°C, 15 minutes later, slowly lower the temperature to 10°C and incubate.
  • the PCR reaction program of PfAgo enrichment system includes:
  • PfAgo-gDNA complex distinguishes ssDNA, dsDNA and SNV of dsDNA under the PCR working system.
  • the PfAgo-gDNA complex was tested for distinguishing shearing of wild-type and mutant dsDNA substrates and enrichment of mutant dsDNA under the PCR system.
  • the gDNAs and primers were synthesized by Shanghai Shenggong Biological Engineering Co., Ltd. KRAS gene gDNAs are phosphorylated at the 5'end.
  • This example optimizes the PfAgo-gDNA complex under the PCR system to distinguish between the shearing of wild-type and mutant dsDNA substrates and the enrichment conditions for mutant dsDNA.
  • the components and working conditions involved in this example mainly include: 2 ⁇ PCR Taq Master Mix, forward and reverse primers, forward and reverse gDNAs, PfAgo, MnCl 2 , template (10nM 1.0%mut KRAS-G12D), etc. , As shown in Table 8.
  • the primers corresponding to each target gene in Table 7 are used.
  • the preferred gDNAs pairs corresponding to each target gene in Table 6 are used.
  • the concentration of the PfAgo mother solution stored after the pre-purification is 5 ⁇ M. In actual use, it should be diluted with the prepared Reaction Buffer in an ice bath in advance: first dilute the 5 ⁇ MPfAgo mother solution to 1 ⁇ M, and then dilute it to 0.3 ⁇ M with the Reaction Buffer.
  • Reaction Buffer 1 component 15mM Tris-Cl, 250mM NaCl, pH 8.0.
  • PfAgo's PCR reaction procedures for the enrichment of 10nM 1.0%mut KRAS-G12D mutant genes include:
  • PfAgo-gDNA complex under PCR system for the enrichment conditions of mutant DNA fragments in 10 nM 1.0% mut KRAS-G12D samples, preferably: PfAgo concentration in the range of 20-100 nM; gDNAs concentration in the range of 200 to 1000 nM; PfAgo: The gDNAs concentration ratio is in the range of 1:10 to 1:20; the pretreatment time of the PfAgo-gDNA complex at 94°C is 3 minutes to 5 minutes; the number of cycles of enrichment PCR is preferably 10 to 30 cycles.
  • the PfAgo-gDNA complex can differentiate and shear KRAS-G12D wild-type and mutant dsDNA substrates, thereby achieving the enrichment of mutant dsDNA.
  • the samples after the enrichment reaction were subjected to first-generation sequencing (Sanger sequencing), and the results showed that the mutation point position of KRAS-G12D (gGt/gAt) was 1.0% mut
  • the mutation point A base after KRAS-G12D sample processing There is an obvious raised peak, that is, the low-abundance mutation 1.0%mut KRAS-G12D DNA is obviously enriched.
  • the enrichment factor F1b/F1a is about 78.
  • a low-abundance tumor gene KRAS-G12D mutation gene (0.1% mut, 0.01% mut) detection method According to the experimental steps of the low-abundance mutant DNA detection system described in the present invention, firstly, specific amplification primers, gDNAs and detection probes are designed and screened for the sequence characteristics of the KRAS-G12D gene fragment. See Table 2 sequence for details.
  • KRAS gene gDNAs are equipped with phosphorylation modification at the 5'end; the nucleotide sequence of the G12D mutant probe has a FAM fluorescent label at the 5'end, and the quenching group BHQ1 is modified at the 3'end; the nucleoside of the KRAS wild-type gene probe
  • the 5'end of the acid sequence is provided with a VIC fluorescent label, and the 3'end is modified with the quenching group BHQ1.
  • This method uses the horizontal reference standards standard products from HORIZON DISCOVERY to verify and analyze the KRAS-G12D Expected Allelic Frequency (AF%) mutation allele frequencies (AF%) of the standard products are 5%mut and 1% respectively mut, 0.1% mut, 0.01% mut and 100% wt.
  • the 0.1% mut and 0.01% mut standards were used to verify the sensitivity and specificity of the low-abundance mutant DNA enrichment and detection method described in this invention.
  • the pre-amplification system is prepared as follows:
  • the volume of the reaction system can be 25.0 ⁇ L, and the components in the 50.0 ⁇ L reaction system can be halved during preparation.
  • PCR program 94°C for 3 minutes; 10-30 cycles (94°C for 10s, 55°C for 30s, 72°C for 20s), 72°C for 1 minute.
  • the final product can be quantified by TaqMan-qPCR to determine whether the target concentration required in the next enrichment system is met.
  • PfAgo-gDNA complex enriches the low-abundance KRAS-G12D mutant genes in the pre-amplified product.
  • the optimized PfAgo-gDNA complex enrichment conditions for KRAS-G12D mutant genes are preferably used: PfAgo concentration is 20-100 nM, gDNAs concentration is 200-2000 nM, and PfAgo:gDNAs concentration ratio is 1. : 5 ⁇ 1:20, 94°C The pretreatment time of PfAgo-gDNA complex is 1 ⁇ 5 minutes, and the number of cycles of enrichment PCR is 10 ⁇ 30 cycles.
  • the components and working conditions involved in this embodiment mainly include: 2X PCR Precision TM MasterMix, forward and reverse primers, forward and reverse gDNAs, PfAgo, MnCl 2 , Standard Target (0.1% mut, 0.01% mut) Etc., as shown in Table 8.
  • the primers corresponding to each target gene in Table 7 are used.
  • the preferred gDNAs pairs corresponding to each target gene in Table 6 are used.
  • the concentration of the PfAgo mother solution stored after the pre-purification is 5 ⁇ M. In actual use, it needs to be diluted with the prepared Reaction Buffer in an ice bath in advance: first dilute the 5 ⁇ M PfAgo mother solution to 1 ⁇ M, and then dilute it to 0.3 ⁇ M with the Reaction Buffer.
  • Reaction Buffer components 15mM Tris-Cl, 250mM NaCl, pH 8.0.
  • PfAgo enrichment PCR reaction procedures for the pre-amplified products of standard 0.1%mut, 0.01%mut KRAS-G12D mutant genes include:
  • the detection of the enriched product involved in the present invention can adopt various methods, such as Sanger sequencing qualitative analysis, second-generation sequencing quantitative analysis, TaqMan fluorescent quantitative PCR method quantitative analysis, fluorescence method real-time detection, high-resolution melting curve method quantitative analysis, etc. .
  • Sanger sequencing qualitative analysis and TaqMan fluorescent quantitative PCR method are used to analyze the enriched products.
  • the inventors Before using the TaqMan fluorescent quantitative PCR method for quantitative analysis, the inventors first designed and measured the standard curve of the double TaqMan probe method for detecting the KRAS-G12D low-abundance mutant DNA (0.01%) substrate. The standard curve is shown in Figure 5.
  • the KRAS-G12D gene standard curve determination method of this embodiment includes:
  • the conditions of the TaqMan-qPCR detection system are as follows: Take 20 ⁇ L system as an example,
  • the TaqMan-qPCR program is as follows:
  • the wild-type double-probe the mutant mother solution was prepared in a ratio of 1:1, and the wild signal (Figure 5A) was detected by a standard sample of 10.0pM gradient dilution. The repeatability was good, and three replicates were set for each concentration.
  • the rightmost curve in Figure 5A is the signal of the wild probe of ddH 2 O.
  • the signal of the wild probe combined with ddH 2 O can be obtained.
  • the signal threshold of the wild-type probe should be 9000.
  • the CT ddH2O is about 37. -39, and the lowest concentration of aM-level CT of the sample is around 38, as shown in the standard curve part of Figure 5C.
  • FIG. 5B rightmost curve of ddH 2 O mutated probe signal, the combined signal probe wild ddH 2 O to obtain the wild-type probe signal threshold to be more appropriate action in 9000, when about 38 CT ddH2O -40, and the lowest concentration of aM-level CT of the sample is around 37, as shown in the standard curve part of Figure 5D.
  • the KRAS-G12D enriched sample of this example is subjected to quantitative analysis by TaqMan fluorescent quantitative PCR method.
  • the components of the enriched sample are prepared in the following order before determination:
  • the conditions of the TaqMan-qPCR detection system are as follows: Take 20 ⁇ L system as an example,
  • the TaqMan-qPCR program is as follows:
  • the low-abundance tumor gene EGFR delE746-A750 fragment deletion mutant gene (0.1% mut, 0.01% mut) was enriched and detected.
  • gDNAs, primers and probes were all synthesized by Shanghai Shenggong Biological Engineering Co., Ltd.
  • EGFR gene gDNAs have phosphorylation modification at the 5'end;
  • the nucleotide sequence of delE746-A750 mutant probe has a VIC fluorescent label at the 5'end, and the quenching group BHQ1 is modified at the 3'end;
  • EGFR wild-type gene probe The 5'end of the nucleotide sequence is provided with FAM fluorescent label, and the 3'end is modified with the quenching group BHQ2.
  • This method uses the horizon reference standards standard products from HORIZON DISCOVERY company to verify and analyze.
  • the EGFR delE746-A750 Expected Allelic Frequency (AF%) mutation allele frequencies (AF%) of the standard products are 5%mut and 1% respectively. mut, 0.1% mut, 0.01% mut and 100% wt. The 0.1% mut and 0.01% mut standards were used to verify the sensitivity and specificity of the low-abundance mutant DNA enrichment and detection method described in this invention.
  • the EGFR delE746-A750 gene pre-amplification reaction system of this embodiment includes:
  • the pre-amplification system is prepared as follows:
  • the volume of the reaction system can be 25.0 ⁇ L, and the components in the 50.0 ⁇ L reaction system can be halved during preparation.
  • PCR conditions of the EGFR delE746-A750 gene pre-amplification reaction in this embodiment are as follows:
  • PCR program 94°C for 3 minutes; 10-30 cycles (94°C for 10s, 55°C for 30s, 72°C for 20s), 72°C for 1 minute.
  • the final product can be quantified by TaqMan-qPCR to determine whether the target concentration required in the next enrichment system is met.
  • PfAgo-gDNA complex enriches low-abundance EGFR delE746-A750 mutant genes in the pre-amplified product.
  • the optimized PfAgo-gDNA complex enrichment conditions for EGFR delE746-A750 mutant genes are preferably used: PfAgo concentration is 20-80 nM, gDNAs concentration is 800 nM, and the ratio of PfAgo:gDNAs concentration is 1: 5 ⁇ 1:20, 94°C PfAgo-gDNA complex pretreatment time is 3 minutes, and the number of cycles of enrichment PCR is 10-30 cycles.
  • the components and working conditions involved in this embodiment mainly include: 2 ⁇ PCR Taq Master Mix, forward and reverse primers, forward and reverse gDNAs, PfAgo, MnCl 2 , Standard Target (0.1% mut, 0.01% mut) Etc., as shown in Table 8.
  • the concentration of the PfAgo mother solution stored after the pre-purification is 5 ⁇ M. In actual use, it needs to be diluted with the prepared Reaction Buffer in advance under ice bath conditions: first dilute the 5 ⁇ MPfAgo mother solution to 1 ⁇ M, and then use the Reaction Buffer to dilute to 0.3 ⁇ M.
  • Reaction Buffer components 15mM Tris-Cl, 250mM NaCl, pH 8.0.
  • PfAgo enrichment PCR reaction procedures for the pre-amplified products of standard 0.1% MUT, 0.01% MUT EGFR delE746-A750 mutant gene include:
  • multiple (triple) low-abundance (0.01% mut) tumor genes KRAS-G12D, PIK3CA-E545K and EGFR-delE746-A750 mutant genes (0.1% mut, 0.01% mut) were enriched and detected .
  • gDNAs, primers and probes were all synthesized by Shanghai Shenggong Biological Engineering Co., Ltd.
  • the 5'end of gDNAs is equipped with phosphorylation modification; the 5'end of the nucleotide sequence of the probe is equipped with different fluorescent labels, and the 3'end is modified with the quenching group BHQ.
  • This method uses the horizon reference standards standard products from HORIZON DISCOVERY company to verify and analyze the standard products.
  • AF% mutant allele frequencies 0.01%. mut and 100% wt.
  • the pre-amplification system is prepared as follows:
  • the volume of the reaction system can be 25.0 ⁇ L, and the components in the 50.0 ⁇ L reaction system can be halved during preparation.
  • PCR program 94°C for 3 minutes; 24-30 cycles (94°C for 30s, 55°C for 30s, 72°C for 20s); 72°C for 1 minute.
  • the final product can be quantified by TaqMan-qPCR to determine whether it meets the required target concentration in the next enrichment system.
  • the optimized PfAgo-gDNA complex enrichment conditions for KRAS-G12D, PIK3CA-E545K, and EGFR-delE746-A750 mutant genes are preferably used: PfAgo concentration is 10-800 nM, and gDNAs concentration is 100- 4000nM; PfAgo:gDNAs concentration ratio is 1:5 ⁇ 1:20, 94°C The pretreatment time of PfAgo-gDNA complex is 3 minutes, and the cycle number of enrichment PCR is 10-30 cycles.
  • the components and working conditions involved in this embodiment mainly include: 2X PCR Precision TM MasterMix, forward and reverse primers, forward and reverse gDNAs, PfAgo, MnCl 2 , Standard Target (0.01% mut), etc., as shown in Table 8. Shown.
  • the primers corresponding to each target gene in Table 7 are used.
  • the preferred gDNAs pairs corresponding to each target gene in Table 6 are used.
  • the concentration of the PfAgo mother solution stored after the pre-purification is 5 ⁇ M. In actual use, it needs to be diluted with the prepared Reaction Buffer in an ice bath in advance: first dilute 5 ⁇ M PfAgo mother solution to 1 ⁇ M, and then use Reaction Buffer to dilute to 0.3 ⁇ M.
  • Reaction Buffer components 15mM Tris-Cl, 250mM NaCl, pH 8.0.
  • the PCR reaction procedure for enrichment of the standard product 0.01%mut KRAS-G12D mutant gene pre-amplification product by PfAgo includes:
  • the detection of the enriched product involved in the present invention can adopt various methods, such as Sanger sequencing qualitative analysis, second-generation sequencing quantitative analysis, TaqMan fluorescent quantitative PCR method quantitative analysis, fluorescence method real-time detection, high-resolution melting curve method quantitative analysis, etc. .
  • the TaqMan fluorescent quantitative PCR method was used to analyze the enriched products.
  • the KRAS-G12D, PIK3CA-E545K and EGFR-delE746-A750 enriched samples of this example were subjected to TaqMan fluorescent quantitative PCR method for quantitative analysis.
  • the components of the enriched sample are prepared in the following order before determination:
  • the conditions of the TaqMan-qPCR detection system are as follows: Take the 20 ⁇ L system as an example,
  • the TaqMan-qPCR program is as follows:
  • the percentage of mutants KRAS-G12D, EGFR-delE746-A750 and PIK3CA-E545K increased to 79% after processing 0.01% mut at the mutation positions of KRAS-G12D, PIK3CA-E545K and EGFR-delE746-A750. , 79%, 41%. That is, the triple low-abundance mutation 0.01% mut tumor gene was also significantly enriched, and the enrichment factor F1b/F1a was about 7900, 7900 and 4100, respectively.
  • F Forward primer
  • R Reverse primer
  • P TaqMan-MGB probe
  • GF Guide Forward DNA
  • GR Guide Reverse DNA
  • F Forward primer
  • R Reverse primer
  • P TaqMan-MGB probe
  • GF Guide Forward DNA
  • GR Guide Reverse DNA
  • F Forward primer
  • R Reverse primer
  • P TaqMan-MGB probe
  • GF Guide Forward DNA
  • GR Guide Reverse DNA
  • F Forward primer
  • R Reverse primer
  • P TaqMan-MGB probe
  • GF Guide Forward DNA
  • GR Guide Reverse DNA

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Abstract

一种基于核酸酶偶联PCR原理富集低丰度单核苷酸突变型基因的检测体系。一种提高目标核酸的相对丰度的方法，包括：(a)提供一核酸样本，含有目标核酸和非目标核酸，所述目标核酸在核酸样本中的丰度为F1a；(b)以核酸样本中的核酸为模板，在扩增-切割反应体系中进行PCR和核酸切割反应，从而获得扩增-切割反应产物；所述目标核酸在扩增-切割反应产物中的丰度为F1b，F1b/F1a的比值≥10。

Description

基于核酸酶偶联PCR原理富集低丰度DNA突变的检测技术体系及应用 技术领域
本发明属于生物技术领域，具体涉及一种基于核酸酶偶联PCR原理富集低丰度单核苷酸突变型基因(Single nucleotide variant，SNV)的检测体系。
背景技术
近年来“液体活检”的概念正在兴起，其基本思想为运用血液等体液样本替代肿瘤组织样本行病理学、分子生物学的检测，通过检测患者体液样本(主要是血液)中的肿瘤循环DNA来获取肿瘤基因突变信息已经成为一种趋势。与目前标准的组织活检相比，革命性的液体活检具有下列不可替代的优势：创伤小、可重复性、均化异质性、实时判断疗效，并随肿瘤的发展而动态调整治疗决策。因此，2015年MIT Technology Review发布的年度十大突破技术(Breakthrough Technologies2015)，ASCO年度进展(Clinical cancer advance 2015)中对未来十年的期许，液体活检均榜上有名。通过检测ctDNA以追踪整个病程中肿瘤的特异性基因改变，对肿瘤筛查、诊断、疗效监测及预后判断等具有重要价值，同时可从中探索肿瘤转移复发及耐药的分子机制，识别新的靶向治疗位点等，因此ctDNA的检测已经成为肿瘤液体活检应用的三大热门方向之一。
在血液中存在着游离的小片段DNA(cell-free DNA,cfDNA)，它们来自死亡的细胞。通常死亡的细胞会被清除掉，因此cfDNA的含量是非常低的，通常一个健康人的1mL血浆中含25ng cfDNA。而癌症患者的cfDNA的含量高出正常几倍，其中一部分是ctDNA(circulating tumor DNA)。ctDNA的相对含量与肿瘤的负荷和对治疗的反应是相关的，可用于鉴定驱动基因、指导临床治疗、监测临床治疗效果及癌症复发、揭示治疗抗性以及检测疾病进展。在有些方面ctDNA方法的灵敏度甚至高传统的手段。例如，与传统的影像学检测相比，追踪早期乳腺癌患者术后血液中的肿瘤DNA，可以提前7.9个月发现乳腺癌复发。在肺癌、肠癌中检测cfDNA的KRAS突变对肺癌也有着重要的诊断价值。ctDNA在癌症早期就可以被检测到。因为cfDNA容易收集，在肺癌中已显示与组织中的变异高度一致，因此ctDNA的液体活检越来越受到关注。
循环肿瘤DNA虽然是一种很好的肿瘤组织替代样本，但是，由于循环肿瘤DNA含量稀少，检测循环肿瘤DNA需要极灵敏的技术。BEAMing扩增法 的应用使得DNA检测技术灵敏度大大提高。2007年，该技术的开发者美国约翰霍普金斯大学的Bert Vogelstein和Kenneth Kinzler对18名结直肠癌患者的循环肿瘤DNA进行了跟踪。研究显示，术后仍能检测到循环肿瘤DNA的患者，基本上都出现了复发，术后未检测到循环肿瘤DNA的患者，结直肠癌无复发，显示了循环肿瘤DNA良好地临床应用前景。BEAMing技术的灵敏度高，可以达到0.1％～0.01％，是一种比较理想的循环肿瘤DNA检测技术。但是由于其操作复杂，仪器昂贵，不适用于大规模临床推广。
目前稀有突变检测方法中，主要有作为“金标准”的基因测序。但是测序灵敏度有限，在大量野生型基因的背景下，测序仅能检测到含有20％的突变，会导致假阴性结果，而且耗时较长。相较于测序，变性高效液相色谱的灵敏度有所提高，但是需要PCR后处理，容易造成实验室污染，容易导致假阳性结果，特异性也有所限制，并且操作步骤繁杂，周期长。基于核酸杂交原理的检测方法，比如TaqMan探针，其选择性检测水平与测序法相当。扩增阻碍突变系统(ARMS)是常用的稀有突变检测方法，基于引物3'末端碱基对不同错配碱基的分辨能力特异性选择扩增突变型模板，但由于分辨能力有限导致选择性一般且不同类型的突变差异性较大。2011年Life technology公司开发出一种高选择性突变检测技术—cast PCR技术，该技术基于ARMS技术，采用MGB探针的高特异性进一步提高了反应的选择性。但是MGB探针合成难度较大，费用较高，不利于广泛应用。数字PCR是近年来出现的另一种高灵敏度检测的技术，这种技术最高也能达到0.01％的灵敏度，但是这种技术极易产生假阳性的结果。同样，高昂的设备和试剂价格、极高的实验操作要求也同样限制了其大规模推广。
因此，本领域迫切需要开发一种高特异性、高灵敏度低丰度突变DNA的富集及检测方法。
发明内容
本发明的目的就是提供一种高特异性、高灵敏度低丰度突变DNA的富集及检测方法。
在本发明的第一方面，提供了一种提高目标核酸的相对丰度的方法，包括步骤：
(a)提供一核酸样本，所述的核酸样本含有第一核酸和第二核酸，其中，所述 的第一核酸为所述目标核酸，而所述的第二核酸为非目标核酸，
并且，所述目标核酸在所述的核酸样本中的丰度为F1a；
(b)对所述核酸样本中的核酸为模板，在扩增-切割反应体系中进行聚合酶链反应(PCR)和核酸切割反应，从而获得扩增-切割反应产物；
其中，所述的核酸切割反应用于特异性切割非目标核酸，但不切割所述目的核酸；
并且，所述的扩增-切割反应体系含有(i)进行PCR反应所需的试剂和(ii)进行核酸切割反应所需的试剂；
其中，所述目标核酸在所述的扩增-切割反应产物中的丰度为F1b，
其中，F1b/F1a的比值≥10。
在另一优选例中，所述的目标核酸和非目标核酸仅相差一个碱基。
在另一优选例中，当1％≤F1a≤10％时，F1b/F1a的比值≥10，当0.1％≤F1a≤0.5％时，F1b/F1a的比值≥100，当F1a≤0.1％时，F1b/F1a的比值≥200。
在另一优选例中，所述的核酸样本包括直接加热裂解的核酸样本、直接裂解酶蛋白酶处理的核酸样本、经过抽提的核酸样本、经PCR预扩增的核酸样本或任意含核酸的样品。
在另一优选例中，所述的经PCR预扩增的核酸样本是经1-30个，较佳地10-20个，更佳地15-30循环的PCR扩增产物。
在另一优选例中，所述的目标核酸为含突变的核苷酸序列。
在另一优选例中，所述的突变选自下组：核苷酸的插入、缺失、取代、或其组合。
在另一优选例中，所述的突变包括SNV。
在另一优选例中，所述的非目标核酸(或第二核酸)为野生型核苷酸序列、高丰度的核苷酸序列、或其组合。
在另一优选例中，所述的非目标核酸在所述的核酸样本中的丰度为F2a。
在另一优选例中，F1a+F2a＝100％。
在另一优选例中，所述的F2a/F1a的比值≥20，较佳地≥50，更佳地≥100，最佳地≥1000或≥5000。
在另一优选例中，所述的非目标核酸在所述的扩增-切割反应产物中的丰度为F2b。
在另一优选例中，F1b+F2b＝100％。
在另一优选例中，所述的F1b/F2b≥0.5，较佳地≥1，更佳地≥2，最佳地≥3或≥5。
在另一优选例中，所述的F1b/F1a的比值≥200，较佳地≥500，更佳地≥1000，最佳地≥2000或≥5000或更高。
在另一优选例中，F1a≤0.5％，较佳地≤0.2％，更佳地≤0.1％，最佳地≤0.01％。
在另一优选例中，F1b≥10％，较佳地≥30％，更佳地≥50％，最佳地≤70％。
在另一优选例中，所述的“进行PCR反应所需的试剂”包括：DNA聚合酶。
在另一优选例中，所述的“进行PCR反应所需的试剂”还包括：dNTP、，1-5Mm Mg 2+,PCR缓冲液。
在另一优选例中，所述的“进行核酸切割反应所需的试剂”包括：核酸切割工具酶和引导DNA(gDNA)。
在另一优选例中，核酸切割工具酶为高温稳定的双链DNA切割工具酶。
在另一优选例中，所述核酸切割工具酶选自但不限于以下来自嗜热微生物(≥60℃)的Argonaute蛋白及其突变体：PfAgo(Pyrococcus furiosus Ago)、MfAgo(Methanocaldococcus fervens Ago)、TcAgo(Thermogladius calderae Ago)、TfAgo(Thermus filiformis Ago)、AaAgo(Aquifex aeolicus Ago)等。
在另一优选例中，所述核酸切割工具酶为PfAgo。
在另一优选例中，所述的gDNA与所述核酸切割工具酶形成复合物，并且所述复合物特异性切割非目标核酸。
在另一优选例中，所述的gDNA与目标核酸(即第一核酸)的靶定区域的核酸序列形成第一互补结合区；并且所述的gDNA还与非目标核酸(即第二核酸)的靶定区域的核酸序列形成第二互补结合区。
在另一优选例中，在第一互补结合区中含有至少2个不匹配的碱基对。
在另一优选例中，在第二互补结合区中含有0或1个不匹配的碱基对。
在另一优选例中，在第二互补结合区中含有1个不匹配的碱基对。
在另一优选例中，在第一互补结合区中含有至少2个不匹配的碱基对，从而导致所述复合物不切割所述目标核酸；而在第二互补结合区中含有1个不匹配的碱基对，从而导致所述复合物切割所述非目标核酸。
在另一优选例中，目标核酸(即第一核酸)的靶定区域与非目标核酸(即第二核 酸)的靶定区域是相对应的。
在另一优选例中，所述的gDNA的长度为15-30nt。
在另一优选例中，在所述gDNA的第7位和/或第10位为错配碱基，所述错配碱基在第一互补结合区和第二互补结合区均用于形成不匹配的碱基对。
在另一优选例中，所述gDNA的第2-8位为“seed region”区，第10、11为PfAgo剪切关键位点。
在另一优选例中，所述核酸切割工具酶和gDNAs的比例(摩尔比)为1：2～1：20。
在另一优选例中，在扩增-切割反应体系中，所述的核酸切割工具酶为30nM,DNA聚合酶为耐高温聚合酶，较佳的为Taq DNA聚合酶、LA Taq DNA聚合酶、Tth DNA聚合酶、Pfu DNA聚合酶、Phusion DNA聚合酶、KOD DNA聚合酶等，更佳的2X PCR Precision TM Master Mix。
在另一优选例中，在扩增-切割反应体系中，作为模板的核酸的数量为0.1-100nM。
在另一优选例中，所述方法还包括：
(c)对所述扩增-切割反应产物进行检测，从而测定所述目标核酸的存在与否和/或数量。
在另一优选例中，步骤(c)中的检测包括定量检测、定性检测、或其组合。
在另一优选例中，所述的定量检测选自下组：q-PCR、ddPCR、化学发光法、高分辨率熔解曲线法、桑格测序、NGS等。
在另一优选例中，所述的第一核酸包括n种不同的核酸序列，其中n为≥1的正整数。
在另一优选例中，n为1、2、3、4、5、6、7、8、9、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、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100或更大。
在另一优选例中，n为2-1000，较佳地3-100，更佳地3-50。
在另一优选例中，在步骤(b)中，进行C个“高温变性-延伸”循环，其中C为≥ 5。
在另一优选例中，所述的高温变性的温度对应于PCR反应的DNA双链的解链温度和所述核酸切割工具酶的切割温度。
在另一优选例中，所述的高温变性的温度为85-95℃。
在另一优选例中，所述的C为5-35。
在另一优选例中，所述方法是非诊断性和非治疗性的。
在另一优选例中，所述的核酸样本包括来自试样的核酸，其中所述试样选自下组：血液、细胞、血清、唾液、体液、血浆、尿液、前列腺液、支气管灌洗液、脑脊液、胃液、胆汁、淋巴液、腹腔液及粪便等或其组合。
在本发明的第二方面，提供了一种扩增-切割反应体系，所述反应体系用于对一核酸样本同时进行聚合酶链反应(PCR)和核酸切割反应，从而获得扩增-切割反应产物；
其中，所述的核酸样本含有第一核酸和第二核酸，其中，所述的第一核酸为所述目标核酸，而所述的第二核酸为非目标核酸；
所述的核酸切割反应用于特异性切割非目标核酸，但不切割所述目的核酸；
所述的扩增-切割反应体系含有(i)进行PCR反应所需的试剂和(ii)进行核酸切割反应所需的试剂。
在另一优选例中，所述的扩增-切割反应体系不含有或含有所述的核酸样本。
在另一优选例中，所述扩增-切割反应体系中，Mn离子的浓度为0.1-1mM。
在另一优选例中，所述扩增-切割反应体系中，Mg离子的浓度为1-3mM。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明技术方案的原理图。
图2显示了gDNA对单链DNA(ssDNA)底物和双链DNA(dsDNA)底物的识别和PfAgo-gDNA复合物的剪切机制。
图3显示了PfAgo-gDNA复合物对ssDNA、dsDNA以及在PCR工作体系下对dsDNA的SNV的区分剪切。图3A-不同正、反向gDNA组合下PfAgo-gDNA复合物对野生型和突变型ssDNA底物的区分剪切；图3B-在优选地正、反向gDNA组合下PfAgo-gDNA复合物对野生型和突变型dsDNA底物的区分剪切；图3C-优选地正、反向gDNA组合下PfAgo-gDNA复合物在PCR体系下对野生型和突变型dsDNA底物的区分剪切及对突变型dsDNA的富集作用。
图4显示了PfAgo-gDNA复合物对KRAS-G12D低丰度突变型dsDNA底物富集条件优化，10nM 1.0％突变比例下最适PfAgo蛋白工作浓度。
图5显示了双TaqMan探针法检测KRAS-G12D低丰度突变型DNA底物的标准曲线。
图6显示了PfAgo-gDNA复合物对KRAS-G12D低丰度突变型DNA(0.1％、0.01％)底物高灵敏度检测。
图7显示了PfAgo-gDNA复合物对EGFR-delE746-A750低丰度突变型DNA(0.1％、0.01％)底物高灵敏度检测及优选富集结果。
图8显示了PfAgo-gDNA复合物对KRAS-G12D、PIK3CA-E545K和EGFR-delE746-A750三重低丰度突变型DNA(0.01％)底物高灵敏度检测及优选富集结果。
具体实施方式
本发明人经过广泛而深入的研究，首次开发了一种灵敏度高、特异性好、通量高的低丰度突变DNA的富集及检测方法。本发明技术体系整体分为PCR预扩增、Ago-PCR富集、目标基因定量检测三步。首先，对不同来源的样品进行前处理，获取含低丰度目标基因的核酸样品后进行PCR预扩增提高目标基因的摩尔浓度以满足Ago-PCR富集所需的样本初始量。其次，对低丰度突变DNA进行特异性富集和扩增，即将所述正、反向gDNAs、PfAgo蛋白、PCR扩增体系与预扩增PCR产物按比例配制低丰度突变DNA富集体系，富集体系在特定条件进行边扩增边富集反应。PfAgo在gDNAs引导下对野生型DNA进行特异性剪切，从而抑制其扩增，以此达到富集低丰度突变型DNA的目的。最后，上述体系中富集的目标产物可联合多终端检测设备和方法，如q-PCR、NGS、化学发光法、高分辨率熔解曲线法、桑格测序、ddPCR等，以此定量检测目标基因突变情况。本发明具有非侵入性、易操作、快速等优势，灵敏度可以达到 0.01％，样本的DNA量可以低至aM级，能更好地进行人液态活检中低丰度突变基因的检测，本发明技术可广泛应用于涉及核酸检测的分子诊断各个领域，如肿瘤液态活检，感染性疾病如重大传染性和病原体感染性疾病(病毒、病原菌)检测领域等领域。在此基础上完成了本发明。
“A-STAR”检测技术
本发明的核心在于开发了具有单点核酸识别特异性的高温稳定性的新型核酸切割工具酶PfAgo，并偶联PCR反应实现边切割-边扩增的过程，建立“A-STAR( Ago-mediated  Specific  Target detection)”技术，原理细节如下：在PCR每一个循环的高温变性步骤，dsDNA变性解链为ssDNA，在此温度下PfAgo在特异设计一对gDNA指引下分别对一对解链野生型基因ssDNA切割，即此过程可专一性剪切野生型基因，而保留突变型基因；在随后的PCR退火步骤，设计的引物位于目标核酸SNV位点的上、下游至少20nt，因此非选择性结合野生型基因及突变基因；在随后的的PCR延伸步骤，由于野生型基因在突变位点处已被剪切，无法作为模板进行延伸，而突变型基因保留原长因此可以作为模板进行扩增。由于此PfAgo高温特异性切割与PCR扩增偶联的反应可在常规PCR(20-35个循环)的每个循环中执行，实现边切割-边扩增从而高效富集低丰度突变型基因。技术优势在于：1)高温区分剪切，操作方便；2)gDNA序列配对靶标序列，具有高特异性；3)可针对任何靶标序列设计，无序列偏好性；4)单个酶对多个核酸靶标实现多重检测；5)可结合多终端检测技术。
“边PCR边剪切”的偶联反应
在本发明中，在采用PfAgo-gDNA复合物进行“边PCR边剪切”的偶联反应时，可以采用相应切割酶和相应扩增酶的合适条件下进行所述反应，只要该条件下所述的切割酶和扩增酶能够发挥其相应功能。
本发明的研究表明，对于通过所述偶联反应来富集突变型dsDNA信号，一些关键因素主要包括以下几个方面：
①富集反应体系中初始模板浓度：野生型(wild type,wt)和突变型(mutant type,mut)总浓度(nM～fM))：优选为0.1-100nM。
②富集反应体系中初始PfAgo蛋白浓度：优选为20-100nM；
③94℃ PfAgo-gDNA复合物前处理时间(Pre-processing time(分钟))：优选 为3-10分钟；
④富集反应体系中初始gDNAs浓度：优选为200-2000nM；
⑤PfAgo蛋白与gDNAs间摩尔浓度比例：优选为1：5～1：20；
⑥富集PCR的循环数循环：优选为10-30；
在本发明的一个实施例中，以KRAS-G12D野生和突变片段为底物，对所述因素进行了实验，参数可见表A。
表A
表PfAgo gDNAs-PCR偶联反应关键影响因素及参数
Figure PCTCN2019115151-appb-000001
本发明的主要优点包括：
1)本发明方法只需少量检测样本，具有很高的检测灵敏度和准确率；
2)本发明所述的低丰度突变基因的快速检测技术可以面向疾病早期痕量核酸标志物检测、疾病驱动基因动态监测以及某些疾病的预后评估等领域；
3)本发明方法还可向感染性疾病，如重大传染性和病原体感染性疾病检测领域拓展应用，做到预测、预防等积极主动管理。
4)本发明方法还可向突变基因相关的遗传代谢病筛查，做到预测及提早治疗等积极主动管理。
5)本发明方法还可向妇产类疾病筛查，产检，新生儿遗传代谢病筛查，做到预测及提早治疗等有效措施。
6)本发明方法还可拓展易感基因检测，提早预测微量的病变风险，在疾病发生之前采取有效的预防措施，最大限度降低患病可能。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring  Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1.
引物及检测探针设计
引物设计遵循原则：引物要求:①引物序列避免一连串的碱基,特别是一连串的G；②Tm一般要求在50～60℃；③(G+C)％的比例控制在28％～80％；④引物3′端的最后5个碱基不可以有超过2个(G+C)；⑤下游引物位置越靠近探针越好，且片段能重叠，扩增片段在75～150bp为佳。
检测探针设计遵循的原则：检测探针与目标基因特异性地结合，其结合位点在目标基因的任意区域。探针的5′端标记有荧光报告基团(Reporter,R)，如FAM、VIC等，3′端标记有淬灭基团(Quencher,Q)。探针设计要求：①探针5′末端不能是G；②探针长度不应小于13bp；③避免一连串的重复碱基序列；④Tm为65～70℃，引物与对应的探针理论退火温度差异最好在5～10℃；⑤要求待检测的SNV位点最好位于探针中间，并尽量靠近3′端。如果在SNV的这段序列中没有合适的探针能达到要求的Tm值，3′端可以引入淬灭基团BHQ等。
在具体实施方案中，优选地，针对不同循环肿瘤DNA(Circulating tumor DNA,ctDNA)设计的用于SNV突变或片段缺失突变扩增的特异性引物对，目标基因包括KRAS-G12D、PIK3CA-E545K、EGFR-delE746-A750、NRAS-A59T等多个肿瘤突变基因及其对应的野生型基因，其分别对应表7组号的引物对。
本发明提供针对不同目标基因设计的用于SNV突变或片段缺失突变富集的正、反向gDNAs及目标基因扩增所需引物，以及特异性检测探针，其包括类似表1中4组目标基因富集及扩增的寡核苷酸序列。
实施例2.
寡核苷酸gDNAs设计及优化
本方法对低丰度突变型目标基因的富集的核心原理在于：一方面通过对gDNAs 5'末端的磷酸化修饰显著提高了PfAgo-gDNA复合物对核酸底物的亲和力。同时本方法在建立之初发现在gDNA上存在一个种子区域，PfAgo-gDNA复合物与底物相互作用的特异性是由gDNAs中的种子序列决定的。本方法通过 探索gDNAs种子区域不同位置(第二至第十五位的核苷酸)，不同核苷酸(碱基)对提高PfAgo-gDNA复合物特异性靶向结合靶DNA底物的作用及其规律，分析归纳了用于识别单核苷酸变异的gDNAs种子区域的设计规律，具体如下：
gDNAs设计遵循的原则：正、反向寡核苷酸gDNAs序列要绝对保守。所述gDNAs种子区域在靶向ss DNA时跨越gDNA的第二至第十五位的核苷酸。其特点与其他Agos报道的种子区域相似，第3、第6、第7、第9位、第10位、第11位的核苷酸对PfAgo-gDNA复合物结合靶ssDNA底物的特异性影响最大。因此，在gDNA设计时首先对gDNAs种子区域影响底物特异性的关键核酸及其位置进行优化，以提高PfAgo-gDNA复合物对单链DNA底物的特异性。根据目标基因野生型与突变型核苷酸的碱基差异，通过在gDNA的第2-15位上引入若干(2个以上)碱基替换(permutations)这种编程方式以区分仅有单个核苷酸差异的ssDNA底物。
其次，在双链DNA底物的特异性识别方面可根据PfAgo-gDNA复合物对单链DNA底物的特异性分布规律，选择合适的正、反向gDNAs，检验双gDNA混合条件下PfAgo-gDNA复合物区分仅有单个核苷酸差异底物的能力。针对特定核酸底物筛选具有高度特异性的PfAgo-gDNA复合物-用于后续低丰度DNA突变的富集。
所述特异性的寡核苷酸gDNA，其特征还包括：5'端，3'端都带有磷酸基团修饰；gDNA长度(≥15nt)，mismatch位置，gDNA上引入的错配位点及数目对PfAgo-gDNA复合物特异性识别核酸底物的影响。
优选地，当寡核苷酸gDNA上的5'端，3'端均为磷酸化修饰，同时gDNA引入的额外错配位点位于gDNA的第7、10、11时，该酶具有很好区分仅有单个核苷酸差异底物的能力，展示对野生型DNA的高特异性剪切，即可以很好的区分野生型和突变型目的基因。
在具体实施方案中，优选地，针对不同循环肿瘤DNA(Circulating tumor DNA,ctDNA)设计的用于SNV突变或片段缺失突变富集的特异性寡核苷酸gDNAs，包括KRAS-G12D、PIK3CA-E545K、EGFR-delE746-A750、NRAS-A59T等多个肿瘤突变基因对应的野生型基因，其分别对应表6中不同的gDNAs对。
实施例3.
PfAgo-gDNA复合物对ssDNA、dsDNA的区分剪切
在本实施例，主要测试PfAgo-gDNA复合物在普通PCR反应缓冲液以及其它成分下是否依然对ssDNA、dsDNA以及在PCR工作体系下对dsDNA存在良好的区分剪切能力。
3.1方法
本实施例中涉及的各组分及工作条件主要包括：2×PCR Taq Master Mix，正、反向引物、正、反向gDNAs、PfAgo、MnCl 2、模板(纯野生、纯突变、以及野生和突变各一半)等，如表8所示。
表8以25μL体系为例，PfAgo富集反应体系组分及配制顺序。
表8中各组分的说明如下：
2×PCR Taq Master Mix反应液由2×PCR缓冲液、dNTPs和热启动酶配制而成。2×PCR缓冲液：KCl，(NH 4) 2SO 4，3mM MgCl 2，Tris-HCl,pH值8.3(25℃)。dNTPs包括dATP,dGTP,dCTP和dTTP，在反应体系中终浓度为0.4mM。热启动酶是使用浓度为5U/μL的Taq DNA聚合酶，在反应体系中终浓度0.1-0.5U/μL。2×PCR Taq Master Mix反应液来自abm生物科技公司(货号：G013)。
正、反向引物使用表6中与各目标基因对应的，优选后的引物。
正、反向gDNAs使用表5中与各目标基因对应的，优选后的gDNAs对。
前期纯化后保存的PfAgo母液浓度在5μM，实际使用时需提前在冰浴条件下使用配制好的Reaction Buffer对其稀释：先将5μMPfAgo母液)稀释至1μM，然后用Reaction Buffer稀释至0.3μM。Reaction Buffer组分：15mM Tris-Cl,250mM NaCl,pH 8.0。
模板中ssDNA和dsDNA分别为60nt的KRAS-G12D ssDNA野生型和突变型片段，以及620bp的KRAS-G12D dsDNA野生型和突变型片段。
PfAgo-gDNA复合物对60nt的KRAS-G12D ssDNA野生型和突变型片段剪切的反应条件：95℃ 15分钟后缓慢降温至10℃保温。
PfAgo-gDNA复合物对620bp的KRAS-G12D dsDNA野生型和突变型片段剪切反应及PCR工作程序：
PfAgo富集体系PCR反应程序包括：
Figure PCTCN2019115151-appb-000002
Figure PCTCN2019115151-appb-000003
3.2结果
如图3所示。PfAgo-gDNA复合物对ssDNA，dsDNA以及在PCR工作体系下对dsDNA的SNV的区分剪切。
实施例4.
PfAgo-gDNA复合物对突变型dsDNA的富集
在本实施例中，测试PfAgo-gDNA复合物在PCR体系下对野生型和突变型dsDNA底物的区分剪切及对突变型dsDNA的富集情况。
4.1方法
按照本发明中描述的低丰度突变型DNA富集体系的实验步骤，首先针对KRAS-G12D基因片段的序列特征，设计、筛选特异性的扩增引物、gDNAs和检测探针。具体见表2序列。
gDNAs、引物均由上海生工生物工程有限公司合成。KRAS基因gDNAs 5'端设有磷酸化修饰。
以KRAS-G12D野生和突变片段为底物，分析PfAgo-gDNA复合物用于边PCR边剪切时影响突变型dsDNA信号富集的关键因素。具体参数见表A。
本实施例优化了PfAgo-gDNA复合物在PCR体系下对野生型和突变型dsDNA底物的区分剪切及对突变型dsDNA的富集条件。本实施例中涉及的各组分及工作条件主要包括：2×PCR Taq Master Mix，正、反向引物、正、反向gDNAs、PfAgo、MnCl 2、模板(10nM 1.0％mut KRAS-G12D)等，如表8所示。
正、反向引物使用表7中与各目标基因对应的，优选后的引物。
正、反向gDNAs使用表6中与各目标基因对应的，优选后的gDNAs对。
前期纯化后保存的PfAgo母液浓度在5μM，实际使用时需提前在冰浴条件下使用配制好的Reaction Buffer对其稀释：先将5μMPfAgo母液稀释至1μM,然后在用Reaction Buffer稀释至0.3μM。Reaction Buffer反应缓冲液1组分：15mM Tris-Cl,250mM NaCl,pH 8.0。
模板：人为配制的10nM 1.0％mut KRAS-G12D样品，采用李记生物公司销售的Pikogreen dsDNA定量试剂盒(超敏)(兼容Qubit 3.0)对配制样品进行定量。
PfAgo-gDNA复合物对620bp的KRAS-G12D dsDNA野生型和突变型片段剪切反应及PCR工作程序：
PfAgo对10nM 1.0％mut KRAS-G12D突变基因的富集PCR反应程序包括：
Figure PCTCN2019115151-appb-000004
PfAgo-gDNA复合物在PCR体系下对KRAS-G12D野生型和突变型dsDNA底物的区分剪切及对突变型dsDNA的富集条件经优化后，检测结果如图4所示。PfAgo-gDNA复合物在PCR体系下对10nM 1.0％mut KRAS-G12D样品中突变型片段DNA的富集条件，优选的是：PfAgo浓度在20～100nM区间；gDNAs浓度在200～1000nM区间；PfAgo：gDNAs浓度比例在1:10～1:20区间；94℃ PfAgo-gDNA复合物前处理时间为3分钟～5分钟；富集PCR的循环数在10～30循环为宜。
4.2结果
PfAgo-gDNA复合物在PCR体系下，可对KRAS-G12D野生型和突变型dsDNA底物的区分剪切，从而实现对突变型dsDNA的富集。
如图4所示，富集反应处理后的样品经一代测序(Sanger测序)后，结果显示在KRAS-G12D(gGt/gAt)突变点位置1.0％mut KRAS-G12D样品处理后突变点A碱基有明显凸起峰，即低丰度突变1.0％mut KRAS-G12D DNA得到了明显的富集。富集倍数F1b/F1a为约78。
实施例5.
对低丰度肿瘤基因KRAS-G12D突变基因的富集和检测
5.1方法
一种低丰度肿瘤基因KRAS-G12D突变基因(0.1％mut、0.01％mut)的检测方法。按照本发明中描述的低丰度突变型DNA检测体系的实验步骤，首先针 对KRAS-G12D基因片段的序列特征，设计、筛选特异性的扩增引物、gDNAs和检测探针。具体见表2序列。
gDNAs、引物及探针均由上海生工生物工程有限公司合成。KRAS基因gDNAs 5'端设有磷酸化修饰；G12D突变型探针的核苷酸序列5'端设有FAM荧光标记，3'端修饰淬灭基团BHQ1；KRAS野生型基因探针的核苷酸序列5'端设有VIC荧光标记，3'端修饰淬灭基团BHQ1。
本方法采对来源于HORIZON DISCOVERY公司的horizon reference standards标准品进行了验证分析，标准品中KRAS-G12D Expected Allelic Frequency(AF％)突变等位基因频率(AF％)分别为5％mut，1％mut,0.1％mut,0.01％mut和100％wt。采用了0.1％mut,0.01％mut标准品对此发明描述的低丰度突变型DNA的富集和检测方法的灵敏度和特异性进行了验证。
本实施例的低丰度KRAS基因突变检测的具体检测步骤如下：
5.1.1、PCR预扩增反应
本实施例的KRAS基因预扩增反应体系包括：
以50μL PCR预扩增体系为例，对标准品中0.1％mut,0.01％mut样品分别预扩增。预扩增的体系配制如下：
Figure PCTCN2019115151-appb-000005
反应体系的体积可以是25.0μL，在配制时将50.0μL反应体系中的组分减半即可。
本实施例的KRAS基因预扩增反应PCR条件如下：
PCR程序：94℃ 3分钟；10-30循环循环(94℃ 10s,55℃ 30s,72℃ 20s)，72℃ 1分钟。
预扩增后可对终产物进行TaqMan-qPCR初步定量，以确定是否满足下步富集体系中所需的target浓度。
5.1.2、PfAgo-gDNA复合物对预扩增产物中低丰度KRAS-G12D突变基因的富集。
本实施例采用优化后的PfAgo-gDNA复合物对KRAS-G12D突变型基因的 富集条件，优选的是：PfAgo浓度为20～100nM，gDNAs浓度为200-2000nM，；PfAgo：gDNAs浓度比例为1：5～1:20，94℃ PfAgo-gDNA复合物前处理时间为1～5分钟，富集PCR的循环数在10～30循环。
本实施例中涉及的各组分及工作条件主要包括：2X PCR Precision TMMasterMix,，正、反向引物、正、反向gDNAs、PfAgo、MnCl 2、Standard Target(0.1％mut,0.01％mut)等，如表8所示。
正、反向引物使用表7中与各目标基因对应的，优选后的引物。
正、反向gDNAs使用表6中与各目标基因对应的，优选后的gDNAs对。前期纯化后保存的PfAgo母液浓度在5μM，实际使用时需提前在冰浴条件下使用配制好的Reaction Buffer对其稀释：先将5μM PfAgo母液稀释至1μM,然后在用Reaction Buffer稀释至0.3μM。Reaction Buffer组分：15mM Tris-Cl,250mM NaCl,pH 8.0。
PfAgo对标准品0.1％mut,0.01％mut KRAS-G12D突变基因预扩增产物的富集PCR反应程序包括：
Figure PCTCN2019115151-appb-000006
5.1.3、KRAS-G12D突变基因富集后野生型和突变型DNA产物的检测。
本发明中涉及的富集后产物检测可采取多种方式，如Sanger测序定性分析、二代测序定量分析、TaqMan荧光定量PCR法定量分析、荧光法实时检测、高分辨率熔解曲线法定量分析等。在本实施例中分别采用了Sanger测序定性分析和TaqMan荧光定量PCR法定量分析两种方法对富集产物进行分析。
本实施例样品富集反应处理后经一代测序(Sanger测序)结果如图6B所示。结果显示在KRAS-G12D(gGt/gAt)突变点位置0.1％mut和0.01％mut KRAS-G12D样品处理后突变点A碱基有明显凸起峰，即低丰度突变0.1％mut和0.01％mut KRAS-G12D DNA得到了明显的富集。
采用TaqMan荧光定量PCR法定量分析前，本发明人首先设计并测定了双 TaqMan探针法检测KRAS-G12D低丰度突变型DNA(0.01％)底物的标准曲线。标准曲线如图5所示。
本实施例的KRAS-G12D基因标准曲线测定方法包括：
标准曲线测定前各组分按以下顺序配制：
①模板(野生型：突变型等于1：1)：158bp wt/mut KRAS G12D 10.0pM线性梯度稀释至(10.0pM、1.0pM、100fM、10fM、1.0fM、100aM、10aM、1.0aM、ddH 2O)
②双探针：SEQ ID No.109(10μM)；SEQ ID No.110(10μM)
③引物对：SEQ ID No.107(10μM)；SEQ ID No.108(10μM)
TaqMan-qPCR检测体系条件如下：以20μL体系为例,
Figure PCTCN2019115151-appb-000007
TaqMan-qPCR程序如下：
Figure PCTCN2019115151-appb-000008
标准曲线如图5所示。标准曲线测定结果分析：
①双探针的野生:突变母液按1:1比例配制，10.0pM梯度稀释的标准样品检测得到野生信号(图5A)重复性良好，每个浓度下设置3个重复。图5A最右侧曲线为ddH 2O的野生探针的信号，结合ddH 2O的野生探针的信号可得到该野生型探针信号阈值应在9000作用较为合适，此时CT ddH2O约为37-39，而样品最低浓度aM级CT在38左右，具体见图5C标准曲线部分。
②双探针野生:突变母液按1:1比例配制，10.0pM梯度稀释的标准样品检测得到突变信号(图5B)重复性良好，每个浓度下设置3个重复。图5B最右侧曲线为ddH 2O的突变探针的信号，结合ddH 2O的野生探针的信号可得到该野生型探针信号阈值应在9000作用较为合适，此时CT ddH2O约为38-40，而样品最 低浓度aM级CT在37左右，具体见图5D标准曲线部分。
标准曲线测定完毕后即对本实施例的KRAS-G12D富集样品进行TaqMan荧光定量PCR法定量分析。
富集样品测定前各组分按以下顺序配制：
TaqMan-qPCR检测体系条件如下：以20μL体系为例,
Figure PCTCN2019115151-appb-000009
TaqMan-qPCR程序如下：
Figure PCTCN2019115151-appb-000010
5.2结果
如图6A所示。在KRAS-G12D(gGt/gAt)突变点位置0.1％mut KRAS-G12D样品处理后突变型占比提高至83％(富集倍数F1b/F1a为830)，0.01％mut KRAS-G12D样品处理后突变型占比提高至78％(富集倍数F1b/F1a为7800)。即低丰度突变0.1％mut和0.01％mut KRAS-G12D DNA得到了极其明显的富集。
实施例6.
低丰度肿瘤基因EGFR delE746-A750片段缺失型突变基因的富集和检测
在本实施例中，对低丰度肿瘤基因EGFR delE746-A750片段缺失型突变基因(0.1％mut、0.01％mut)进行富集和检测。
6.1方法
按照本发明中描述的低丰度片段缺失型突变DNA检测体系的实验步骤(同实施例5)，首先针对EGFR delE746-A750基因片段的序列特征，设计、筛选特 异性的扩增引物、gDNAs和检测探针。具体见表4序列。
gDNAs、引物及探针均由上海生工生物工程有限公司合成。EGFR基因gDNAs 5'端设有磷酸化修饰；delE746-A750突变型探针的核苷酸序列5'端设有VIC荧光标记，3'端修饰淬灭基团BHQ1；EGFR野生型基因探针的核苷酸序列5'端设有FAM荧光标记，3'端修饰淬灭基团BHQ2。
本方法采对来源于HORIZON DISCOVERY公司的horizon reference standards标准品进行了验证分析，标准品中EGFR delE746-A750Expected Allelic Frequency(AF％)突变等位基因频率(AF％)分别为5％mut，1％mut,0.1％mut,0.01％mut和100％wt。采用了0.1％mut,0.01％mut标准品对此发明描述的低丰度突变型DNA的富集和检测方法的灵敏度和特异性进行了验证。
本实施例的低丰度EGFR delE746-A750片段缺失型突变基因检测的具体检测步骤如下：
6.1.1、PCR预扩增反应
本实施例的EGFR delE746-A750基因预扩增反应体系包括：
以50μL PCR预扩增体系为例，对标准品中0.1％MUT,0.01％MUT样品分别预扩增。预扩增的体系配制如下：
Figure PCTCN2019115151-appb-000011
反应体系的体积可以是25.0μL，在配制时将50.0μL反应体系中的组分减半即可。
本实施例的EGFR delE746-A750基因预扩增反应PCR条件如下：
PCR程序：94℃ 3分钟；10-30循环(94℃ 10s,55℃ 30s,72℃ 20s)，72℃ 1分钟。
预扩增后可对终产物进行TaqMan-qPCR初步定量，以确定是否满足下步富集体系中所需的target浓度。
6.1.2、PfAgo-gDNA复合物对预扩增产物中低丰度EGFR delE746-A750突变基因的富集。
本实施例采用优化后的PfAgo-gDNA复合物对EGFR delE746-A750突变型 基因的富集条件，优选的是：PfAgo浓度为20～80nM，gDNAs浓度为800nM，；PfAgo：gDNAs浓度比例为1：5～1:20，94℃ PfAgo-gDNA复合物前处理时间为3分钟，富集PCR的循环数在10～30循环。
本实施例中涉及的各组分及工作条件主要包括：2×PCR Taq Master Mix，正、反向引物、正、反向gDNAs、PfAgo、MnCl 2、Standard Target(0.1％mut,0.01％mut)等，如表8所示。
前期纯化后保存的PfAgo母液浓度在5μM，实际使用时需提前在冰浴条件下使用配制好的Reaction Buffer对其稀释：先将5μMPfAgo母液稀释至1μM，然后用Reaction Buffer稀释至0.3μM。
Reaction Buffer组分：15mM Tris-Cl,250mM NaCl,pH 8.0。
PfAgo对标准品0.1％MUT,0.01％MUT EGFR delE746-A750突变基因预扩增产物的富集PCR反应程序包括：
Figure PCTCN2019115151-appb-000012
6.1.3、EGFR delE746-A750突变基因富集后野生型和突变型DNA产物的检测。
在本实施例中采用了Sanger测序定性分析。
6.2结果
结果如图7所示。在EGFR delE746-A750(fragment deletion)突变位置0.1％mut和0.01％mut样品处理后显示丰富的缺失片段的碱基序列，即低丰度突变0.1％mut和0.01％mut EGFR delE746-A750 DNA得到了明显的富集，富集倍数F1b/F1a分别约为800、7400。
实施例7
三重低丰度突变的富集和检测
在本实施例中，对于多重(三重)低丰度(0.01％mut)肿瘤基因KRAS-G12D、PIK3CA-E545K及EGFR-delE746-A750突变基因(0.1％mut、0.01％mut)进行富 集和检测。
7.1方法
按照本发明中描述的低丰度突变型DNA检测体系的实验步骤，首先针对肿瘤突变基因片段的序列特征，设计、筛选特异性的扩增引物、gDNAs和检测探针。具体见表1、表2及表3的序列。
gDNAs、引物及探针均由上海生工生物工程有限公司合成。gDNAs 5'端设有磷酸化修饰；探针的核苷酸序列5'端设有不同荧光标记，3'端修饰淬灭基团BHQ。
本方法采对来源于HORIZON DISCOVERY公司的horizon reference standards标准品进行了验证分析，标准品中还有上述3种肿瘤基因Expected Allelic Frequency(AF％)突变等位基因频率(AF％)分别为0.01％mut和100％wt。我们采用了0.01％mut标准品对此发明描述的三重低丰度突变型DNA的富集和检测方法的灵敏度和特异性进行了验证。
本实施例的三重低丰度肿瘤基因突变检测的具体检测步骤如下：
7.1.1、PCR预扩增反应
本实施例的肿瘤基因预扩增反应体系包括：
以50μL PCR预扩增体系为例，对标准品中0.01％MUT样品分别预扩增。预扩增的体系配制如下：
Figure PCTCN2019115151-appb-000013
反应体系的体积可以是25.0μL，在配制时将50.0μL反应体系中的组分减半即可。
本实施例的三重低丰度肿瘤基因预扩增反应PCR条件如下：
PCR程序：94℃ 3分钟；24-30循环(94℃ 30s,55℃ 30s,72℃ 20s)；72℃ 1分钟。
预扩增后可对终产物进行TaqMan-qPCR初步定量，以确定是否满足下步 富集体系中所需的target浓度。
7.1.2、PfAgo-gDNA复合物对预扩增产物中三重低丰度肿瘤突变基因的富集。
本实施例采用优化后的PfAgo-gDNA复合物对KRAS-G12D、PIK3CA-E545K及EGFR-delE746-A750突变型基因的富集条件，优选的是：PfAgo浓度为10～800nM，gDNAs浓度为100～4000nM；PfAgo：gDNAs浓度比例为1：5～1:20，94℃ PfAgo-gDNA复合物前处理时间为3分钟，富集PCR的循环数在10～30循环。
本实施例中涉及的各组分及工作条件主要包括：2X PCR Precision TMMasterMix，正、反向引物、正、反向gDNAs、PfAgo、MnCl 2、Standard Target(0.01％mut)等，如表8所示。
正、反向引物使用表7中与各目标基因对应的，优选后的引物。
正、反向gDNAs使用表6中与各目标基因对应的，优选后的gDNAs对。
前期纯化后保存的PfAgo母液浓度在5μM，实际使用时需提前在冰浴条件下使用配制好的Reaction Buffer对其稀释：先将5μM PfAgo母液)稀释至1μM，然后用Reaction Buffer稀释至0.3μM。
Reaction Buffer组分：15mM Tris-Cl,250mM NaCl,pH 8.0。
PfAgo对标准品0.01％mut KRAS-G12D突变基因预扩增产物的富集PCR反应程序包括：
Figure PCTCN2019115151-appb-000014
7.1.3、KRAS-G12D、PIK3CA-E545K及EGFR-delE746-A750突变基因富集后野生型和突变型DNA产物的检测。
本发明中涉及的富集后产物检测可采取多种方式，如Sanger测序定性分析、二代测序定量分析、TaqMan荧光定量PCR法定量分析、荧光法实时检测、高分辨率熔解曲线法定量分析等。在本实施例中采用了TaqMan荧光定量PCR法定量分析法对富集产物进行分析。
采用TaqMan荧光定量PCR法定量分析前，我们首先设计并测定了双TaqMan探针法分别检测KRAS-G12D、PIK3CA-E545K及EGFR-delE746-A750低丰度突变型DNA底物的标准曲线。
标准曲线测定完毕后即对本实施例的KRAS-G12D、PIK3CA-E545K及EGFR-delE746-A750富集样品进行TaqMan荧光定量PCR法定量分析。
富集样品测定前各组分按以下顺序配制：
TaqMan-qPCR检测体系条件如下：以20μL体系为例,
Figure PCTCN2019115151-appb-000015
TaqMan-qPCR程序如下：
Figure PCTCN2019115151-appb-000016
7.2结果
如图8所示，在KRAS-G12D、PIK3CA-E545K及EGFR-delE746-A750突变位置0.01％mut样品处理后突变型占比KRAS-G12D、EGFR-delE746-A750及PIK3CA-E545K分别提高至79％、79％、41％。即三重低丰度突变0.01％mut肿瘤基因也得到了明显的富集，富集倍数F1b/F1a分别为约7900、7900和4100。
表1
Figure PCTCN2019115151-appb-000017
Figure PCTCN2019115151-appb-000018
表2
Figure PCTCN2019115151-appb-000019
Figure PCTCN2019115151-appb-000020
F:正向引物；R:反向引物；P:TaqMan-MGB探针；GF:Guide Forward DNA；GR:Guide Reverse DNA
表3
Figure PCTCN2019115151-appb-000021
Figure PCTCN2019115151-appb-000022
F:正向引物；R:反向引物；P:TaqMan-MGB探针；GF:Guide Forward DNA；GR:Guide Reverse DNA
表4
Figure PCTCN2019115151-appb-000023
Figure PCTCN2019115151-appb-000024
F:正向引物；R:反向引物；P:TaqMan-MGB探针；GF:Guide Forward DNA；GR:Guide Reverse DNA
表5
Figure PCTCN2019115151-appb-000025
Figure PCTCN2019115151-appb-000026
F:正向引物；R:反向引物；P:TaqMan-MGB探针；GF:Guide Forward DNA；GR:Guide Reverse DNA
表6
Figure PCTCN2019115151-appb-000027
表7
Target 引物序列(5'-3') SEQ ID No.
KRAS G12D-158F 5'-GTGACATGTTCTAATATAGTC-3' SEQ ID No.3
KRAS G12D-158R 5'-GGATCATATTCGTCCACAAA-3' SEQ ID No.4
PIK3CA E545K-139F 5'-GAGACAATGAATTAAGGGAA-3' SEQ ID No.13
PIK3CA E545K-139R 5'-GAAACAGAGAATCTCCATT-3' SEQ ID No.14
EGFR delE746-157F 5'-CTGTCATAGGGACTCTGGAT-3' SEQ ID No.23
EGFR delE746-157R 5'-GCCTGAGGTTCAGAGCCAT-3' SEQ ID No.24
NRAS A59T-154F 5'-CCAGGATTCTTACAGAAAACAAGT-3' SEQ ID No.33
NRAS A59T-154R 5'-GCTATTATTGATGGCAAATACACAG-3' SEQ ID No.34
表8.PfAgo富集反应25μL体系组分及配制顺序
加样顺序 组分 体积
1 2×PCR Taq Master Mix 12.5μL
2 dd H 2O 3.75μL
3 FW/RV primer(10μM) 各0.5μL
4 MnCl 2(10mM) 1.25μL
5 Template 2μL
6 Ago(XX nM) XμL
7 gDNA mix XμL
注：以上样品在体系配制时请遵循先后顺序添加。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
以上所述的具体实施例，对本发明的解决的技术问题、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种提高目标核酸的相对丰度的方法，其特征在于，包括步骤：
    (a)提供一核酸样本，所述的核酸样本含有第一核酸和第二核酸，其中，所述的第一核酸为所述目标核酸，而所述的第二核酸为非目标核酸，
    并且，所述目标核酸在所述的核酸样本中的丰度为F1a；
    (b)对所述核酸样本中的核酸为模板，在扩增-切割反应体系中进行聚合酶链反应(PCR)和核酸切割反应，从而获得扩增-切割反应产物；
    其中，所述的核酸切割反应用于特异性切割非目标核酸，但不切割所述目的核酸；
    并且，所述的扩增-切割反应体系含有(i)进行PCR反应所需的试剂和(ii)进行核酸切割反应所需的试剂；
    其中，所述目标核酸在所述的扩增-切割反应产物中的丰度为F1b，
    其中，F1b/F1a的比值≥10。
  2. 如权利要求1所述的方法，其特征在于，所述的目标核酸和非目标核酸仅相差一个碱基。
  3. 如权利要求1所述的方法，其特征在于，所述的目标核酸为含突变的核苷酸序列。
  4. 如权利要求1所述的方法，其特征在于，所述核酸切割工具酶选自但不限于以下来自嗜热微生物(≥60℃)的Argonaute蛋白(Ago)及其突变体：PfAgo(Pyrococcus furiosus Ago)、MfAgo(Methanocaldococcus fervens Ago)、TcAgo(Thermogladius calderae Ago)、TfAgo(Thermus filiformis Ago)、AaAgo(Aquifex aeolicus Ago)等。
  5. 如权利要求1所述的方法，其特征在于，所述的guide DNA与目标核酸(即第一核酸)的靶定区域的核酸序列形成第一互补结合区；并且所述的gDNA还与非目标核酸(即第二核酸)的靶定区域的核酸序列形成第二互补结合区。
  6. 如权利要求1所述的方法，其特征在于，所述核酸切割工具酶和gDNAs的比例(摩尔比)为1：2～1：20。
  7. 如权利要求1所述的方法，其特征在于，所述方法还包括：
    (c)对所述扩增-切割反应产物进行检测，从而测定所述目标核酸的存在与否 和/或数量。
  8. 如权利要求1所述的方法，其特征在于，所述的第一核酸包括n种不同的核酸序列，其中n为≥1的正整数。
  9. 如权利要求1所述的方法，其特征在于，在步骤(b)中，进行C个“高温变性-延伸”循环，其中C为≥5。
  10. 一种扩增-切割反应体系，其特征在于，所述反应体系用于对一核酸样本同时进行聚合酶链反应(PCR)和核酸切割反应，从而获得扩增-切割反应产物；
    其中，所述的核酸样本含有第一核酸和第二核酸，其中，所述的第一核酸为所述目标核酸，而所述的第二核酸为非目标核酸；
    所述的核酸切割反应用于特异性切割非目标核酸，但不切割所述目的核酸；
    所述的扩增-切割反应体系含有(i)进行PCR反应所需的试剂和(ii)进行核酸切割反应所需的试剂。
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