WO2023174391A1 - 一种对低丰度突变dna进行富集及检测的方法 - Google Patents

一种对低丰度突变dna进行富集及检测的方法 Download PDF

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WO2023174391A1
WO2023174391A1 PCT/CN2023/082033 CN2023082033W WO2023174391A1 WO 2023174391 A1 WO2023174391 A1 WO 2023174391A1 CN 2023082033 W CN2023082033 W CN 2023082033W WO 2023174391 A1 WO2023174391 A1 WO 2023174391A1
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wild
amplification
type
probe
gene
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French (fr)
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申洪杰
刘浩
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广州迪澳基因科技有限公司
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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
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention relates to the field of biological detection technology, and specifically relates to a method for enriching and detecting low-abundance mutant DNA.
  • DNA often undergoes random mutations during the replication process, and the mutation frequency often increases significantly in some disadvantageous environments (such as radiation, chemical mutagens, etc.).
  • Gene mutation is one of the important factors in biological evolution and the fundamental cause of biological genetic diversity; however, genetic mutation can also cause some adverse effects, such as the occurrence of cancer, drug resistance of pathogenic microorganisms, etc.
  • These mutations are often mixed with a large number of normal wild-type genes, and their low proportion of mutation content often brings great difficulties to clinical detection. Therefore, highly sensitive mutation detection technology is of great significance in guiding medication.
  • technologies for enriching and/or detecting low-abundance mutant DNA mainly include sequencing, digital PCR, low denaturation temperature co-amplification PCR (COLD-PCR), primer amplification hindered mutation system (ARMS-PCR), wild-type hindered mutation system Time-lag amplification PCR (WTB-PCR), denaturing high-performance liquid chromatography (dHPLC), etc.
  • COLD-PCR low denaturation temperature co-amplification PCR
  • ARMS-PCR primer amplification hindered mutation system
  • WTB-PCR wild-type hindered mutation system Time-lag amplification PCR
  • dHPLC denaturing high-performance liquid chromatography
  • the purpose of the present invention is to provide a method for enriching and detecting low-abundance mutant DNA.
  • the technical solution provided by the present invention is optimized and improved on the basis of wild-type blocked amplification PCR technology, so that the system can enrich low-abundance mutant DNA.
  • Abundant DNA mutations and detection of multiple base mutation types have the advantages of high sensitivity, high accuracy, low cost, short time consumption, and easy operation.
  • the present invention provides a wild-type amplification retarder, the Tm value of the wild-type amplification retarder that specifically binds to the wild-type template of the gene to be tested is T m1 ; the wild-type amplification retarder has a Tm value of The Tm value of the wild-type amplification retarder that specifically binds to the mutant template of the gene to be tested is T m2 ; T m1 > T m2 ; the wild-type amplification retarder has a blocking group at its 3' end.
  • the binding position of the wild-type amplification retarder and the gene to be tested covers the mutation site of the gene to be tested. Since T m1 > T m2 , when in a specific annealing temperature range , the wild-type amplification retarder only binds to the wild-type template, and because its 3' end is modified with a blocking group, it cannot continue to extend, thereby blocking the amplification of the wild-type template.
  • the blocking group includes at least one selected from the group consisting of dideoxycytidine, reverse dT, amino, phosphate group, inter-arm (such as inter-arm C3, inter-arm C5, etc.) or others that can block all
  • the wild-type amplification retarder serves as the base for primer extension.
  • T m1 and T m2 are 5-20°C, such as about 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, etc.
  • the T m1 is 60-75°C, such as about 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, etc.
  • the wild-type amplification retarder is completely complementary to the wild-type template of the gene to be tested.
  • At least one, at least two, at least three, at least four, at least five, at least six, at least seven, of the wild-type amplification retarder is complementary to the gene to be tested.
  • At least eight or at least nine bases are RNA bases.
  • the difference between T m1 and T m2 can be further increased.
  • the base on the wild-type amplification blocker corresponding to the mutation position base of the gene to be tested is located in the middle, 5' end or 3' end of the wild-type amplification blocker.
  • the wild-type amplification repressor is an oligonucleotide chain with a length of 20-45bp, such as 20bp, 25bp, 30bp, 35bp, 40bp, 45bp, etc.
  • the present invention provides an amplification-blocking mutation system, which system includes a forward primer, a reverse primer, and at least one blocker-probe combination; the blocker-probe combination includes a probe. and the wild-type amplification retarder of the present invention;
  • the forward primer and the reverse primer are used to amplify the gene to be tested, and the binding positions of the forward primer and the reverse primer to the gene to be tested are respectively located between the wild-type amplification blocker and the gene to be tested. upstream and downstream of the binding position;
  • the probe competitively binds to the gene to be tested with the wild-type amplification blocker; the binding position of the probe to the gene to be tested covers all Describe the mutation site of the gene to be tested.
  • the wild-type amplification blocker is located between the forward primer and the reverse primer.
  • the technical solution of the present invention avoids the above-mentioned competitive binding that affects the ability of the wild-type amplification retarder to block the amplification of the wild-type allele, as well as the efficiency of primer annealing and binding to the mutant template. Therefore, the technical solution of the present invention further improves the sensitivity of detecting mutant alleles.
  • the amplification-blocking mutation system includes more than two groups of blocker-probe combinations, such as two groups, three groups, four groups, five groups, six groups, seven groups, eight groups, nine groups Groups, groups of ten, etc.
  • the number of blocker-probe combinations can be determined based on the number and location of mutation sites of the gene to be tested. This enables simultaneous detection of multiple mutation sites of the gene to be tested in one detection system.
  • the length of the probe is 15-40 bp.
  • the probe is a fluorescent probe.
  • the 5' end of the fluorescent probe is modified with a fluorescent group selected from one or more of FAM, HEX, ROX, TAMRA, Texas RED, CY5, Cy3, TET, JOE, and VIC; the molecule
  • the 3' end of the beacon probe is modified with a quenching group selected from one or more of BHQ1, BHQ2, Dabcy1, TAMRA, MGB, and ECLIPSE.
  • the probe is a molecular beacon probe.
  • the probe has one or both of the following modifications: modified to peptide nucleic acid (PNA), locked nucleic acid (LNA).
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • the probe and the wild-type amplification retarder have base overlap, and the number of overlapping bases is no less than 90%, 80%, 70%, 60%, and 50 of the total number of bases in the probe. %, 40%, 30% or 20%.
  • the Tm value of the probe that specifically binds to the wild-type template of the gene to be tested is T m3 , T m1 > T m3 > T m2 .
  • the difference between T m1 and T m3 is 5-15°C, such as 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, etc.
  • Tm values of the forward primer and the reverse primer that specifically bind to the gene to be tested are T m4-F and T m4-R respectively, T m1 > T m4-F , T m4-R, further, 55°C ⁇ T m4-F , T m4-R ⁇ 70°C.
  • a third aspect of the present invention provides a nucleic acid detection system, which includes the amplification-impeding mutation system described in the second aspect of the present invention, and also includes RCR buffer, DNA polymerase, and dNTPs.
  • the PCR buffer includes (NH 4 ) 2 SO 4 , MgCl 2 , KCl, Tris-HCl, etc.
  • the concentration of (NH 4 ) 2 SO 4 is 5-20 mM, such as 5 mM, 10 mM, 15 mM, 20 mM, etc.; the concentration of MgCl 2 is 2 -3mM, such as 2mM, 2.5mM, 3mM, etc.; the concentration of KCl is 5-20mM, such as 5mM, 10mM, 15mM, 20mM, etc.; the concentration of Tris-HCl is 10- 30 mM, such as 10 mM, 20 mM, 30 mM, etc.; the pH value of Tris-HCl is 8-8.5.
  • the final concentration of the wild-type amplification retarder is 0.02-0.5 ⁇ M, such as 0.02 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.15 ⁇ M, 0.2 ⁇ M, 0.25 ⁇ M, 0.3 ⁇ M, 0.35 ⁇ M, 0.4 ⁇ M, 0.45 ⁇ M, 0.5 ⁇ M, etc.
  • the final concentration of the probe is 0.02-0.5 ⁇ M, such as 0.02 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.15 ⁇ M, 0.2 ⁇ M, 0.25 ⁇ M, 0.3 ⁇ M, 0.35 ⁇ M, 0.4 ⁇ M, 0.45 ⁇ M, 0.5 ⁇ M, etc.
  • the final concentration of the forward primer and reverse primer is 0.02-0.5 ⁇ M, such as 0.02 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.15 ⁇ M, 0.2 ⁇ M, 0.25 ⁇ M, 0.3 ⁇ M, 0.35 ⁇ M, 0.4 ⁇ M, 0.45 ⁇ M, 0.5 ⁇ M, etc.
  • the final concentration of dNTPs is 0.2-0.5 mM, such as 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, etc.
  • the DNA polymerase does not have 5' ⁇ 3' exonuclease activity and 3' ⁇ 5' endonuclease activity.
  • a fourth aspect of the present invention provides a nucleic acid detection kit, which includes the nucleic acid detection system described in the third aspect of the present invention.
  • a fifth aspect of the present invention provides a nucleic acid detection method, which includes the following steps: adding the template of the gene to be tested into the nucleic acid detection system of the present invention, and performing PCR amplification and melting curve detection;
  • the reaction procedure of the PCR amplification includes: denaturation at 92-98°C for 10-30 s; first annealing at T m1 temperature condition for 10-30 s; and third annealing at T m4-F or T m4-R temperature condition. Secondary annealing for 10-30 s; extension at a temperature greater than T m3 and less than T m1 for 0.5-2 min; perform the above reaction for a total of 45-55 cycles.
  • pre-denaturation at 92-98°C for 1-10 minutes.
  • the PCR amplification is asymmetric PCR amplification.
  • reaction program for melting curve detection includes: 92-98°C, 1 min; T m1 temperature condition, 1 min; 45-50°C, 1 min; heating to 85°C, with a heating rate of 0.01-0.05°C/s; heating process Collect fluorescence signals.
  • fluorescence signal collection is performed every 1°C during the temperature rise process.
  • the wild-type amplification repressor provided by the present invention can enrich a large amount of low-abundance mutant DNA without affecting the amplification efficiency of the mutant template, improving the specificity and sensitivity of detection to meet the actual needs of clinical detection of mutant genes. need;
  • the probe provided by the present invention can perform melting curve detection on amplified low-abundance mutant DNA to distinguish its mutation types.
  • different wavelength fluorescent labeling groups can be used to achieve multi-channel and multi-target single-tube detection. .
  • Figure 1 is the FAM channel melting curve detection result of Example 1
  • Figure 2 is the VIC channel melting curve detection result of Example 1
  • Figure 3 shows the detection results of the Texas RED channel melting curve in Example 1
  • Figure 4 is the detection result of the CY5 channel melting curve in Example 1;
  • Figure 5 is the FAM channel melting curve detection result of Example 2; wherein, the 95A position is mutated to 95G;
  • Figure 6 is the detection result of Texas RED melting curve in Example 2; wherein the 1376G site is mutated to 1376T;
  • Figure 7 is the detection result of Texas RED melting curve in Example 2; wherein, the 1388G site is mutated to 1388A;
  • Figure 8 is the detection result of the BRAF gene mutation melting curve in Example 3.
  • Figure 9 is the detection result of CYP2C9-CC type melting curve detected by Texas channel in Example 4.
  • Figure 10 is the detection result of CYP2C9-AA type melting curve detected by Texas channel in Example 4.
  • Figure 11 is the detection result of CYP2C9-CA type melting curve detected by Texas channel in Example 4.
  • Figure 12 is the detection result of VKROC1-GG type melting curve detected by CY5 channel in Example 4.
  • Figure 13 is the detection result of VKROC1-AA type melting curve detected by CY5 channel in Example 4.
  • Figure 14 is the detection result of VKROC1-GA type melting curve detected by CY5 channel in Example 4.
  • Figure 15 is the melting curve detection result of Texas channel detection of HPV16 in Example 5.
  • Figure 16 is the melting curve detection result of Texas channel detection of HPV18 in Example 5.
  • Figure 17 is the melting curve detection result of HPV31 detected by Texas channel in Example 5.
  • Figure 18 is the melting curve detection result of HPV33 detected by Texas channel in Example 5.
  • Figure 19 is the melting curve detection result of FAM channel detection of HPV35 in Example 5.
  • Figure 20 is the melting curve detection result of FAM channel detection of HPV39 in Example 5.
  • Figure 21 is the melting curve detection result of FAM channel detection of HPV45 in Example 5.
  • Figure 22 is the melting curve detection result of FAM channel detection of HPV51 in Example 5.
  • Figure 23 is the melting curve detection result of CY5 channel detection of HPV52 in Example 5.
  • Figure 24 is the melting curve detection result of CY5 channel detection of HPV53 in Example 5.
  • Figure 25 is the melting curve detection result of CY5 channel detection of HPV56 in Example 5.
  • Figure 26 is the melting curve detection result of CY5 channel detection of HPV58 in Example 5.
  • Figure 27 is the melting curve detection result of VIC channel detection of HPV59 in Example 5.
  • Figure 28 is the melting curve detection result of VIC channel detection of HPV66 in Example 5.
  • Figure 29 is the melting curve detection result of VIC channel detection of HPV68 in Example 5.
  • Figure 30 is the melting curve detection result of VIC channel detection of HPV73 in Example 5.
  • Figure 31 is the melting curve detection result of VIC channel detection of HPV82 in Example 5.
  • the invention provides a wild-type amplification blocker (hereinafter referred to as Blocker), an amplification-blocking mutation system and a corresponding nucleic acid detection system and detection method.
  • Tm value (T m1 ) of the Blocker provided by the invention that specifically binds to the wild-type template of the gene to be tested is greater than the Tm value (T m2 ) that it specifically binds to the mutant template, and has a blocking group at the 3' end. .
  • the probe and the Blocker competitively bind to the template of the gene to be tested, and the Tm value of the specific binding of the probe to the wild-type template is T m3 (T m1 > T m3 > T m2 ); the Tm values of forward primer and reverse primer specifically binding to the gene to be tested are T m4-F and T m4-R respectively (T m1 > T m4-F , T m4-R ).
  • the Blocker When there is only wild-type template in the reaction system, perform the first annealing at T m1 temperature condition, and the Blocker will preferentially bind to the wild-type allele completely complementary; perform the second annealing at T m4-F or T m4-R temperature condition.
  • Annealing the amplification primer is complementary to the template to be tested; extension is performed at a temperature greater than T m3 and less than T m1 .
  • the amplification primer extends to the Blocker position, the polymerase used in the system does not have a 5' ⁇ 3' outer Due to the Dicer activity, the Blocker cannot be digested and terminates extension, thereby blocking the amplification of the wild-type template.
  • Blocker can often only inhibit the amplification of wild-type alleles to a certain extent.
  • the starting amount of wild-type DNA is too high, there will be a certain amount of false positive amplification.
  • the present invention also uses probes for subsequent melting curve detection.
  • the initial temperature condition is 92-98°C, so that the PCR product is completely denatured into a single strand; when the temperature drops to T m1 , the Blocker in the system competes with the probe and preferentially denatures the possible wild-type amplified strands.
  • the amplification products are completely complementary to each other, which means that under subsequent temperature reduction conditions, since the wild-type amplification products that may exist in the system have been significantly reduced, the probe is almost entirely bound to the mutant amplification products. This effectively reduces the interference caused by false positive amplification and improves the accuracy of melting curve detection results.
  • the first annealing is performed under T m1 temperature conditions, and the Blocker preferentially binds to the wild-type allele completely complementary; it is performed under T m4-F or T m4-R temperature conditions.
  • the amplification primer fully complements and extends the wild-type and mutant templates at the same time; it terminates when it extends the wild-type template to the Blocker position, and when it extends, the mutant template can be fully extended and complete amplification, thereby enriching a large amount of Collect low-abundance mutant DNA.
  • Primer Premier6.0, DNAMAN, ClustalX and other software were used to design primers based on the core region of the rifampicin resistance mutation on the rpoB gene of Mycobacterium tuberculosis.
  • the length of the amplified fragment was 145 bp; four probes were designed to completely cover the core region of the mutation. .
  • four Blockers were designed to compete with the probe.
  • the upstream and downstream primer sequence regions were selected to synthesize the wild-type plasmid, and the 511CCG, 516GTC, 526TAC and 531TTG mutated codons were selected to synthesize the mutant plasmid based on clinical high-frequency mutations.
  • Designed primers F-rpoB, R-rpoB
  • probes P1-rpoB, P2-rpoB, P3-rpoB, P4-rpoB
  • blockers B1-rpoB, B2-rpoB, B3-rpoB, B4- rpoB
  • Each primer, probe and plasmid were synthesized by professional companies. The specific sequence is shown in Table 1.
  • 1% group Take 10 ⁇ l of the mixed solution of the 10% group and mix it with 90 ⁇ l of 10 4 copies/ ⁇ l wild-type plasmid, and shake to mix.
  • 0.5% group Take 10 ⁇ l of the mixed solution of the 5% group and mix it with 90 ⁇ l of 10 4 copies/ ⁇ l wild-type plasmid, and shake to mix.
  • 0.1% group Take 10 ⁇ l of the mixed solution of the 1% group and mix it with 90 ⁇ l of 10 4 copies/ ⁇ l wild-type plasmid, and shake to mix.
  • 0.01% group Take 10 ⁇ l of the mixed solution of the 0.1% group and mix it with 90 ⁇ l of 10 4 copies/ ⁇ l wild-type plasmid, and shake to mix.
  • Wild type control 10 4 copies/ ⁇ l of wild plasmid.
  • the reaction system is 25 ⁇ l.
  • the system includes dNTPs 0.2 mM, (NH 4 ) 2 SO 4 10 mM, MgCl 2 3 mM, KCl 10 mM, Tris-HCl 20 mM with pH 8.3, 1U DNA polymerase, and upstream primer 0.02 ⁇ M. , downstream primer 0.2 ⁇ M, probe and blocker each 0.1 ⁇ M, 5 ⁇ l DNA template, and the rest are made up with water;
  • the program of PCR amplification is: 95°C, 10min; (95°C, 15s; 82°C, 20s, 61°C, 15s; 75°C, 30s reaction for 50 cycles).
  • the program of the melting curve reaction is: 95°C, 1 min; 82°C, 1 min; 45°C, 1 min.
  • the temperature is raised to 85°C at a rate of 0.05°C/s.
  • the fluorescence signal is collected every 1°C.
  • the Tm value of the wild-type control of the FAM channel is 74.1°C ⁇ 1°C; the Tm value of the wild-type control of the VIC channel is 73.5°C ⁇ 1°C; the Tm value of the wild-type control of the Texas channel is 74.4°C ⁇ 1°C; the wild-type control Tm value of the CY5 channel is 71.6°C ⁇ 1°C. If the melting curve result of the sample to be tested differs ⁇ 2°C from the Tm value of the wild-type control, it is determined to be a mutant type.
  • Blocker When only wild-type DNA exists in the reaction system, the Blocker is completely complementary to the wild-type allele, and the amplification primer DNA template cannot continue to extend after being combined with the DNA template and extending to the Blocker position, so no fluorescence signal is generated. However, in actual testing, it was found that Blocker can only inhibit the amplification of wild-type alleles to a certain extent. When the initial dosage of wild-type DNA is too high, there will be a certain amount of false positive amplification. The melting curve detection results showed that the Tm value of each fluorescence channel was consistent with the wild-type control.
  • the annealing temperature of the Blocker and the mutant allele (68°C) is lower than the extension temperature of the amplification primer and the DNA template (75°C), so the amplification primer is extended at 75°C Extend with the mutant template to obtain a fluorescent signal.
  • FIG. 1-4 A wild melting peak appears in the wild control, but no melting peak appears in the negative control, indicating that the detection system can effectively amplify without contamination. Only mutation peaks appeared in the pure mutant type, while wild peaks and mutation peaks appeared in the 5%, 1%, 0.5%, 0.1% and 0.01% groups.
  • 1 is the melting peak of the wild-type control
  • 2 is the melting peak of the 100% mutant sample
  • 3 is the melting peak of the mixed sample with 5% mutation content
  • 4 is the melting peak of the mixed sample with 1% mutation content
  • 5 is 0.5 % mutation content mixed sample melting peak
  • 6 is the melting peak of 0.1% mutation content mixed sample
  • 7 is the melting peak of 0.01% mutation content mixed sample
  • 8 is the negative control melting peak.
  • G6PD Glucose-6-phosphate dehydrogenase
  • G6PD deficiency is a sex-linked incomplete dominant genetic disease. G6PD deficiency will greatly increase the chance of newborns suffering from jaundice. If not treated or not treated promptly, it may lead to kernicterus and permanent neurological disease. Injury or hemolytic anemia. Studies have shown that mutations at different sites in the G6PD gene will lead to varying degrees of reduction in G6PD enzyme activity.
  • G6PD examination is clinically listed as one of the items such as premarital examination, prenatal examination and newborn examination, so as to achieve eugenics and postnatal care. Since there are different proportions of mutant nucleic acids and wild-type nucleic acids in actual clinical samples, in order to better amplify low-abundance mutant nucleic acids in mixed samples, this embodiment provides a method that can achieve low-abundance G6PD mutant nucleic acids. A method for detecting samples with different sizes, thereby improving detection sensitivity.
  • This example detects glucose-6-phosphate dehydrogenase (G6PD) gene mutations.
  • the specific steps are as follows:
  • Primer Premier6.0, DNAMAN, ClustalX and other software were used to design primers for the mutation regions of G6PD gene 95A>G, 1376G>T, and 1388G>A.
  • the amplified fragment lengths were 141bp and 150bp respectively; two probes were designed to completely Cover the core region of the mutation.
  • two Blockers and probes were designed to compete.
  • the G6PD-M wild-type plasmid was synthesized by a professional company. This plasmid contains the unmutated original sequences of 95A, 1376G, and 1388G.
  • the selected vector was pUC-GW-Amp; and the 95A>G, 1376G>T, and 1388G>A point mutation plasmids were synthesized respectively.
  • G6PD-95G-M, G6PD-1376T-M, and G6PD-1388A-M respectively.
  • the G6PD-M wild-type plasmid sequence and mutant plasmid sequence are shown in Table 3.
  • the reaction system is 25 ⁇ l, and the system includes dNTPs 0.2 mM, (NH 4 ) 2 SO 4 10 mM, MgCl 2 3 mM, KCl 10 mM, Tris-HCl 20 mM with a pH of 8.3, 2 ⁇ l DNA polymerase, and upstream primer 0.02 ⁇ M, downstream primer 0.2 ⁇ M, probe and blocker 0.1 ⁇ M, 5 ⁇ l DNA template, and the rest are made up with water;
  • the PCR amplification program is: 95°C, denaturation for 10 minutes; (95°C, 15s; 81°C, 20s, 63°C, 15s; 72°C, 30s reaction for 50 cycles).
  • the program of the melting curve reaction is: 95°C, 1 min; 81°C, 1 min; 45°C, 1 min.
  • the temperature is raised to 85°C at a rate of 0.05°C/s.
  • the fluorescence signal is collected every 1°C.
  • the plasmid nucleic acid provided in this example (2) is a plasmid nucleic acid in which the 95A site of the G6PD gene is mutated to 95G (G6PD-95G-M, FAM channel, Figure 5), and a plasmid nucleic acid in which the 1376G site of the G6PD gene is mutated to 1376T (G6PD- 1376T-M, Texas RED channel, Figure 6), and plasmid nucleic acid in which the 1388G site of the G6PD gene was mutated to 1388A (G6PD-1388A-M, Texas RED channel, Figure 7), and the results are shown in Figure 5-7 .
  • Line 1 is the melting curve of 95A wild type, with a Tm value of 77°C
  • line 2 is the melting curve of the 95A site mutated to 95G mutant, with a Tm value of 62°C
  • line 3 is the 95G mutant proportion of 50%.
  • Melting curve of mixed sample test results line 4 is the negative control
  • line 1 is the melting curve of 1376G wild type, Tm value is 77.6°C
  • line 2 is the melting curve of 1376T mutant
  • Tm value is 64°C
  • line 3 It is the melting curve of the mixed sample test result with 50% 95G mutant proportion.
  • Line 4 is the negative control; in Figure 7: Line 1 is the melting curve of 1388G wild type with a Tm value of 77.6°C, and line 2 is the melting curve of 1388A mutant. , the Tm value is 68.5°C, the 3rd line is the melting curve of the mixed sample test result with a 95G mutant proportion of 50%, and the 4th line is the negative control.
  • the BRAF gene is an oncogene located on the long arm of chromosome 7. It contains 18 exons and encodes a B-RAF protein containing 783 amino acids. It is a member of the EGFR pathway RAS/RAF/MEK/MRK/MAPK. It is an important transduction factor involved in various physiological processes such as cell growth, differentiation and apoptosis. Studies have shown that BRAF gene mutations exist in various malignant tumors such as non-small cell lung cancer and colorectal cancer, among which the V600E mutation in exon 15 is the most common, accounting for more than 90%. This mutation causes the B-RAF protein to be abnormally activated, causing patients to fail treatment with EGFR-TKI drugs and EGFR monoclonal antibodies.
  • This example detects human BRAF gene mutations.
  • the specific steps are as follows:
  • Primer Premier6.0, DNAMAN, ClustalX and other software were used to design primers for the mutation region of BRAF gene 1799T>A.
  • the length of the amplified fragment was 200 bp; a probe was designed to completely cover the core region of the mutation.
  • a Blocker is designed to compete with the probe.
  • BRAF-1799T-M wild-type plasmid and BRAF-1799A-M mutant plasmid were synthesized by a professional company, and the selected vector was pUC-GW-Amp; the wild-type plasmid sequence and mutant plasmid sequence are shown in Table 4.
  • the reaction system is 25 ⁇ l.
  • the system includes dNTPs 0.2 mM, (NH 4 ) 2 SO 4 10 mM, MgCl 2 3 mM, KCl 10 mM, Tris-HCl 20 mM with pH 8.3, 2 ⁇ l DNA polymerase, and upstream primer. 0.02 ⁇ M, downstream primer 0.2 ⁇ M, probe and blocker 0.1 ⁇ M, 5 ⁇ l DNA template, and the rest are made up with water;
  • the PCR amplification program is: 95°C, denaturation for 10 minutes; (95°C, 15s; 80°C, 20s, 63°C, 15s; 70°C, 30s reaction for 50 cycles).
  • the program of the melting curve reaction is: 95°C, 1 min; 80°C, 1 min; 45°C, 1 min.
  • the temperature is raised to 85°C at a rate of 0.05°C/s.
  • the fluorescence signal is collected every 1°C.
  • the BRAF-1799T-M wild-type plasmid and BRAF-1799A-M mutant plasmid provided in this example, and a mixed sample with a mutant ratio of 1 ⁇ were tested.
  • the results are shown in Figure 8.
  • Line 1 in the figure is BRAF-1799T-M wild-type melting curve, Tm value is 70°C;
  • Line 2 is the melting curve of BRAF-1799A-M mutant, Tm value is 62°C;
  • Line 3 is BRAF-1799A-M mutant with a ratio of 1 ⁇ Mix the samples, and line 4 is the negative control.
  • Warfarin is a commonly used clinical drug for the prevention and treatment of thromboembolic diseases. It is a coumarin anticoagulant. Excessive dosage of warfarin can cause bleeding, and its dosage is affected by many factors (mainly genetics and environment). There are large individual differences in clinical efficacy and adverse reactions, and the dosage is difficult to control. Therefore, the dosage needs to be determined according to different individuals.
  • VKORC1 promoter polymorphism -1639G/A
  • CYP2C9 1075A/C
  • this example detects CYP2C9 (rs1057910) and VKORC1 (rs9923231) gene mutations.
  • the specific steps are as follows:
  • CYP2C9 rs1057910
  • VKORC1 rs9923231
  • four primers, two probes and two blocker sequences were designed. These primers can specifically amplify the two genes respectively. Design probes for the mutant type in the amplified target region. These probes can specifically bind to the mutant target sequence and use the difference in Tm value of the melting curve for typing detection.
  • blockers were designed for wild type, which can enrich mutant samples to a certain extent. All sequences are synthesized by professional companies.
  • the reaction system is 25 ⁇ l.
  • the system includes dNTPs 0.3 mM, (NH 4 ) 2 SO 4 8 mM, MgCl 2 3 mM, KCl 10 mM, Tris-HCl 20 mM with pH 8.3, 2 ⁇ l DNA polymerase, and upstream primer 0.02 ⁇ M. , downstream primer 0.2 ⁇ M, probe and Blocker 0.1 ⁇ M, 5 ⁇ l DNA template, and the rest are made up with water;
  • the program of PCR amplification is: 95°C, 10min; (95°C, 15s; 75°C, 20s, 61°C, 15s; 70°C, 30s reaction for 50 cycles).
  • the program of the melting curve reaction is: 95°C, 1 min; 75°C, 1 min; 45°C, 1 min.
  • the temperature is raised to 90°C at a rate of 0.05°C/s.
  • the fluorescence signal is collected every 1°C.
  • the Texas channel detects CYP2C9.
  • the Tm value of the CC type control is 69.8°C ⁇ 1°C ( Figure 9).
  • the Tm value of the AA type control is 65.4°C ⁇ 1°C ( Figure 10).
  • the AC type control has a double peak, one high and one low.
  • the Tm value is 65.6°C ⁇ 1°C and the Tm value is 70.2°C ⁇ 1°C ( Figure 11); CY5 channel detects VKROC1, the Tm value of the GG type control is 76.4°C ⁇ 1°C ( Figure 12), and the Tm value of the AA type control is 69.8°C ⁇ 1°C ( Figure 13), the GA type has a double peak with one high and one low, with a Tm value of 70.2°C ⁇ 1°C and a Tm value of 75.6°C ⁇ 1°C ( Figure 14). If the difference between the melting curve result of the sample to be tested and the Tm value of the control is ⁇ 1°C, it is determined to be the type.
  • HPV Human Papilloma Virus
  • E region early transcription region
  • Late Region L region
  • LCR regulatory region
  • HPV6 HPV11, HPV42, HPV43, HPV44, etc.
  • Low-risk types generally do not induce cancer.
  • Common high-risk types include HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV49, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, and HPV82.
  • cervical cancer and other malignant female reproductive tracts The occurrence of tumors is related to repeated infection with high-risk HPV viruses.
  • This embodiment detects HPV typing.
  • the specific steps are as follows:
  • plasmid powder synthesized by a professional company adding a marked volume of water, measuring the nucleic acid concentration and then calculating the plasmid copy number, constructing plasmid standards by gradient dilution, and obtaining concentrations of 10 7 copies/ ⁇ l and 10 6 copies / ⁇ l, 10 5 copies/ ⁇ l, 10 4 copies/ ⁇ l, 10 3 copies/ ⁇ l, 10 2 copies/ ⁇ l, 10 copies/ ⁇ l and 1 copies/ ⁇ l plasmid standards of different types.
  • Negative control dilute normal healthy human genomic DNA to 10ng/ ⁇ l with buffer containing 0.9% NaCl, 1mmol/L EDTA, 10mmol/L Tris-HCl and 0.1% Triton X-100.
  • Preparation of low-abundance mixed sample Use the dilution used above to prepare a plasmid mixture containing 1 high-risk type and 5 low-risk types, so that the final concentration of each high-risk plasmid is 10 copies/ ⁇ l.
  • HPV high-risk types were classified and identified.
  • the specific steps are as follows:
  • Designed primers F1-HPVL1, F2-HPVL1, F3-HPVL1, R-HPVL1
  • probes P16-HPVL1, P18-HPVL1, P31-HPVL1, P33-HPVL1, P35-HPVL1, P39-HPVL1, P45 -HPVL1, P51-HPVL1, P52-HPVL1, P53-HPVL1, P56-HPVL1, P58-HPVL1, P59-HPVL1, P66-HPVL1, P68-HPVL1, P73-HPVL1, P82-HPVL1) and Blocker (B6-HPVL1, B11-HPVL1, B42-HPVL1, B43-HPVL1, B44-HPVL1) were all subjected to homology comparison through BLAST to ensure their specificity. Since actual clinical samples are difficult to obtain, artificially synthesized plasmids were used for verification. Synthesized by professional companies. The specific sequence is shown in Table 7.
  • the reaction system is 25 ⁇ l.
  • the system includes dNTPs 0.4 mM, (NH 4 ) 2 SO 4 10 mM, MgCl 2 5 mM, KCl 15 mM, Tris-HCl 20 mM with pH 8.3, 3.5 ⁇ l DNA polymerase, and upstream primer 0.02 ⁇ M, downstream primer 0.2 ⁇ M, probe and blocker 0.1 ⁇ M, 5 ⁇ l DNA template, and the rest are made up with water;
  • the program of PCR amplification is: 95°C, 10min; (95°C, 15s; 75°C, 20s, 61°C, 15s; 70°C, 30s reaction for 50 cycles).
  • the program of the melting curve reaction is: 95°C, 1 min; 75°C, 1 min; 45°C, 1 min.
  • the temperature is increased to 85°C at a rate of 0.05°C/s.
  • the fluorescence signal is collected every 1°C.
  • the Tm value of the Texas channel HPV16 control is 75.3°C ⁇ 1°C
  • the Tm value of the HPV18 control is 70.5°C ⁇ 1°C
  • the Tm value of the HPV31 control is 65.3°C ⁇ 1°C
  • the Tm value of the HPV33 control is 60.2°C ⁇ 1°C
  • the Tm value of the FAM channel HPV35 control is 86.9°C ⁇ 1°C
  • the Tm value of the HPV39 control is 79.5°C ⁇ 1°C
  • the Tm value of the HPV45 control is 63.3°C ⁇ 1°C
  • the Tm value of the HPV51 control is 70.9°C ⁇ 1°C
  • the Tm value of the CY5 channel HPV52 control is 60.4°C ⁇ 1°C
  • the Tm value of the HPV53 control is 65.4°C ⁇ 1°C
  • the Tm value of the HPV56 control is 70.1°C ⁇ 1°C
  • the Tm value of the HPV58 control is 7

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Abstract

提供一种对低丰度突变DNA进行富集及检测的方法，还提供一种野生型扩增阻滞物，其与野生型模板特异性结合的Tm值（T m1）大于其与突变型模板特异性结合的Tm值（T m2），且该野生型扩增阻滞物的3'末端具有阻滞基团。还提供了对应的扩增阻碍突变系统和核酸检测体系，其中包括与野生型扩增阻滞物竞争性结合模板的探针（结合野生型模板的Tm值为T m3，T m1＞T m3＞T m2）以及上下游的引物。提供的技术方案使得体系可以富集低丰度DNA突变并对多种碱基突变类型进行检测，具有灵敏度高、准确度高、成本较低、耗时较短、操作简便等优点。

Description

一种对低丰度突变DNA进行富集及检测的方法 技术领域
本发明涉及生物检测技术领域，具体涉及一种对低丰度突变DNA进行富集及检测的方法。
 背景技术
DNA在复制过程中往往会发生随机突变，在一些劣势环境中（如辐射、化学诱变剂等）突变频率往往会大幅增加。基因突变是生物进化的重要因素之一，也是生物遗传多样性的根本原因；但基因突变同时也会造成一些不良影响，如癌症的发生、病原微生物的耐药等等。这些突变往往混杂有大量的正常野生型基因，其低比例的突变含量往往给临床检测带来很大的困难，因此高灵敏度的突变检测技术在指导用药上具有很重要的意义。
近年来富集和/或检测低丰度突变DNA的技术主要有测序、数字PCR、低变性温度共扩增PCR（COLD-PCR）、引物扩增受阻突变系统（ARMS-PCR）、野生型阻滞扩增PCR（WTB-PCR）、变性高效液相色谱法（dHPLC）等。但这些方法在灵敏度、准确度、成本、耗时、操作等方面各有优缺点。
本领域技术人员希望开发一种新的对低丰度突变DNA进行富集及检测的方法，以克服现有技术中存在的在灵敏度、准确度、成本、耗时、操作等方面的不足。
 发明内容
本发明的目的在于提供一种对低丰度突变DNA进行富集及检测的方法，本发明提供的技术方案在野生型阻滞扩增PCR技术基础上进行优化改良得到，使得体系可以富集低丰度DNA突变并对多种碱基突变类型进行检测，具有灵敏度高、准确度高、成本较低、耗时较短、操作简便等优点。
为此，第一方面，本发明提供一种野生型扩增阻滞物，所述野生型扩增阻滞物与待测基因的野生型模板特异性结合的Tm值为T m1；所述野生型扩增阻滞物与待测基因的突变型模板特异性结合的Tm值为T m2；T m1＞T m2；所述野生型扩增阻滞物的3’末端具有阻滞基团。
根据本发明的技术方案，所述野生型扩增阻滞物与所述待测基因的结合位置覆盖所述待测基因的突变位点，由于T m1＞T m2，当在特定的退火温度范围内，所述野生型扩增阻滞物只与野生型模板结合，且由于其3’末端修饰有阻滞基团，从而无法继续延伸，从而阻断野生型模板的扩增。
进一步，所述阻滞基团包括选自双脱氧胞苷、反向dT、氨基、磷酸基团、间臂（例如间臂C3、间臂C5等）中的至少一种或其他可阻断所述野生型扩增阻滞物作为引物延伸的基团。
进一步，所述T m1与T m2的差值为5-20℃，例如约5℃、6℃、7℃、8℃、9℃、10℃、11℃、12℃、13℃、14℃、15℃、16℃、17℃、18℃、19℃、20℃等。
进一步，所述T m1为60-75℃，例如约60℃、61℃、62℃、63℃、64℃、65℃、66℃、67℃、68℃、69℃、70℃、71℃、72℃、73℃、74℃、75℃等。
在一些实施方式中，所述野生型扩增阻滞物与待测基因的野生型模板完全互补配对。
在一些实施方式中，所述野生型扩增阻滞物与待测基因所互补配对的至少一个、至少两个、至少三个、至少四个、至少五个、至少六个、至少七个、至少八个或至少九个碱基为RNA碱基。
根据本发明的技术方案，通过将野生型扩增阻滞物上与突变位置碱基互补配对的碱基替换为RNA碱基，可以进一步增大T m1和T m2的差值。
进一步，所述野生型扩增阻滞物上与待测基因的突变位置碱基所相对应的碱基位于所述野生型扩增阻滞物的中间、5’末端或3’末端。
进一步，所述野生型扩增阻遏物为寡核苷酸链，其长度为20-45bp，例如20bp、25bp、30bp、35bp、40bp、45bp等。
第二方面，本发明提供一种扩增阻碍突变系统，所述系统包括正向引物、反向引物、至少一组阻滞物-探针组合；所述阻滞物-探针组合包括探针和本发明所述的野生型扩增阻滞物；
所述正向引物和反向引物用于扩增所述待测基因，所述正向引物和反向引物与待测基因的结合位置分别位于所述野生型扩增阻滞物与待测基因的结合位置的上游和下游；
在每组阻滞物-探针组合中，所述探针与所述野生型扩增阻滞物竞争性结合所述待测基因；所述探针与所述待测基因的结合位置覆盖所述待测基因的突变位点。
根据本发明的技术方案，野生型扩增阻滞物位于正向引物和反向引物之间，与现有技术中野生型扩增阻滞物与扩增引物竞争性结合模板的技术方案相比，本发明的技术方案避免了因上述竞争性结合而影响野生型扩增阻滞物阻断野生型等位基因扩增的能力，以及引物退火结合突变型模板的效率。从而，本发明的技术方案进一步提高了对突变型等位基因进行检测的灵敏度。
在一些实施方式中，所述扩增阻碍突变系统包括两组以上所述阻滞物-探针组合，例如两组、三组、四组、五组、六组、七组、八组、九组、十组等。所述阻滞物-探针组合的数量可以根据待测基因的突变位点数量及位置等进行确定。从而实现在一个检测体系中对待测基因的多个突变位点实现同步检测。
进一步，所述探针的长度为15-40bp。
进一步，所述探针为荧光探针。
进一步，所述荧光探针的5’端修饰有荧光基团，选自FAM、HEX、ROX、TAMRA、Texas RED、CY5、Cy3、TET、JOE、VIC中的一种或多种；所述分子信标探针的3’端修饰有淬灭基团，选自BHQ1、BHQ2、Dabcy1、TAMRA、MGB、ECLIPSE中的一种或多种。
在一些实施方式中，所述探针为分子信标探针。
在一些实施方式中，所述探针具有以下修饰中的一种或两种：修饰为肽核酸（PNA）、锁核酸（LNA）。
进一步，所述探针与所述野生型扩增阻滞物存在碱基重叠，重叠的碱基数量不少于所述探针碱基总数的90%、80%、70%、60%、50%、40%、30%或20%。
进一步，所述探针与待测基因的野生型模板特异性结合的Tm值为T m3，T m1＞T m3＞T m2。
在某实施方式中，所述T m1与T m3的差值为5-15℃，例如5℃、6℃、7℃、8℃、9℃、10℃、11℃、12℃、13℃、14℃、15℃等。
进一步，所述正向引物、反向引物与待测基因特异性结合的Tm值分别为T m4-F和T m4-R，T m1＞T m4-F、T m4-R，进一步，55℃≤T m4-F、T m4-R≤70℃。
本发明的第三方面，提供一种核酸检测体系，其包括本发明第二方面所述的扩增阻碍突变系统，还包括RCR缓冲液、DNA聚合酶、dNTPs。
进一步，所述PCR缓冲液包括(NH 4) 2SO 4、MgCl 2、KCl、Tris-HCl等。
在一些实施方式中，所述PCR缓冲液中，(NH 4) 2SO 4的浓度为5-20 mM，例如5 mM、10 mM、15 mM、20 mM等；所述MgCl 2的浓度为2-3 mM，例如2 mM、2.5 mM、3 mm等；所述KCl的浓度为5-20 mM，例如5 mM、10 mM、15 mM、20 mM等；所述Tris-HCl的浓度为10-30 mM，例如10 mM、20 mM、30 mM等；所述Tris-HCl的pH值为8-8.5。
进一步，所述核酸检测体系中，所述野生型扩增阻滞物的终浓度为0.02-0.5 μM，例如0.02 μM、0.05 μM、0.1 μM、0.15 μM、0.2 μM、0.25 μM、0.3 μM、0.35 μM、0.4 μM、0.45 μM、0.5 μM等。
进一步，所述核酸检测体系中，所述探针的终浓度为0.02-0.5 μM，例如0.02 μM、0.05 μM、0.1 μM、0.15 μM、0.2 μM、0.25 μM、0.3 μM、0.35 μM、0.4 μM、0.45 μM、0.5 μM等。
进一步，所述核酸检测体系中，所述正向引物、反向引物的终浓度为0.02-0.5 μM，例如0.02 μM、0.05 μM、0.1 μM、0.15 μM、0.2 μM、0.25 μM、0.3 μM、0.35 μM、0.4 μM、0.45 μM、0.5 μM等。
进一步，所述核酸检测体系中，dNTPs的终浓度为0.2-0.5 mM，例如0.2 mM、0.3 mM、0.4 mM、0.5 mM等。
进一步，所述DNA聚合酶不具备5’→3’外切酶活性和3’→5’内切酶活性。
本发明的第四方面，提供一种核酸检测试剂盒，其包括本发明第三方面所述的核酸检测体系。
本发明的第五方面，提供一种核酸检测方法，其包括以下步骤：将待测基因的模板加入本发明所述的核酸检测体系中，进行PCR扩增和熔解曲线检测；
所述PCR扩增的反应程序包括：92-98℃变性10-30 s；在T m1温度条件下进行第一退火10-30 s；在T m4-F或T m4-R温度条件下进行第二退火10-30 s；在大于T m3且小于T m1的温度条件下延伸0.5-2 min；进行上述反应共45-55个循环。
进一步，在所述PCR扩增中，在首次变性之前还包括以下步骤：92-98℃预变性1-10 min。
在一些实施方式中，所述PCR扩增为不对称PCR扩增。
进一步，所述熔解曲线检测的反应程序包括：92-98℃，1min；T m1温度条件，1min；45-50℃，1min；升温至85℃，升温速率为0.01-0.05℃/s；升温过程中进行荧光信号采集。
在一些实施方式中，升温过程中每1℃进行荧光信号采集。
与现有技术相比，本发明的技术方案具有以下有益效果：
（1）本发明提供的野生型扩增阻遏物可大量富集低丰度突变DNA且不影响突变型模板的扩增效率，提高了检测的特异性和灵敏度，以满足临床检测突变基因的实际需求；
（2）本发明提供的探针可对扩增得到的低丰度突变DNA进行熔解曲线检测，区分其突变类型，同时可使用不同波长荧光标记基团，实现多通道、多靶标的单管检测。
 附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。在附图中：
图1为实施例1 FAM通道熔解曲线检测结果；
图2为实施例1 VIC通道熔解曲线检测结果；
图3为实施例1 Texas RED通道熔解曲线检测结果；
图4为实施例1 CY5通道熔解曲线检测结果；
图5为实施例2 FAM通道熔解曲线检测结果；其中，95A位点突变为95G；
图6为实施例2 Texas RED熔解曲线检测结果；其中1376G位点突变为1376T；
图7为实施例2 Texas RED熔解曲线检测结果；其中，1388G位点突变为1388A；
图8为实施例3 BRAF基因突变熔解曲线检测结果；
图9为实施例4 Texas通道检测CYP2C9-CC型熔解曲线检测结果；
图10为实施例4 Texas通道检测CYP2C9-AA型熔解曲线检测结果；
图11为实施例4 Texas通道检测CYP2C9-CA型熔解曲线检测结果；
图12为实施例4 CY5通道检测VKROC1-GG型熔解曲线检测结果；
图13为实施例4 CY5通道检测VKROC1-AA型熔解曲线检测结果；
图14为实施例4 CY5通道检测VKROC1-GA型熔解曲线检测结果；
图15为实施例5 Texas通道检测HPV16的熔解曲线检测结果；
图16为实施例5 Texas通道检测HPV18的熔解曲线检测结果；
图17为实施例5 Texas通道检测HPV31的熔解曲线检测结果；
图18为实施例5 Texas通道检测HPV33的熔解曲线检测结果；
图19为实施例5 FAM通道检测HPV35的熔解曲线检测结果；
图20为实施例5 FAM通道检测HPV39的熔解曲线检测结果；
图21为实施例5 FAM通道检测HPV45的熔解曲线检测结果；
图22为实施例5 FAM通道检测HPV51的熔解曲线检测结果；
图23为实施例5 CY5通道检测HPV52的熔解曲线检测结果；
图24为实施例5 CY5通道检测HPV53的熔解曲线检测结果；
图25为实施例5 CY5通道检测HPV56的熔解曲线检测结果；
图26为实施例5 CY5通道检测HPV58的熔解曲线检测结果；
图27为实施例5 VIC通道检测HPV59的熔解曲线检测结果；
图28为实施例5 VIC通道检测HPV66的熔解曲线检测结果；
图29为实施例5 VIC通道检测HPV68的熔解曲线检测结果；
图30为实施例5 VIC通道检测HPV73的熔解曲线检测结果；
图31为实施例5 VIC通道检测HPV82的熔解曲线检测结果。
 本发明的实施方式
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
本发明提供一种野生型扩增阻滞物（以下简称Blocker）、一种扩增阻碍突变系统及对应的核酸检测体系和检测方法。
本发明提供的Blocker与待测基因的野生型模板特异性结合的Tm值（T m1）大于其与突变型模板特异性结合的Tm值（T m2），并且在3’末端具有阻滞基团。在本发明提供的扩增阻碍突变系统中，探针与Blocker竞争性结合待测基因的模板，探针与野生型模板的特异性结合的Tm值为T m3（T m1＞T m3＞T m2）；正向引物、反向引物与待测基因特异性结合的Tm值分别为T m4-F和T m4-R（T m1＞T m4-F、T m4-R）。
当反应体系中只存在野生型模板时，在T m1温度条件下进行第一退火，Blocker优先与野生型等位基因完全互补结合；在T m4-F或T m4-R温度条件下进行第二退火，扩增引物与待测模板互补结合；在大于T m3且小于T m1的温度条件下进行延伸，扩增引物延伸至Blocker位置时，由于体系使用的聚合酶不具备5'→3'外切酶活性，Blocker无法被酶切而终止延伸，从而阻断野生型模板的扩增。
然而，实际检测中发现Blocker往往只能在一定程度上抑制野生型等位基因的扩增，当野生型DNA起始量过高时，存在一定的假阳性扩增。为了克服这一缺陷，本发明还利用探针进行后续的熔解曲线检测。在进行熔解曲线检测时，初始温度条件为92-98℃，使得PCR产物完全变性为单链；当温度降至T m1时，体系中的Blocker与探针竞争并且优先与可能存在的野生型扩增产物完全互补结合，这使得在后续温度降低的条件下，由于体系中可能存在的野生型扩增产物已显著减少，从而探针几乎全部与突变型扩增产物结合。这有效减少了假阳性扩增带来的干扰，提升了熔解曲线的检测结果的准确性。
当反应体系中只存在突变型模板时，在大于T m3且小于T m1的温度条件下进行延伸时，Blocker和探针都无法与模板结合，扩增引物与模板完全互补结合并延伸，从而使突变型模板得到大量扩增。
当体系中同时存在野生型和突变型模板时，在T m1温度条件下进行第一退火，Blocker优先与野生型等位基因完全互补结合；在T m4-F或T m4-R温度条件下进行第二退火，扩增引物同时与野生型和突变型模板完全互补结合并延伸；当其延伸野生型模板至Blocker位置时终止，当其延伸突变型模板可完全延伸并完成扩增，从而大量富集低丰度突变型DNA。
实施例
结核分枝杆菌的RNA聚合酶β亚单位编码基因（rpoB）第507-533位密码子出现氨基酸突变，就会导致对一线药物利福平（RIF）的耐药。本实施例以该突变区域为例，考察了野生型扩增阻滞物用于低丰度突变检测的能力，具体步骤如下：
（1）引物、探针、Blocker与野生型和突变型质粒的设计与合成
采用Primer Premier6.0、DNAMAN、ClustalX等软件，根据结核分枝杆菌rpoB基因上的利福平耐药突变核心区域设计引物，扩增片段长度为145bp；设计四条探针以完全覆盖该突变核心区域。为阻遏野生型模板的扩增，对应设计四条Blocker和探针竞争。
选取上、下游引物序列区域合成野生型质粒，并根据临床高频突变选择511CCG、516GTC、526TAC和531TTG突变密码子合成突变型质粒。
设计好的引物（F-rpoB、R-rpoB）、探针（P1-rpoB、P2-rpoB、P3-rpoB、P4-rpoB）和Blocker（B1-rpoB、B2-rpoB、B3-rpoB、B4-rpoB）均通过BLAST进行同源性比对以保证其特异性，各引物、探针与质粒均由专业公司合成。具体序列见表1所示。
表1 引物、探针、Blocker及质粒序列
名称 序列（5'→3'） SEQ ID NO：
F-rpoB GGCCGGTGGTCGCCGCGATCAAG 1
R-rpoB CCGGCACGCTCACGTGACAGA 2
P1-rpoB (FAM)-CATTGGCA CCTG CCAGCT GAGCCAATG-(BHQ1) 3
P2-rpoB (VIC)-CCTGCT CATA GAC CAGAACAACCCGCTGGCAGG-(BHQ1) 4
P3-rpoB (Texas)-CCTGCTCGGGGTAGACGCACAAGCGCGCAGG-(BHQ2) 5
P4-rpoB (CY5)-CTGCCCGACTGTCGGCGCTGGGCAG-(BHQ3) 6
B1-rpoB CGGCACCAGCCAGCUGAGCCAAU 7
B2-rpoB TCATGGACCAGAACAACCCGCUG 8
B3-rpoB TCGGGGTUGACCCACAAGCGC 9
B4-rpoB CCGACTGTCGGCGCTGGGG 10
野生型质粒（涉及耐药核心区的部分序列） GGAGCGGATGACCACCCAGGACGTGGAGGCGATCACACCGCAGACGTTGATCAACATCCGGCCGGTGGTCGCCGCGATCAAGGAGTTCTTCGGCACCAGCCAGCTGAGCCAATTCATGGACCAGAACAACCCGCTGTCGGGGTTGACCCACAAGCGCCGACTGTCGGCGCTGGGGCCCGGCGGTCTGTCACGTGAGCGTGCCGGGCTGGAGGTCCGCGACGTGCACCCGTCGCACTACGGCCGGATGTGCCCGATCGAAACCCCTGAGGGGCCCAACATCGGTCTGATCGGCTCGCTGTCGGT 11
突变型质粒（涉及耐药核心区的部分序列） GGAGCGGATGACCACCCAGGACGTGGAGGCGATCACACCGCAGACGTTGATCAACATCCGGCCGGTGGTCGCCGCGATCAAGGAGTTCTTCGGCACCAGCCAGCCGAGCCAATTCATGGTCCAGAACAACCCGCTGTCGGGGTTGACCTACAAGCGCCGACTGTTGGCGCCCGGGCCCGGCGGTCTGTCACGTGAGCGTGCCGGGCTGGAGGTCCGCGACGTGCACCCGTCGCACTACGGCCGGATGTGCCCGATCGAAACCCCTGAGGGGCCCAACATCGGTCTGATCGGCTCGCTGTCGGT 12
表1中，下划线示出了LNA修饰碱基，着重号示出了RNA碱基。
把野生型和突变型质粒分别调整到10 4 copies/μl。按下述方法配制为不同浓度的模板：
10%组：取10μl 10 4 copies/μl的突变型质粒并混入90μl同浓度的野生型质粒，振荡混匀。
5%组：取5μl 10 4 copies/μl的突变型质粒并混入95 μl同浓度的野生型质粒，振荡混匀。
1%组：取10μl 10%组的混合液并混入90μl 10 4 copies/μl的野生型质粒，振荡混匀。
0.5%组：取10μl 5%组的混合液并混入90μl 10 4 copies/μl的野生型质粒，振荡混匀。
0.1%组：取10μl 1%组的混合液并混入90μl 10 4 copies/μl的野生型质粒，振荡混匀。
0.01%组：取10μl 0.1%组的混合液并混入90μl 10 4 copies/μl的野生型质粒，振荡混匀。
野生型对照组：10 4 copies/μl的野生质粒。
（2）实时荧光PCR扩增体系的构建
反应体系为25μl，体系中包括dNTPs 0.2 mM、(NH 4) 2SO 4 10 mM、MgCl 2 3 mM、KCl 10 mM、pH为8.3的Tris-HCl 20 mM、1U DNA聚合酶、上游引物0.02 μM，下游引物0.2 μM，探针和Blocker各0.1 μM、5μl DNA模板，其余用水补足；
（3）PCR扩增与熔解曲线反应
将DNA模板在25μl反应体系中进行不对称PCR，扩增出大量单链DNA，之后再进行熔解曲线分析，具体反应程序如下：
PCR扩增的程序为：95℃，10min；（95℃，15s；82℃，20s，61℃，15s；75℃，30s反应50个循环）。
熔解曲线反应的程序为：95℃，1min；82℃，1min；45℃，1min，以0.05℃/s速率升温至85℃，升温过程中每1℃采集荧光信号。
结果与分析：
FAM通道野生型对照Tm值为74.1℃±1℃；VIC通道野生型对照Tm值为73.5℃±1℃；Texas通道野生型对照Tm值为74.4℃±1℃；CY5通道野生型对照Tm值为71.6℃±1℃。待检样本熔解曲线结果若与野生型对照Tm值相差≥2℃，则判定为突变型。
当反应体系中只存在野生型DNA时，Blocker与野生型等位基因完全互补结合，扩增引物DNA模板结合、延伸至Blocker位置后无法继续延伸，从而无荧光信号产生。但实际检测中发现Blocker只能在一定程度上抑制野生型等位基因的扩增，当野生型DNA起始用量过高时，存在一定的假阳性扩增。熔解曲线检测结果各荧光通道Tm值与野生型对照一致。
当反应体系中只存在突变型DNA时，Blocker与突变型等位基因的退火温度（68℃）低于扩增引物与DNA模板的延伸温度（75℃），因此在75℃延伸时扩增引物与突变型模板延伸，从而获得荧光信号。
结果如图1-4所示，野生对照出现野生熔解峰，阴性对照没有熔解峰出现，说明检测体系可以有效扩增，且没有污染。纯突变型只出现突变峰，5%、1%、0.5%、0.1%和0.01%组同时出现野生峰和突变峰。在图1-4中，1为野生型对照熔解峰，2为100%突变型样本熔解峰，3为5%突变含量混合样本熔解峰，4为1%突变含量混合样本熔解峰，5为0.5%突变含量混合样本熔解峰，6为0.1%突变含量混合样本熔解峰，7为0.01%突变含量混合样本熔解峰，8为阴性对照熔解峰。
实施例
葡萄糖-6-磷酸脱氢酶（G6PD）存在于人体各种组织及细胞中，参与磷酸戊糖途径。葡萄糖-6-磷酸脱氢酶缺乏症是一种伴性不完全显性遗传病，G6PD缺乏会大大增加新生儿患黄疸几率，如果不进行治疗或治疗不及时，可能导致核黄疸和永久性神经损伤或出现溶血性贫血。有研究表明，G6PD基因不同位点发生突变会导致G6PD酶活不同程度降低，其中95A>G，1376G>T，1388G>A突变是我国最常见的三种基因突变类型（占比70%），目前临床上将G6PD检查列为婚检，产检及新生儿检查等项目之一，从而实现优生优育。由于实际临床样本中存在突变型核酸与野生型核酸不同比例的情况，为能在混合样本中较好地扩增低丰度的突变型核酸，本实施例提供了可实现对G6PD突变型低丰度样本进行检测的方法，从而提高了检测灵敏度。
本实施例对葡萄糖-6-磷酸脱氢酶（G6PD）基因突变进行了检测，具体步骤如下：
（1）引物、探针与Blocker的设计与合成
采用Primer Premier6.0、DNAMAN、ClustalX等软件，分别对G6PD基因95A>G和1376G>T，1388G>A所在突变区域设计引物，扩增片段长度分别为141bp和150bp；设计两条探针以完全覆盖该突变核心区域。为阻遏野生型模板的扩增，对应设计两条Blocker和探针竞争。
表2 引物、探针及Blocker序列
名称 序列（5'→3'） SEQ ID NO：
F1-G6PD GGATCCTGCGGGAAGAGC 13
R1-G6PD GAAGGCCATCCCGGAACAG 14
F2-G6PD GCCTCATCCTGGACGTCTTC 15
R2-G6PD GGCCTCGGCTGCCATAAATA 16
P1-G6PD (FAM)-CGGATACACACATATTCATCATC-(BHQ1) 17
P2-G6PD (VIC)-AATACGCCAGGCCTCACGGAG-(BHQ1) 18
B1-G6PD TCGGATACACACATATT CAT CATCATGGG 19
B2-G6PD AAAATACG CCAGGCCTCACGGAGCT 20
表2中，下划线示出了LNA修饰碱基，着重号示出了RNA碱基。
（2）G6PD基因野生型和突变型质粒合成
由于实际临床样本难以获得，因此通过人工合成质粒进行验证。由专业公司合成G6PD-M野生型质粒，该质粒含95A、1376G、1388G未突变原始序列，选用载体为pUC-GW-Amp；并分别合成95A>G，1376G>T，1388G>A点突变质粒，分别命名为G6PD-95G-M、G6PD-1376T-M、G6PD-1388A-M。G6PD-M野生型质粒序列、突变型质粒序列如表3所示。
表3 G6PD野生型和突变型质粒序列
名称 序列（5'→3'） SEQ ID NO：
G6PD-M ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCGGGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATCATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGGTGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCCGTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGCCACCCCAGAGGAGAAGCTCTGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCGAGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCCCTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTTCGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCACCAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGCCCCACGGAGGCAGACGAGCTGATGAAGAGA 21
G6PD-95G-M ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCGGGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACAC GCATATTCATCATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGGTGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCCGTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGCCACCCCAGAGGAGAAGCTCTGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCGAGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCCCTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTTCGTGCGCAGCGACGAGCTCCGTGAGGCCTGGCGTATTTTCACCCCACTGCTGCACCAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGCCCCACGGAGGCAGACGAGCTGATGAAGAGA 22
G6PD-1376T-M ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCGGGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATCATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGGTGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCCGTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGCCACCCCAGAGGAGAAGCTCTGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCGAGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCCCTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTTCGTGCGCAGCGACGAGCTCC TTGAGGCCTGGCGTATTTTCACCCCACTGCTGCACCAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGCCCCACGGAGGCAGACGAGCTGATGAAGAGA 23
G6PD-1388A-M ATGGCAGAGCAGGTGGCCCTGAGCCGGACCCAGGTGTGCGGGATCCTGCGGGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGGATACACACATATTCATCATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCATCTGGTGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCCGTTCCCGCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCTTCAAGGCCACCCCAGAGGAGAAGCTCTGATGACCAAGAAGCCGGGCATGTTCTTCAACCCCGAGGAGTCGGAGCTGGACCTGACCTACGGCAACAGATACAAGAACGTGAAGCTCCCTGACGCCTATGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTTCGTGCGCAGCGACGAGCTCCGTGAGGCCTGGC ATATTTTCACCCCACTGCTGCACCAGATTGAGCTGGAGAAGCCCAAGCCCATCCCCTATATTTATGGCAGCCGAGGCCCCACGGAGGCAGACGAGCTGATGAAGAGA 24
（3）实时荧光PCR扩增体系
反应体系为25 μl，体系中包括dNTPs 0.2 mM、(NH 4) 2SO 4 10 mM、MgCl 2 3 mM、KCl 10 mM、pH为8.3的Tris-HCl 20 mM、2μl DNA聚合酶、上游引物0.02 μM，下游引物0.2 μM，探针和Blocker 0.1 μM、5μl DNA模板，其余用水补足；
（4）PCR扩增与熔解曲线反应程序
PCR扩增的程序为：95℃，变性10min；（95℃，15s；81℃，20s，63℃，15s；72℃，30s反应50个循环）。
熔解曲线反应的程序为：95℃，1min；81℃，1min；45℃，1min，以0.05℃/s速率升温至85℃，升温过程中每1℃采集荧光信号。
（5）结果分析
对本实施例（2）中提供的G6PD基因第95A位点突变为95G的质粒核酸（G6PD-95G-M，FAM通道，图5）、G6PD基因第1376G位点突变为1376T的质粒核酸（G6PD-1376T-M，Texas RED通道，图6）、G6PD基因第1388G位点突变为1388A的质粒核酸（G6PD-1388A-M，Texas RED通道，图7）进行检测，其结果如图5-7所示。图5中：1线为95A野生型熔解曲线，Tm值为77℃，2线为95A位点突变为95G突变型熔解曲线，Tm值为62℃，3线为95G突变型比例为50%的混合样本检测结果的熔解曲线，4线为阴性对照；图6中：1线为1376G野生型熔解曲线，Tm值为77.6℃，2线为1376T突变型熔解曲线，Tm值为64℃，3线为95G突变型比例为50%的混合样本检测结果的熔解曲线，4线为阴性对照；图7中：1线为1388G野生型熔解曲线，Tm值为77.6℃，2线为1388A突变型熔解曲线，Tm值为68.5℃，3线为95G突变型比例为50%的混合样本检测结果的熔解曲线，4线为阴性对照。
实施例
BRAF基因是位于7号染色体长臂上的一种癌基因，含有18个外显子，可编码一种含783个氨基酸的B-RAF蛋白，是EGFR通路RAS/RAF/MEK/MRK/MAPK中重要的转导因子，参与了细胞的生长、分化和凋亡等多种生理过程。研究表明，在非小细胞肺癌、结直肠癌等多种恶性肿瘤中存在BRAF基因突变，其中第15外显子上V600E突变最常见，约占90%以上。该突变导致B-RAF蛋白被被异常激活，从而使患者接受EGFR-TKI药物和EGFR单抗类药物治疗失败。根据中国2010版《肿瘤临床实践指南》建议对KRAS基因检测正常的非小细胞肺癌患者应进一步检查B-RAF基因突变状态，以指导靶向药物治疗方案。由于在癌症发生和发展过程中，少量的癌细胞从原发肿瘤组织脱落进入血液循环，取人外周血进行DNA提取时，提取的总DNA中突变基因所占比例较低，因此，采用本发明提供的技术方案可实现对低丰度B-RAF突变基因的富集和对突变碱基的检测。
本实施例对人BRAF基因突变进行了检测，具体步骤如下：
（1）引物、探针与Blocker的设计与合成
采用Primer Premier6.0、DNAMAN、ClustalX等软件，对BRAF基因1799T＞A所在突变区域设计引物，扩增片段长度为200bp；设计一条探针以完全覆盖该突变核心区域。为阻遏野生型模板的扩增，对应设计一条Blocker和探针竞争。
表4 BRAF引物、探针及Blocker序列
名称 序列（5'→3'） SEQ ID NO：
F1-BRAF ACTCTTCATAATGCTTGCTCTG 25
R1-BRAF CCTCAATTCTTACCATCCACAA 26
P1-BRAF (FAM)-CCACTCCATCGAGATTTCACTGTAGCTAGACC-(BHQ1) 27
B1-BRAF CCACTCCAUCGAGATTT CACTGTAGCTAGACC 28
表4中，单下划线示出了LNA修饰碱基，着重号示出了RNA碱基。
（2）BRAF基因野生型和突变型质粒合成
由于实际临床样本难以获得，因此通过人工合成质粒进行试验验证。由专业公司合成BRAF-1799T-M野生型质粒和BRAF-1799A-M突变型质粒，选用载体为pUC-GW-Amp；野生型质粒序列和突变型质粒序列如表4所示。
表5 BRAF野生型和突变型质粒序列
名称 序列（5'→3'） SEQ ID NO：
BRAF-1799T-M TATATAGGCTAAATAGAACTAATCATTGTTTTAGACATACTTATTGACTCTAAGAGGAAAGATGAAGTACTATGTTTTAAAGAATATTATATTACAGAATTATAGAAATTAGATCTCTTACCTAAACTCTTCATAATGCTTGCTCTGATAGGAAAATGAGATCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGTGAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCTGAGATGCTGCTGAGTTACTAGAAAGTCATTGAAGGTCTCAACTATAGTATTTTCATAGTTCCCAGTATTCACAAAAATCAGTGTTCTTATTTTTTATGTCCGAAGGCAGTTTGAACAGTTGTCTGGATCCCCTTCGG 29
BRAF-1799A-M TATATAGGCTAAATAGAACTAATCATTGTTTTAGACATACTTATTGACTCTAAGAGGAAAGATGAAGTACTATGTTTTAAAGAATATTATATTACAGAATTATAGAAATTAGATCTCTTACCTAAACTCTTCATAATGCTTGCTCTGATAGGAAAATGAGATCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG AGAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCTGAGATGCTGCTGAGTTACTAGAAAGTCATTGAAGGTCTCAACTATAGTATTTTCATAGTTCCCAGTATTCACAAAAATCAGTGTTCTTATTTTTTATGTCCGAAGGCAGTTTGAACAGTTGTCTGGATCCCCTTCGG 30
（3）实时荧光PCR扩增体系
反应体系为25 μl，体系中包括dNTPs 0.2 mM、(NH 4) 2SO 4 10 mM、MgCl 2 3 mM、KCl 10 mM、pH为8.3的Tris-HCl 20 mM、2 μl DNA聚合酶、上游引物0.02 μM，下游引物0.2 μM，探针和Blocker 0.1 μM、5 μl DNA模板，其余用水补足；
（4）PCR扩增与熔解曲线反应程序
PCR扩增的程序为：95℃，变性10min；（95℃，15s；80℃，20s，63℃，15s；70℃，30s反应50个循环）。
熔解曲线反应的程序为：95℃，1min；80℃，1min；45℃，1min，以0.05℃/s速率升温至85℃，升温过程中每1℃采集荧光信号。
（5）结果分析
对本实施例提供的的BRAF-1799T-M野生型质粒和BRAF-1799A-M突变型质粒，及其突变型比例为1‰的混合样本进行检测，结果如图8所示，图中1线为BRAF-1799T-M野生型熔解曲线，Tm值为70℃；2线为BRAF-1799A-M突变型熔解曲线，Tm值为62℃；3线为BRAF-1799A-M突变型比例为1‰的混合样本，4线为阴性对照。
实施例
华法林是一种临床上常用的适用于预防和治疗血栓栓塞性疾病的药物，是一种香豆素类抗凝剂。华法林用量过大会导致出血，而它的用量又受许多因素（主要是遗传和环境）影响，临床疗效和不良反应间个体差异大，剂量难以掌握，因此需要根据不同个体确定用量。
大量的研究表明，VKORC1启动子的多态性（-1639G/A）和CYP2C9（1075A/C）的突变是影响华法林用药的重要因素。
因此，本实施例对CYP2C9（rs1057910）和VKORC1（rs9923231）基因突变进行了检测，具体步骤如下：
针对CYP2C9（rs1057910）和VKORC1（rs9923231）所在的区域，设计4条引物、2条探针和2个阻断物序列，这些引物可以分别对两个基因进行特异性扩增。在扩增靶标区域针对突变型设计探针，这些探针能够特异性地结合到突变型的靶标序列上，利用熔解曲线Tm值的不同进行分型检测。另外，为了提高突变型的检测灵敏度，针对野生型设计了阻断物，能够一定程度上富集突变型样本。所有序列由专业公司合成。
表6. 引物、探针及Blocker序列
名称 序列（5'→3'） SEQ ID NO：
CYP2C9-1075A/C-F ATGCAAGACAGGAGCCACAT 31
CYP2C9-1075A/C-R TGGGAATGAGATAGTTTCTG 32
VKORC1-rs9923231-F agggatccctctgggaagtc 33
VKORC1-rs9923231-R ccacctcggcctcccaaaat 34
P-CYP2C9 (Texas)-ACGAG GTCCA GAGATACCTTGACCTTCT-(BHQ2) 35
P-VKORC1 (CY5)-acaaccatt ggccAggt gcggtggct-(BHQ3) 36
B-CYP2C9 GTGCACGAGGTCCAGAGATACATTGACCTTCTCCC 37
B-VKORC1 gaaaaacaaccattggccgggtgcggtggctcac 38
CYP2C9（1075C/C） ATGCAAGACAGGAGCCACATGCCCTACACAGATGCTGTGGTGCACGAGGTCCAGAGATACCTTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCCCA 39
CYP2C9（1075A/A ATGCAAGACAGGAGCCACATGCCCTACACAGATGCTGTGGTGCACGAGGTCCAGAGATACATTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCATTCCCA 40
VKORC1（-1639G/G） Agggatccctctgggaagtcaagcaagagaagacctgaaaaacaaccattggccgggtgcggtggctcacgcctataatcctagcattttgggaggccgaggtgg 41
VKORC1（-1639A/A） AgggatccctctgggaagtcaagcaagagaagacctgaaaaacaaccattggccAggtgcggtggctcacgcctataatcctagcattttgggaggccgaggtgg 42
表6中，下划线示出了LNA修饰碱基，着重号示出了RNA碱基。
（2）实时荧光PCR扩增体系的构建
反应体系为25μl，体系中包括dNTPs 0.3 mM、(NH 4) 2SO 4 8 mM、MgCl 2 3 mM、KCl 10 mM、pH为8.3的Tris-HCl 20 mM、2μl DNA聚合酶、上游引物0.02 μM，下游引物0.2 μM，探针和Blocker 0.1 μM、5μl DNA模板，其余用水补足；
（3）PCR扩增与熔解曲线反应
将DNA模板在25μl反应体系中进行不对称PCR，扩增出大量单链DNA，之后再进行熔解曲线分析，具体反应程序如下：
PCR扩增的程序为：95℃，10min；（95℃，15s；75℃，20s，61℃，15s；70℃，30s反应50个循环）。
熔解曲线反应的程序为：95℃，1min；75℃，1min；45℃，1min，以0.05℃/s速率升温至90℃，升温过程中每1℃采集荧光信号。
结果与分析：
Texas通道检测CYP2C9，CC型对照Tm值为69.8℃±1℃（图9），AA型对照的Tm值为65.4℃±1℃（图10），AC型对照出现一高一低的双峰，Tm值为65.6℃±1℃和Tm值为70.2℃±1℃（图11）；CY5通道检测VKROC1，GG型对照的Tm值为76.4℃±1℃（图12），AA型对照的Tm值为69.8℃±1℃（图13），GA型为一高一低的双峰，Tm值为70.2℃±1℃和Tm值为75.6℃±1℃（图14）。待检样本熔解曲线结果若与对照Tm值相差≤1℃，则判定为该分型。
实施例
人乳头状瘤病毒（Human Papilloma Virus，HPV）是一种双链DNA病毒，基因组编码3个区，分别为早期转录（Early Region，E区），晚期转录区（Late Region，L区）和上游调控区（LCR）。
目前已经发现的HPV类型有100多种，其中常见的低危型有HPV6、HPV11、HPV42、HPV43、HPV44等，低危型一般不会诱发癌症。常见的高危型有HPV16、HPV18、HPV31、HPV33、HPV35、HPV39、HPV49、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV68、HPV73、HPV82，大量的研究表明宫颈癌等女性生殖道恶性肿瘤的发生与高危型的HPV病毒反复感染有关。
本实施例对HPV分型进行了检测，具体步骤如下：
针对HPV较为保守的L1基因，设计3条上游引物和1条下游引物，这些引物可以特异性地扩增HPV的L1基因约150bp的片段。在扩增靶标区域设计17种特异性的探针，这些探针能够特异性地结合到对应分型的HPV扩增片段上，利用熔解曲线Tm值的不同进行分型检测。另外，为了提高低丰度样本的检测灵敏度，针对常见的低危型HPV（6、11、42、43、44）设计了5种阻断物，能够一定程度上富集低丰度样本。
灵敏度分析和特异性分析：
不同梯度阳性标准品的获得：经由专业公司合成的质粒粉末，加入标识体积的水，测定核酸浓度后计算质粒拷贝数，梯度稀释构建质粒标准品，获得浓度为10 7 copies/μl、10 6 copies/μl、10 5 copies/μl、10 4 copies/μl、10 3 copies/μl、10 2 copies/μl、10 copies/μl和1 copies/μl的不同分型的质粒标准品。
阴性对照：用含有0.9% NaCl，1mmol/L EDTA，10mmol/L Tris-HCl和0.1% Triton X-100的缓冲液稀释正常健康人类基因组DNA至10ng/μl。
低丰度混合样本的制备：用上述所用的稀释液制备出含1种高危型以及5种低危型的质粒混合液，使每种高危型质粒的最终浓度为10 copies/μl。
4、低丰度混合样本的检测：以不加阻断物为对照组，发现在不加阻断物的情况下，无法检测出10 copies/μl的质粒，但是加入阻断物的实验组依然可以检出，说明本发明所述方法可以提高低灵敏度样本的检出率。
本实施例对HPV高危型进行了分型鉴定，具体步骤如下：
（1）引物、探针与Blocker的设计与合成
采用Primer Premier6.0、DNAMAN、ClustalX等软件，根据HPV相对保守的L1基因设计通用引物，包括3条上游引物和1条下游引物，扩增片段长度约为145bp；设计17条对应每个分型的特异性探针。为阻遏常见的5种低危型的模板扩增，对应设计了五条Blocker和探针竞争。
设计好的引物（F1-HPVL1、F2-HPVL1、F3-HPVL1、R-HPVL1）、探针（P16-HPVL1、P18-HPVL1、P31-HPVL1、P33-HPVL1、P35-HPVL1、P39-HPVL1、P45-HPVL1、P51-HPVL1、P52-HPVL1、P53-HPVL1、P56-HPVL1、P58-HPVL1、P59-HPVL1、P66-HPVL1、P68-HPVL1、P73-HPVL1、P82-HPVL1）和Blocker（B6-HPVL1、B11-HPVL1、B42-HPVL1、B43-HPVL1、B44-HPVL1）均通过BLAST进行同源性比对以保证其特异性。由于实际临床样本难以获得，因此通过人工合成质粒进行验证。由专业公司合成。具体序列见表7所示。
表7. 引物、探针及Blocker序列
名称 序列（5'→3'） SEQ ID NO：
F1-HPVL1 TTTSTTACTGTDGTDGATACHAC 43
F2-HPVL1 TTTNTHACHGTHGTHGAYACYAC 44
F3-HPVL1 TTTTTATTACTTGTGTTGACAC 45
R-HPVL1 GAAAAAYAAAYTGTAADTCAWAYTC 46
P16-HPVL1 (Texas)-tgtg ctgccatat ctactt cagaaacta-(BHQ2) 47
P18-HPVL1 (Texas)-ttctacacagt ctcctgtac ctggg-(BHQ2) 48
P31-HPVL1 (Texas)-tgcaattg caaa cagtgatactaca-(BHQ2) 49
P33-HPVL1 (Texas)-acaagtaa ctagtga cagtacatat-(BHQ2) 50
P35-HPVL1 (FAM)-tg ctgtgt ctacta gt ga cagtaca-(BHQ1) 51
P39-HPVL1 (FAM)-tctatagagt cttc catac cttctaca-(BHQ1) 52
P45-HPVL1 (FAM)-tctaca caaaatcctgtgccaaatac-(BHQ1) 53
P51-HPVL1 (FAM)-tgccactgctgcggtttccccaaca-(BHQ1) 54
P52-HPVL1 (CY5)-tgaggttaaaaaggaaag cacatat-(BHQ3) 55
P53-HPVL1 (CY5)-aaccacacagt ctatgt ctacatat-(BHQ3) 56
P56-HPVL1 (CY5)-tg cta cagaa cagttaagtaaatatg-(BHQ3) 57
P58-HPVL1 (CY5)-tgaagtaa ctaag gaag gta catat-(BHQ3) 58
P59-HPVL1 (VIC)-ttctactacgtctt ctattc ctaatg-(BHQ1) 59
P66-HPVL1 (VIC)-ag ctaaaagca cattaa ctaaatatga-(BHQ1) 60
P68-HPVL1 (VIC)-tactacaga ctcta ctgta ccagct-(BHQ1) 61
P73-HPVL1 (VIC)-aggtacacagg ctagtag ct cta ct-(BHQ1) 62
P82-HPVL1 (VIC)-actgctgctactccatcagttg ca c-(BHQ1) 63
B6-HPVL1 tatgtgcatccgtaactacatcttccacatacacc 64
B11-HPVL1 tatgtgcatctgtgtctaaatctgctacatacact 65
B42-HPVL1 tgtgtgccactgcaacatctggtgatacatataca 66
B43-HPVL1 tatgtgcctctactgaccctactgtgcccagtaca 67
B44-HPVL1 tatgtgctgccactacacagtcccctccgtctaca 68
HPV16 tttgttactgttgttgatactacacgcagtacaaatatgtcattatgtgctgccatatctacttcagaaactacatataaaaatactaactttaaggagtacctacgacatggggaggaatatgatttacagtttatttttc 69
HPV18 TTTGTTACTGTGGTAGATACCACTCCCAGTACCAATTTAACAATATGTGCTTCTACACAGTCTCCTGTACCTGGGCAATATGATGCTACCAAATTTAAGCAGTATAGCAGACATGTTGAGGAATATGATTTGCAGTTTATTTTTC 70
HPV31 Tttgttactgtggtagataccacacgcagtaccaatatgtctgtttgtgctgcaattgcaaacagtgatactacatttaaaagtagtaattttaaagagtatttaagacatggtgaggaatttgatttacaatttatatttc 71
HPV33 Tttgttactgtggtagataccactcgcagtactaatatgactttatgcacacaagtaactagtgacagtacatataaaaatgagaattttaaagaatatataagacatgttgaagaatatgatctacagtttgtttttc 72
HPV35 Tttgttactgtagttgatacaacccgtagtacaaatatgtctgtgtgttctgctgtgtctactagtgacagtacatataaaaatgacaattttaaggaatatttaaggcatggtgaagaatatgatttacagtttatttttc 73
HPV39 Tttcttactgtagtggacactacccgtagtaccaactttacattatctacctctatagagtcttccataccttctacatatgatccttctaagtttaaggaatataccaggcacgtggaggagtatgatttacaatttatatttc 74
HPV45 Tttgttactgtagtggacactacccgcagtactaatttaacattatgtgcctctacacaaaatcctgtgccaaatacatatgatcctactaagtttaagcactatagtagacatgtggaggaatatgatttacagtttatttttc 75
HPV51 Tttattacctgtgttgatactaccagaagtacaaatttaactattagcactgccactgctgcggtttccccaacatttactccaagtaactttaagcaatatattaggcatggggaagagtatgaattgcaatttatttttc 76
HPV52 Tttgtcacagttgtggataccactcgtagcactaacatgactttatgtgctgaggttaaaaaggaaagcacatataaaaatgaaaattttaaggaataccttcgtcatggcgaggaatttgatttacaatttatttttc 77
HPV53 Tttgtaactgttgtggataccaccaggaatacaaacatgactctttccgcaaccacacagtctatgtctacatataattcaaagcaaattaaacagtatgttagacatgcagaggaatatgaattacaatttgtgtttc 78
HPV56 Tttgttactgtagtagatactactagaagtactaacatgactattagtactgctacagaacagttaagtaaatatgatgcacgaaaaattaatcagtaccttagacatgtggaggaatatgaattacaatttgtttttc 79
HPV58 Tttgttaccgtcgttgataccactcgtagcactaatatgacattatgcactgaagtaactaaggaaggtacatataaaaatgataattttaaggaatatgtacgtcatgttgaagaatatgacttacagtttgtttttc 80
HPV59 Tttttaacagttgtagatactactcgcagcaccaatctttctgtgtgtgcttctactacgtcttctattcctaatgtatacacacctaccagttttaaagaatatgccagacatgtggaggaatttgatttgcagtttatatttc 81
HPV66 Tttgttactgttgtggatactaccagaagcaccaacatgactattaatgcagctaaaagcacattaactaaatatgatgcccgtgaaatcaatcaataccttcgccatgtggaggaatatgaactacagtttgtgtttc 82
HPV68 Tttcttaccgttgtggatacaacgcgcagtactaattttacattgtccactactacagactctactgtaccagctgtttatgattctaataaatttaaggaatatgttaggcatgttgaggaatatgatttgcagtttatatttc 83
HPV73 Tttttaactgttgtagatactactagaagcactaatttttctgtatgtgtaggtacacaggctagtagctctactacaacgtatgccaactctaattttaaagaatatttaagacatgcagaagagtatgatttacagtttgtttttc 84
HPV82 tttttattacttgtgttgacactaccagaagtactaatttaaccattagcactgctgctactccatcagttgcacagacattcactccaacaaactttaagcagtatattaggcacggggaagaatatgaattacaatttatttttc 85
表7中，下划线示出了LNA修饰碱基，着重号示出了RNA碱基。
（2）实时荧光PCR扩增体系的构建
反应体系为25μl，体系中包括dNTPs 0.4 mM、(NH 4) 2SO 4 10 mM、MgCl 2 5 mM、KCl 15 mM、pH为8.3的Tris-HCl 20 mM、3.5μl DNA聚合酶、上游引物0.02 μM，下游引物0.2 μM，探针和Blocker 0.1 μM、5μl DNA模板，其余用水补足；
（3）PCR扩增与熔解曲线反应
将DNA模板在25μl反应体系中进行不对称PCR，扩增出大量单链DNA，之后再进行熔解曲线分析，具体反应程序如下：
PCR扩增的程序为：95℃，10min；（95℃，15s；75℃，20s，61℃，15s；70℃，30s反应50个循环）。
熔解曲线反应的程序为：95℃，1min；75℃，1min；45℃，1min，以0.05℃/s速率升温至85℃，升温过程中每1℃采集荧光信号。
结果与分析：
Texas通道HPV16对照的Tm值为75.3℃±1℃，HPV18对照的Tm值为70.5℃±1℃，HPV31对照的Tm值为65.3℃±1℃，HPV33对照的Tm值为60.2℃±1℃；FAM通道HPV35对照的Tm值为86.9℃±1℃，HPV39对照的Tm值为79.5℃±1℃；HPV45对照的Tm值为63.3℃±1℃，HPV51对照的Tm值为70.9℃±1℃；CY5通道HPV52对照的Tm值为60.4℃±1℃，HPV53对照的Tm值为,65.4℃±1℃,HPV56对照的Tm值为70.1℃±1℃,HPV58对照的Tm值为76.2℃±1℃；VIC通道HPV59对照的Tm值为71.7℃±1℃，HPV66对照的Tm值为66.90℃±1℃，HPV68对照的Tm值为75.6℃±1℃，HPV73对照的Tm值为82.5℃±1℃，HPV82对照的Tm值为61.8℃±1℃。待检样本熔解曲线结果若与对照Tm值相差≤2℃，则判定为该分型的HPV。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (10)

  1. 一种野生型扩增阻滞物，其特征在于，所述野生型扩增阻滞物与待测基因的野生型模板特异性结合的Tm值为T m1；所述野生型扩增阻滞物与待测基因的突变型模板特异性结合的Tm值为T m2；T m1＞T m2；所述野生型扩增阻滞物的3’末端具有阻滞基团。
  2. 如权利要求1所述的野生型扩增阻滞物，其特征在于，所述野生型扩增阻滞物与待测基因的野生型模板完全互补配对；
    优选地，所述野生型扩增阻滞物与待测基因互补配对的至少一个、至少两个、至少三个、至少四个、至少五个、至少六个、至少七个、至少八个或至少九个碱基为RNA碱基；
    优选地，所述T m1与T m2的差值为5-20℃；
    优选地，所述T m1为65-87℃；
    优选地，所述阻滞基团包括选自双脱氧胞苷、反向dT、氨基、磷酸基团、间臂中的至少一种。
  3. 一种扩增阻碍突变系统，其特征在于，所述扩增阻碍突变系统包括正向引物、反向引物、至少一组阻滞物-探针组合；所述阻滞物-探针组合包括探针和权利要求1或2所述的野生型扩增阻滞物；
    所述正向引物和反向引物用于扩增所述待测基因，所述正向引物和反向引物与待测基因的结合位置分别位于所述野生型扩增阻滞物与待测基因的结合位置的上游和下游；
    在每组阻滞物-探针组合中，所述探针与所述野生型扩增阻滞物竞争性结合所述待测基因；所述探针与所述待测基因的结合位置覆盖所述待测基因的突变位点。
  4. 如权利要求3所述的扩增阻碍突变系统，其特征在于，所述探针与所述野生型扩增阻滞物存在碱基重叠，重叠的碱基数量不少于所述探针碱基总数的90%、80%、70%、60%、50%、40%、30%或20%；
    优选地，所述探针与待测基因的野生型模板特异性结合的Tm值为T m3，T m1＞T m3＞T m2；
    优选地，所述T m1与T m3的差值为5-15℃。
  5. 如权利要求3所述的扩增阻碍突变系统，其特征在于，所述探针为荧光探针；
    优选地，所述荧光探针的5’端修饰有荧光基团，选自FAM、HEX、ROX、TAMRA、Texas RED、CY5、Cy3、TET、JOE、VIC中的一种或多种；所述分子信标探针的3’端修饰有淬灭基团，选自BHQ1、BHQ2、Dabcy1、TAMRA、MGB、ECLIPSE中的一种或多种；
    优选地，所述探针为分子信标探针；
    优选地，所述探针具有以下修饰中的一种或两种：修饰为肽核酸、锁核酸。
  6. 如权利要求3所述的扩增阻碍突变系统，其特征在于，所述正向引物、反向引物与待测基因特异性结合的Tm值分别为T m4-F和T m4-R，T m1＞T m4-F、T m4-R；
    优选地，55℃≤T m4-F、T m4-R≤70℃。
  7. 一种核酸检测体系，其特征在于，包括权利要求3-6任一项所述的扩增阻碍突变系统，还包括RCR缓冲液、DNA聚合酶、dNTPs。
  8. 如权利要求7所述的核酸检测体系，其特征在于，所述PCR缓冲液包括(NH 4) 2SO 4、MgCl 2、KCl、Tris-HCl；
    优选地，所述核酸检测体系中，所述野生型扩增阻滞物的终浓度为0.02-0.5 μM；
    优选地，所述核酸检测体系中，所述探针的终浓度为0.02-0.5 μM；
    优选地，所述核酸检测体系中，所述正向引物、反向引物的终浓度为0.02-0.5 μM；
    优选地，所述核酸检测体系中，dNTPs的终浓度为0.2-0.5 mM；
    优选地，所述DNA聚合酶不具备5’→3’外切酶活性和3’→5’外切酶活性。
  9. 一种核酸检测试剂盒，其特征在于，其包括权利要求7或8所述的核酸检测体系。
  10. 一种核酸检测方法，其特征在于，包括以下步骤：将待测基因的模板加入权利要求7所述的核酸检测体系中，进行PCR扩增和熔解曲线检测；
    所述PCR扩增的反应程序包括：92-98℃变性10-30 s；在T m1温度条件下进行第一退火10-30 s；在T m4-F或T m4-R温度条件下进行第二退火10-30 s；在大于T m3且小于T m1的温度条件下延伸0.5-2 min；进行上述反应共45-55个循环
    优选地，所述熔解曲线检测的反应程序包括：92-98℃，1min；T m1温度条件，1min；45-50℃，1min；升温至85℃，升温速率为0.01-0.05℃/s；升温过程中进行荧光信号采集。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113122629A (zh) * 2019-12-30 2021-07-16 香港城市大学深圳研究院 用于基因突变检测的发夹式扩增阻断法的引物和探针及其应用
CN117025765A (zh) * 2023-02-01 2023-11-10 珠海圣美生物诊断技术有限公司 多重数字pcr检测试剂盒及其检测方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114592046A (zh) * 2022-03-18 2022-06-07 广州迪澳基因科技有限公司 一种对低丰度突变dna进行富集及检测的方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102242211A (zh) * 2011-07-08 2011-11-16 无锡锐奇基因生物科技有限公司 检测突变核酸序列的方法及试剂盒
WO2013123031A2 (en) * 2012-02-15 2013-08-22 Veridex, Llc Highly sensitive method for detecting low frequency mutations
CN105420349A (zh) * 2014-09-17 2016-03-23 吉复生物科技有限公司 确定突变核酸碱基的方法及试剂盒
CN107034277A (zh) * 2017-04-27 2017-08-11 厦门大学 一种检测低丰度基因突变的方法
CN107604053A (zh) * 2017-08-16 2018-01-19 金绍莲 一种野生型扩增阻滞物在制备检测基因痕量突变试剂中的应用及其试剂盒和检测方法
CN111004839A (zh) * 2019-12-18 2020-04-14 杭州瑞普基因科技有限公司 基于锁核酸修饰的阻滞物进行阻滞取代扩增富集检测目标突变的方法
CN111118119A (zh) * 2019-12-18 2020-05-08 杭州瑞普基因科技有限公司 基于引入额外碱基错配的阻滞物进行阻滞取代扩增富集检测目标突变的方法
WO2020151283A1 (zh) * 2019-01-21 2020-07-30 北京大学 一种基于选择性消除野生链背景干扰的基因突变检测方法
CN114592046A (zh) * 2022-03-18 2022-06-07 广州迪澳基因科技有限公司 一种对低丰度突变dna进行富集及检测的方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4505839B2 (ja) * 2004-05-18 2010-07-21 アークレイ株式会社 Ｃｙｐ２ｄ６＊４の変異の検出法ならびにそのための核酸プローブおよびキット
CN101580879B (zh) * 2009-04-30 2012-04-11 广西医科大学 检测结核杆菌耐药基因膜芯片
WO2018014682A1 (zh) * 2016-07-20 2018-01-25 广州易活生物科技有限公司 Pcr技术与efirm技术结合检测靶标基因的方法
CN108048531B (zh) * 2017-11-16 2021-06-18 苏州吉玛基因股份有限公司 一种高灵敏度检测稀有突变的超阻滞荧光定量pcr方法
CN107841576A (zh) * 2017-12-07 2018-03-27 广州凯普医药科技有限公司 14种高危型人乳头状瘤病毒E6/E7区mRNA检测试剂盒
CN111020031A (zh) * 2019-12-10 2020-04-17 银丰基因科技有限公司 序列特异性阻断剂结合特定pcr程序检测肿瘤基因突变的方法

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102242211A (zh) * 2011-07-08 2011-11-16 无锡锐奇基因生物科技有限公司 检测突变核酸序列的方法及试剂盒
WO2013123031A2 (en) * 2012-02-15 2013-08-22 Veridex, Llc Highly sensitive method for detecting low frequency mutations
CN105420349A (zh) * 2014-09-17 2016-03-23 吉复生物科技有限公司 确定突变核酸碱基的方法及试剂盒
CN107034277A (zh) * 2017-04-27 2017-08-11 厦门大学 一种检测低丰度基因突变的方法
CN107604053A (zh) * 2017-08-16 2018-01-19 金绍莲 一种野生型扩增阻滞物在制备检测基因痕量突变试剂中的应用及其试剂盒和检测方法
WO2020151283A1 (zh) * 2019-01-21 2020-07-30 北京大学 一种基于选择性消除野生链背景干扰的基因突变检测方法
CN111004839A (zh) * 2019-12-18 2020-04-14 杭州瑞普基因科技有限公司 基于锁核酸修饰的阻滞物进行阻滞取代扩增富集检测目标突变的方法
CN111118119A (zh) * 2019-12-18 2020-05-08 杭州瑞普基因科技有限公司 基于引入额外碱基错配的阻滞物进行阻滞取代扩增富集检测目标突变的方法
CN114592046A (zh) * 2022-03-18 2022-06-07 广州迪澳基因科技有限公司 一种对低丰度突变dna进行富集及检测的方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113122629A (zh) * 2019-12-30 2021-07-16 香港城市大学深圳研究院 用于基因突变检测的发夹式扩增阻断法的引物和探针及其应用
CN117025765A (zh) * 2023-02-01 2023-11-10 珠海圣美生物诊断技术有限公司 多重数字pcr检测试剂盒及其检测方法

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