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WO2022042399A1 - Procédé d'amplification génique et son application - Google Patents

Procédé d'amplification génique et son application Download PDF

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Publication number
WO2022042399A1
WO2022042399A1 PCT/CN2021/113340 CN2021113340W WO2022042399A1 WO 2022042399 A1 WO2022042399 A1 WO 2022042399A1 CN 2021113340 W CN2021113340 W CN 2021113340W WO 2022042399 A1 WO2022042399 A1 WO 2022042399A1
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Prior art keywords
seq
competitor
amplification
kit
sequence
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Chinese (zh)
Inventor
何逖
覃嘉嘉
田进
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Shanghai Genechem Clinical Laboratory Inc
SHANGHAI GENECHEM CO Ltd
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Shanghai Genechem Clinical Laboratory Inc
SHANGHAI GENECHEM CO Ltd
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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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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

Definitions

  • the invention relates to the field of genes, and more particularly to a gene amplification method involving competitors, wherein the amplification efficiency of the competitors is controllable.
  • Designing an amplification method that includes competitors can solve the problem of misjudgment caused by differences in detection signals caused by differences in sample quality, and can also infer the copy number of the target gene or the content of the target gene based on the amplification of the competitor. Therefore, it is often necessary to amplify the gene of interest using an amplification method involving a competitor.
  • the competitor used is an artificially synthesized sequence
  • the artificially synthesized sequence has high purity and low amplification steric hindrance, which is incompatible with the amplification efficiency of gDNA.
  • the amplification efficiency is more than a thousand times that of gDNA. Thus, it is not easy to debug to find the most suitable competitor concentration.
  • the amplification efficiency is generally reduced by continuously diluting the competitor, which is not only cumbersome and time-consuming, but also has poor reproducibility at concentrations as low as one-tenth of a pg level, and the current technology cannot Accurate quantification leads to problems such as large gaps between batches and difficulty in stable storage.
  • problems due to the high amplification efficiency of the competitor, problems such as accidental mixing into the negative control and the failure of the experiment often occur during operation.
  • the purpose of the present invention is to provide a gene amplification method in which the amplification efficiency of the competitor is controllable, and is effectively used for amplification.
  • the present invention provides an amplification method, comprising using a pair of locked nucleic acid-modified amplification primers to simultaneously amplify a target gene and its competitors.
  • it is used to control the amplification efficiency of the competitor, so that the amplification efficiency of the competitor is close to or preferably equal to the amplification efficiency of the target gene.
  • one or two primers in the pair of amplification primers are modified by locked nucleic acid; preferably, one or more bases of one or two primers in the pair of amplification primers are Locked nucleic acid modification; more preferably wherein the base at the 3' end of one or both primers in the pair of amplification primers is modified with locked nucleic acid.
  • the target gene is selected from the following group of genes or a combination thereof: SMN1, SMN2, ALK, RET, ROS1, NTRK1, NTRK2, NTRK3.
  • amplification primer pair is selected from the sequences described in SEQ ID NOs: 1-72 or different combinations thereof.
  • the competitor comprises a nucleotide sequence with different bases compared with the target gene sequence, and the number of different bases is 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10.
  • At least one of the different bases is located in the nucleotide sequence of the competitor corresponding to the amplification primer pair; preferably, at least one of the different bases is located in the competitor corresponding to the nucleotide sequence The nucleotide site of the base that is modified by the locked nucleic acid in the amplification primer pair.
  • the bases at the 3' ends of one or both primers in the pair of amplification primers are modified by locked nucleic acid, and the bases modified by the locked nucleic acid in the pair of amplification primers correspond to all The bases of the target gene are different from those of its competitors.
  • the competitor is an artificially synthesized single-stranded or double-stranded nucleic acid molecule; preferably an artificially synthesized single-stranded or double-stranded DNA molecule; more preferably an artificially synthesized plasmid.
  • the competitor is selected from the sequences described in SEQ ID NOs: 113-145 or different combinations thereof.
  • the final concentration of the competitor is 0.05-250ng/ ⁇ l, 0.1-200ng/ ⁇ l, 0.1-150ng/ ⁇ l, 0.1-100ng/ ⁇ l, 0.1-90ng/ ⁇ l, 0.1-80ng/ ⁇ l, 0.1-70ng/ ⁇ l, 0.1-60ng/ ⁇ l, 0.1-50ng/ ⁇ l, 0.1-40ng/ ⁇ l, 0.1-30ng/ ⁇ l, 0.1-20ng/ ⁇ l, 0.1-10ng/ ⁇ l, 0.5-100ng/ ⁇ l, 0.5- 90ng/ ⁇ l, 0.5-80ng/ ⁇ l, 0.5-70ng/ ⁇ l, 0.5-60ng/ ⁇ l, 0.5-50ng/ ⁇ l, 0.5-40ng/ ⁇ l, 0.5-30ng/ ⁇ l, 0.5-20ng/ ⁇ l, 0.5-10ng/ ⁇ l, 1-100ng/ ⁇ l, 1-90ng/ ⁇ l, 1-80ng/ ⁇ l, 1-70ng/ ⁇ l, 1-60ng/ ⁇ l, 1-50ng/ ⁇ l, 1-40ng/ ⁇ l, 0.1
  • the sample loading amount of the competitor is 5-80ng, 10-70ng, 15-60ng, 20-50ng, 20-40ng or 20-30ng, preferably 20-40ng.
  • the amplification method is used in technologies such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Taqman-PCR, next-generation sequencing, capillary electrophoresis analysis, or digital PCR.
  • the present invention provides a kit comprising a pair of locked nucleic acid-modified amplification primers and a competitor of the target gene.
  • the kit further comprises amplification reagents selected from the group consisting of dNTP, DNA polymerase, MgCl 2 and combinations thereof.
  • one or two primers in the pair of amplification primers are modified by locked nucleic acid; preferably, one or more bases of one or two primers in the pair of amplification primers are Locked nucleic acid modification; more preferably wherein the base at the 3' end of one or both primers in the pair of amplification primers is modified with locked nucleic acid.
  • amplification primer pair is selected from the sequences described in SEQ ID NOs: 1-72 or different combinations thereof.
  • the competitor comprises a nucleotide sequence with different bases compared with the target gene sequence, and the number of different bases is 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10.
  • At least one of the different bases is located in the nucleotide sequence of the competitor corresponding to the amplification primer pair; preferably, at least one of the different bases is located in the competitor corresponding to the nucleotide sequence The nucleotide site of the base that is modified by the locked nucleic acid in the amplification primer pair.
  • the bases at the 3' ends of one or both primers in the pair of amplification primers are modified by locked nucleic acid, and the bases modified by the locked nucleic acid in the pair of amplification primers correspond to all The bases of the target gene are different from those of its competitors.
  • the competitor is an artificially synthesized single-stranded or double-stranded nucleic acid molecule; preferably an artificially synthesized single-stranded or double-stranded DNA molecule; more preferably an artificially synthesized plasmid.
  • the competitor is selected from the sequences described in SEQ ID NOs: 113-145 or different combinations thereof.
  • the final concentration of the competitor is 0.05-250ng/ ⁇ l, 0.1-200ng/ ⁇ l, 0.1-150ng/ ⁇ l, 0.1-100ng/ ⁇ l, 0.1-90ng/ ⁇ l, 0.1-80ng/ ⁇ l, 0.1-70ng/ ⁇ l, 0.1-60ng/ ⁇ l, 0.1-50ng/ ⁇ l, 0.1-40ng/ ⁇ l, 0.1-30ng/ ⁇ l, 0.1-20ng/ ⁇ l, 0.1-10ng/ ⁇ l, 0.5-100ng/ ⁇ l, 0.5- 90ng/ ⁇ l, 0.5-80ng/ ⁇ l, 0.5-70ng/ ⁇ l, 0.5-60ng/ ⁇ l, 0.5-50ng/ ⁇ l, 0.5-40ng/ ⁇ l, 0.5-30ng/ ⁇ l, 0.5-20ng/ ⁇ l, 0.5-10ng/ ⁇ l, 1-100ng/ ⁇ l, 1-90ng/ ⁇ l, 1-80ng/ ⁇ l, 1-70ng/ ⁇ l, 1-60ng/ ⁇ l, 1-50ng/ ⁇ l, 1-40ng/ ⁇ l, 0.1
  • the sample loading amount of the competitor is 5-80ng, 10-70ng, 15-60ng, 20-50ng, 20-40ng or 20-30ng, preferably 20-40ng.
  • the kit is used to detect spinal muscular atrophy in a subject.
  • the kit is used to detect cancer in a subject; preferably, lung cancer, hematological tumor, thyroid cancer, bile duct cancer, soft tissue sarcoma, breast cancer, gastric cancer, esophageal cancer or colorectal cancer.
  • the present invention also provides the use of the kit for controlling the amplification efficiency of the competitor in the simultaneous amplification of the target gene and its competitor.
  • the kit is used to amplify a gene selected from the group consisting of SMN1, SMN2, ALK, RET, ROS1, NTRK1, NTRK2, and NTRK3.
  • the present invention also provides the use of locked nucleic acid to control the amplification efficiency of the competitor in the simultaneous amplification of the target gene and its competitor.
  • the locked nucleic acid is used to modify the primer pair for amplifying the target gene and its competitor.
  • the target gene is selected from the following group of genes or a combination thereof: SMN1, SMN2, ALK, RET, RO1, NTRK1, NTRK2, NTRK3.
  • the primer pair is selected from the primer sequences described in SEQ ID NO: 1-72.
  • the present invention also provides the use of the locked nucleic acid modified primer pair to control the amplification efficiency of the competitor in the simultaneous amplification of the target gene and its competitor.
  • the amplification primer pair is selected from the sequences described in SEQ ID NOs: 1-72 or a combination thereof.
  • the competitor sequence (5' ⁇ 3') of the target gene is selected from the sequences described in SEQ ID NOs: 113-145 or a combination thereof.
  • the pair of amplification primers, competitors and/or extension primers may be further preferably combined, for example:
  • the amplification primer pair is selected from the sequences described in SEQ ID NO: 1-10 or a combination thereof
  • the competitor is selected from the sequences described in SEQ ID NO: 113-117 or The combination thereof; wherein the preferred example is used to detect gene mutation and/or copy number of SMN1 and/or SMN2 gene.
  • an extension primer is also included, and the extension primer is selected from the sequences described in SEQ ID NOs: 73-79 or a combination thereof.
  • the amplification primer pair is selected from the sequences described in SEQ ID NO: 17-44 or a combination thereof
  • the competitor is selected from the sequences described in SEQ ID NO: 118-131 or The combination thereof; wherein the preferred example is used to detect the gene fusion mutation of ALK and/or RET and/or ROS1 gene.
  • an extension primer is also included, and the extension primer is selected from the sequences described in SEQ ID NOs: 85-98 or a combination thereof.
  • the amplification primer pair is selected from the sequences described in SEQ ID NOs: 45-72 or a combination thereof
  • the competitor is selected from the sequences described in SEQ ID NOs: 132-145 or The combination thereof; wherein the preferred embodiment is used to detect gene fusion mutations of NTRK1 and/or NTRK2 and/or NTRK3 genes.
  • an extension primer is also included, and the extension primer is selected from the sequences described in SEQ ID NOs: 99-112 or a combination thereof.
  • the sequence of the amplification primer pair is SMN1-2_E5_F (SEQ ID NO:3) and SMN1-2_E5_R (SEQ ID NO:4), and the sequence of the competitor of the target gene (5 ' ⁇ 3') is SMN1-2_E5_QC (SEQ ID NO: 114).
  • the extension primer sequence is SMN1-2_E5_W1_E (SEQ ID NO:74).
  • the sequence of the amplification primer pair is SMN1-2_E6_F (SEQ ID NO:5), SMN1-2_E6_R (SEQ ID NO:6), and the sequence of the competitor of the target gene ( 5' ⁇ 3') is SMN1-2_E6_QC (SEQ ID NO: 115).
  • the extension primer sequence is SMN1-2_E6_W1_E (SEQ ID NO:75).
  • the sequence of the amplification primer pair is SMN1-2_E7_TY_TYI_W1_F (SEQ ID NO:7), SMN1-2_E7_TY_TYI_W1_R (SEQ ID NO:8), and the sequence of the competitor of the target gene ( 5' ⁇ 3') is SMN1-2_E7_QC (SEQ ID NO: 116).
  • the extension primer sequence is SMN1-2_E7_TY_W1_E (SEQ ID NO:76) or SMN1-2_E7_TYI_W1_E (SEQ ID NO:77).
  • the sequence of the amplification primer pair is SMN1-2_E8_TY_TYI_W1_F (SEQ ID NO:9), SMN1-2_E8_TY_TYI_W1_R (SEQ ID NO:10), and the sequence of the competitor of the target gene ( 5' ⁇ 3') is SMN1-2_E8_QC (SEQ ID NO: 117).
  • the extension primer sequence is SMN1-2_E8_TY_W1_E (SEQ ID NO:78) or SMN1-2_E8_TYI_W1_E (SEQ ID NO:79).
  • the sequence of the amplification primer pair is RPP40_F (SEQ ID NO: 1), RPP40_R (SEQ ID NO: 2), and the competitor sequence of the target gene (5' ⁇ 3 ') is RPP40_QC (SEQ ID NO: 113).
  • the extension primer sequence is RPP40#2_W1_E (SEQ ID NO:73).
  • sequence of the amplification primer pair is ALK_01-02_F (SEQ ID NO: 21), ALK_01-02_R (SEQ ID NO: 22), and the sequence of the competitor of the target gene (5' ⁇ 3') is ALK_01-02_QC (SEQ ID NO: 120).
  • the extension primer sequence is ALK_01-02_E (SEQ ID NO:87).
  • sequence of the amplification primer pair is ALK_21-22_F (SEQ ID NO:23), ALK_21-22_R (SEQ ID NO:24), and the sequence of the competitor of the target gene (5' ⁇ 3') is ALK_21-22_QC (SEQ ID NO: 121).
  • the extension primer sequence is ALK_21-22_E (SEQ ID NO:88).
  • the amplification primer pair sequence is ALK_22-23_F (SEQ ID NO:25), and ALK_22-23_R (SEQ ID NO:26), and the competitor sequence of the target gene (5' ⁇ 3') is ALK_22-23_QC (SEQ ID NO: 122).
  • the extension primer sequence is ALK_22-23_E (SEQ ID NO:89).
  • sequence of the amplification primer pair is ALK_23-24_F (SEQ ID NO:27), ALK_23-24_R (SEQ ID NO:28), and the sequence of the competitor of the target gene (5' ⁇ 3') is ALK_23-24_QC (SEQ ID NO: 123).
  • the extension primer sequence is ALK_23-24_E (SEQ ID NO:90).
  • the sequence of the amplification primer pair is RET_02-03_F (SEQ ID NO:29), RET_02-03_R (SEQ ID NO:30), and the sequence of the competitor of the target gene (5' ⁇ 3') is RET_02-03_QC (SEQ ID NO: 124).
  • the extension primer sequence is RET_02-03_E (SEQ ID NO: 91).
  • the sequence of the amplification primer pair is RET_04-05_F (SEQ ID NO:31), RET_04-05_R (SEQ ID NO:32), and the sequence of the competitor of the target gene (5' ⁇ 3') is RET_04-05_QC (SEQ ID NO: 125).
  • the extension primer sequence is RET_04-05_E (SEQ ID NO:92).
  • the amplification primer pair sequence is RET_12-13_F (SEQ ID NO:33), and RET_12-13_R (SEQ ID NO:34), and the competitor sequence of the target gene (5' ⁇ 3') is RET_12-13_QC (SEQ ID NO: 126).
  • the extension primer sequence is RET_12-13_E (SEQ ID NO:93).
  • the amplification primer pair sequence is RET_13-14_F (SEQ ID NO:35), and RET_13-14_R (SEQ ID NO:36), and the competitor sequence of the target gene (5' ⁇ 3') is RET_13-14_QC (SEQ ID NO: 127).
  • the extension primer sequence is RET_13-14_E (SEQ ID NO:94).
  • the amplification primer pair sequence is ROS1_01-02_F (SEQ ID NO:37), and ROS1_01-02_R (SEQ ID NO:38), and the competitor sequence of the target gene (5' ⁇ 3') is ROS1_01-02_QC (SEQ ID NO: 128).
  • the extension primer sequence is ROS1_01-02_E (SEQ ID NO:95).
  • the amplification primer pair sequence is ROS1_04-05_F (SEQ ID NO:39), and ROS1_04-05_R (SEQ ID NO:40), and the competitor sequence of the target gene (5' ⁇ 3') is ROS1_04-05_QC (SEQ ID NO: 129).
  • the extension primer sequence is ROS1_04-05_E (SEQ ID NO:96).
  • the amplification primer pair sequence is ROS1_35-36_F (SEQ ID NO:41), and ROS1_35-36_R (SEQ ID NO:42), and the competitor sequence of the target gene (5' ⁇ 3') is ROS1_35-36_QC (SEQ ID NO: 130).
  • the extension primer sequence is ROS1_35-36_E (SEQ ID NO:97).
  • the amplification primer pair sequence is ROS1_37-38_F (SEQ ID NO:43), and ROS1_37-38_R (SEQ ID NO:44), and the competitor sequence of the target gene (5' ⁇ 3') is ROS1_37-38_QC (SEQ ID NO: 131).
  • the extension primer sequence is ROS1_37-38_E (SEQ ID NO:98).
  • sequence of the amplification primer pair is EML4_11-12_F (SEQ ID NO: 17), EML4_11-12_R (SEQ ID NO: 18), and the sequence of the competitor of the target gene (5' ⁇ 3') is EML4_11-12_QC (SEQ ID NO: 118).
  • the extension primer sequence is EML4_11-12_E (SEQ ID NO:85).
  • sequence of the amplification primer pair is EML4_13-14_F (SEQ ID NO: 19), EML4_13-14_R (SEQ ID NO: 20), and the sequence of the competitor of the target gene (5' ⁇ 3') is EML4_13-14_QC (SEQ ID NO: 119).
  • the extension primer sequence is EML4_13-14_E (SEQ ID NO:86).
  • the sequence of the amplification primer pair is NTRK1_07-08_F (SEQ ID NO:49), and NTRK1_07-08_R (SEQ ID NO:50), and the sequence of the competitor of the target gene (5' ⁇ 3') is NTRK1_07-08_QC (SEQ ID NO: 134).
  • the extension primer sequence is NTRK1_07-08_E (SEQ ID NO: 101).
  • sequence of the amplification primer pair is NTRK1_08-09_F (SEQ ID NO:51), and NTRK1_08-09_R (SEQ ID NO:52), and the sequence of the competitor of the target gene (5' ⁇ 3') is NTRK1_08-09_QC (SEQ ID NO: 135).
  • the extension primer sequence is NTRK1_08-09_E (SEQ ID NO: 102).
  • the sequence of the amplification primer pair is NTRK1_13-14_F (SEQ ID NO:53), and NTRK1_13-14_R (SEQ ID NO:54), and the sequence of the competitor of the target gene (5' ⁇ 3') is NTRK1_13-14_QC (SEQ ID NO: 136).
  • the extension primer sequence is NTRK1_13-14_E (SEQ ID NO: 103).
  • the sequence of the amplification primer pair is NTRK1_14-15_F (SEQ ID NO:55), NTRK1_14-15_R (SEQ ID NO:56), and the sequence of the competitor of the target gene (5' ⁇ 3') is NTRK1_14-15_QC (SEQ ID NO: 137).
  • the extension primer sequence is NTRK1_14-15_E (SEQ ID NO: 104).
  • the sequence of the amplification primer pair is NTRK2_09-10_F (SEQ ID NO:57), NTRK2_09-10_R (SEQ ID NO:58), and the sequence of the competitor of the target gene (5' ⁇ 3') is NTRK2_09-10_QC (SEQ ID NO: 138).
  • the extension primer sequence is NTRK2_09-10_E (SEQ ID NO: 105).
  • the sequence of the amplification primer pair is NTRK2_10-11_F (SEQ ID NO:59), and NTRK2_10-11_R (SEQ ID NO:60), and the sequence of the competitor of the target gene (5' ⁇ 3') is NTRK2_10-11_QC (SEQ ID NO: 139).
  • the extension primer sequence is NTRK2_10-11_E (SEQ ID NO: 106).
  • the sequence of the amplification primer pair is NTRK2_13-14_F (SEQ ID NO:61), and NTRK2_13-14_R (SEQ ID NO:62), and the sequence of the competitor of the target gene (5' ⁇ 3') is NTRK2_13-14_QC (SEQ ID NO: 140).
  • the extension primer sequence is NTRK2_13-14_E (SEQ ID NO: 107).
  • the sequence of the amplification primer pair is NTRK2_14-15_F (SEQ ID NO:63), NTRK2_14-15_R (SEQ ID NO:64), and the sequence of the competitor of the target gene (5' ⁇ 3') is NTRK2_14-15_QC (SEQ ID NO: 141).
  • the extension primer sequence is NTRK2_14-15_E (SEQ ID NO: 108).
  • the sequence of the amplification primer pair is NTRK3_09-10_F (SEQ ID NO:65), and NTRK3_09-10_R (SEQ ID NO:66), and the sequence of the competitor of the target gene (5' ⁇ 3') is NTRK3_09-10_QC (SEQ ID NO: 142).
  • the extension primer sequence is NTRK3_09-10_E (SEQ ID NO: 109).
  • the sequence of the amplification primer pair is NTRK3_10-11_F (SEQ ID NO:67), and NTRK3_10-11_R (SEQ ID NO:68), and the sequence of the competitor of the target gene (5' ⁇ 3') is NTRK3_10-11_QC (SEQ ID NO: 143).
  • the extension primer sequence is NTRK3_10-11_E (SEQ ID NO: 110).
  • the sequence of the amplification primer pair is NTRK3_14-15_F (SEQ ID NO:69), and NTRK3_14-15_R (SEQ ID NO:70), and the sequence of the competitor of the target gene (5' ⁇ 3') is NTRK3_14-15_QC (SEQ ID NO: 144).
  • the extension primer sequence is NTRK3_14-15_E (SEQ ID NO: 111).
  • the amplification primer pair sequence is NTRK3_15-16_F (SEQ ID NO:71), and NTRK3_15-16_R (SEQ ID NO:72), and the competitor sequence of the target gene (5' ⁇ 3') is NTRK3_15-16_QC (SEQ ID NO: 145).
  • the extension primer sequence is NTRK3_15-16_E (SEQ ID NO: 112).
  • the amplification primer pair sequence is TPM3_08-09_F (SEQ ID NO:45), and TPM3_08-09_R (SEQ ID NO:46), and the competitor sequence of the target gene (5' ⁇ 3') is TPM3_08-09_QC (SEQ ID NO: 132).
  • the extension primer sequence is TPM3_08-09_E (SEQ ID NO:99).
  • the amplification primer pair sequence is TPM3_10-11_F (SEQ ID NO:47), and TPM3_10-11_R (SEQ ID NO:48), and the competitor sequence of the target gene (5' ⁇ 3') is TPM3_10-11_QC (SEQ ID NO: 133).
  • the extension primer sequence is TPM3_10-11_E (SEQ ID NO: 100).
  • the combination of amplification primer pairs, competitors and/or extension primers may be further combined into a combination comprising multiple amplification primer pairs, multiple competitors and/or multiple extension primers .
  • Figure 1 shows the mass spectrometry results of the amplification products of exon 5 of the SMN1 and SMN2 genes of samples 11, 17, 20, and 21.
  • Figure 2 shows the mass spectrometry results of the amplified products of exon 6 of SMN1 and SMN2 genes of samples 11, 17, 20, and 21.
  • Figure 3 shows the mass spectrometry results of the amplification products of exon 7 of the SMN1 and SMN2 genes of samples 11, 17, 20, and 21.
  • Figure 4 shows the mass spectrometry results of the amplification products of exon 8 of SMN1 and SMN2 genes of samples 11, 17, 20, and 21.
  • Figure 5 shows the mass spectrometry results of the RPP40 gene amplification products of samples 11, 17, 20, and 21.
  • Figure 6 shows mass spectrometry results of amplification products using locked nucleic acid-modified primers and non-locked nucleic acid-modified primers.
  • Figure 7 shows the qPCR amplification curve of SMN1-2_E5 using locked nucleic acid-modified primers and non-locked nucleic acid-modified primers, in which four samples 1, 2, 3, and 4 were performed, and 3 samples were performed for each sample.
  • 1 refers to SMN1-2_E5 feeding 5 ng unlocked nucleic acid primer
  • 2 refers to SMN1-2_E5 feeding 1 ng unlocked nucleic acid primer
  • 3 refers to SMN1-2_E5 feeding 5 ng locked nucleic acid primer
  • 4 refers to SMN1-2_E5 feeding 1 ng locked nucleic acid primer.
  • Figure 8 shows the qPCR amplification curve of RPP40 using locked nucleic acid-modified primers and non-locked nucleic acid-modified primers, in which four samples 1, 2, 3, and 4 were performed, and each sample was performed with 3 replicates , 1 refers to RPP40 feeding 5 ng non-locked nucleic acid primer, 2 refers to RPP40 feeding 1 ng non-locked nucleic acid primer, 3 refers to RPP40 feeding 5 ng locked nucleic acid primer (near the three repeated test curves of number 3), 4 refers to RPP40 feeding 1 ng locked nucleic acid primer (close to number 3) Three replicate test curves for number 4).
  • Figure 9 shows the comparison of MassArray mass spectrometry results of the ALK gene.
  • Figure 10 shows a comparison of MassArray mass spectrometry results of the ROS1 gene.
  • Figure 11 shows a comparison of MassArray mass spectrometry results for the RET gene.
  • Figure 12 shows a comparison of MassArray mass spectrometry results for the EML4 gene.
  • Figure 13 shows a comparison of MassArray mass spectrometry results of the NTRK1 gene.
  • Figure 14 shows a comparison of MassArray mass spectrometry results of the NTRK2 gene.
  • Figure 15 shows a comparison of MassArray mass spectrometry results of the NTRK3 gene.
  • Figure 16 shows a comparison of MassArray mass spectrometry results for the TPM3 gene.
  • Figure 17 shows Sanger sequencing results.
  • the inventors provide an optimized technical solution: for a specific site of the target gene, design a section of artificially synthesized competitor and a pair of 3' Amplification primer pair containing locked nucleic acid at the ends.
  • the inventors use the above competitors, primer pairs and nucleic acid samples to carry out amplification reactions, and then analyze the amplification products in combination with mass spectrometry, and found that this technical solution can simply and significantly reduce the amplification efficiency of competitors, while the amplification of target genes no effect.
  • the inventors found that the copy number variation and hot point mutation of the SMN1 gene and the SMN2 gene can be detected at the same time; in addition, the present invention It also realized the detection of the expression of six genes ALK, ROS1, RET, NTRK1, NTRK2, and NTRK3 using MassArray technology, so as to determine whether the six genes of ALK, ROS1, RET, NTRK1, NTRK2, and NTRK3 have gene fusion mutations. Fusions of six genes were detected.
  • MassARRAY technology system its basic principle is matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology, which has extremely high specificity and sensitivity.
  • the system uses matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to precisely detect DNA molecules. Genetic variants are distinguished by analyzing their individual mass, eliminating the need for fluorescence or labeling.
  • MassARRAY technology Compared with existing technologies such as RT-PCR, MLPA, and NGS, MassARRAY technology has certain advantages.
  • RT-PCR technology uses the amplification of fluorescent signals. It is necessary to use competitive probes and other methods to eliminate the interference of similar sites. It is difficult to effectively eliminate the interference, resulting in data deviation and difficult interpretation.
  • RT-PCR technology can only detect one fragment in one reaction tube, and multiple reaction tubes are required for multiplex detection. It is easy to cause differences between reaction wells, and the cost is not low, and the detection throughput is limited.
  • MLPA technology has strict requirements on the quality of the template.
  • the amplified probe is a fluorescent probe.
  • the signal intensity is obtained by collecting fluorescence, which is costly and causes signal deviation.
  • CNV the adjacent fragments should also be considered.
  • One reaction well of MLPA technology can only judge the copy number, but cannot interpret other sites such as mutations, the throughput is limited, and the process is more complicated.
  • NGS can only be used for primary screening, and suspected positive results need to be verified by methods such as MLPA. In terms of copy number analysis, NGS has low accuracy and reliability and high cost.
  • MassArray technology does not require high sample quality, and genomic DNA extracted from dried blood spots can also be well detected.
  • the difference in molecular weight between different bases is used to distinguish the detection site, and the base type of the detected detection point can be directly and accurately detected. No fluorescent probes are used, which avoids the fluorescence interference of similar sites, and the cost is low.
  • MassaArray technology completes the detection of all sites in one reaction well, with low cost and high detection throughput.
  • SMA spinal muscular atrophy, spinal muscular atrophy or spinal muscular atrophy
  • spinal muscular atrophy is an autosomal recessive progressive motor neuron disease characterized by progressive degeneration of the anterior horn cells of the spinal cord and the motor nuclei of the brainstem. as the main feature.
  • the main clinical manifestations are progressive, symmetrical muscle weakness and atrophy, the proximal end is heavier than the distal end, and the lower extremity is heavier than the upper extremity. Children with this disease account for about 1/10,000 of newborns, and about 1/50 carriers of the disease-causing gene.
  • SMA1 spinal muscular atrophy type I
  • SMA3 childhood-onset or adolescent-onset spinal muscular atrophy type III
  • SMA4 adult-onset Spinal muscular atrophy type IV
  • SMA1 spinal muscular atrophy type 1
  • the SMN1 gene is located on chromosome 5, with a total length of about 20kb and 9 exons. It is highly homologous to its immediate neighbors SMN2 and SMN1, differing by only 5 nucleotides.
  • SMN2 is a regulatory gene whose copy number is inversely proportional to the severity of SMA.
  • ALK Chinese name is Anaplastic Lymphoma Kinase (Anaplastic Lymphoma Kinase), this gene is responsible for encoding a receptor tyrosine kinase (Receptor Tyrosine Kinase, RTK) called ALK, which is a member of the receptor tyrosine kinase family
  • RTK receptor Tyrosine Kinase
  • ALK gene rearrangement allows ALK to undergo phosphorylation before dimerization. Therefore, the ALK fusion protein will continue to be activated and activate its downstream pathways, resulting in excessive cell proliferation and tumorigenesis.
  • ROS1 is a proto-oncogene and is highly expressed in a variety of tumor cell lines.
  • ROS1 belongs to a family of tyrosine kinase insulin receptors.
  • the protein encoded by it is a type I membrane protein with tyrosine kinase activity, which can be used as a receptor for growth factors or differentiation factors; the common pathogenic mutation of ROS1 gene is gene rearrangement, and the resulting ROS1 fusion protein It becomes a persistently activated tyrosine kinase, which activates signaling in its downstream pathways, resulting in cell overgrowth and proliferation.
  • RET is a proto-oncogene, responsible for encoding a transmembrane protein called RET, which is a receptor tyrosine kinase; pathogenic mutations in the RET gene—mutation or rearrangement, activate the RET gene, and It is possible to encode a RET protein with abnormal activity, which will transmit abnormal signals and cause multiple effects: including cell growth, survival, invasion, metastasis, etc. Continued signaling leads to excessive proliferation of cells, thus leading to tumorigenesis and progression.
  • NRTK Neurotrophic receptor tyrosine kinase (Neurotrophic Receptor Kinase).
  • the NTRK gene includes three subtypes: NTRK1, NTRK2, and NTRK3, which are located on different chromosomes.
  • the protein encoded by NTRK is called TRK protein, and NTRK1, NTRK2, and NTRK3 encode three proteins, TRKA, TRKB, and TRKC, respectively.
  • NTRK gene fusion mutations are caused by the fusion of NTRK gene family members (NTRK1, NTRK2, NTRK3) with another unrelated gene (usually a housekeeping gene), and the encoded TRK fusion protein is in a structurally activated state, triggering a persistent signal
  • the cascade reaction drives the malignant proliferation of cells, leading to carcinogenesis.
  • Gene fusion mutation refers to linking the coding regions of two or more genes end to end.
  • a chimeric gene is formed under the control of the same set of regulatory sequences (including promoters, enhancers, ribosome binding sequences, terminators, etc.).
  • the above-mentioned ALK, ROS1, RET, NTRK1, NTRK2, NTRK3 gene fusion mutation detection is of great significance for clinical diagnosis and treatment and prognosis, and also has an important guiding role for clinical selection of targeted drugs.
  • Competitors refers to a substance that differs from the sample to be tested by at least 1 nucleotide and whose molecular weight and/or sequence and/or content are known. It is added to the sample to be tested, and the content of the substance to be tested is compared to determine the content of the sample to be tested. Competitors are also referred to in the art as competitive substrates, internal standards, internal control DNA, competitive oligonucleotides, and the like.
  • Competitors in different regions often have different characteristics. Competitors may consist of natural and/or non-natural nucleotides (eg, labeled nucleotides) or mixtures thereof. Competitors suitable for use in the present invention can be synthesized and labeled using known techniques. Competitors can be chemically synthesized or purified according to any known suitable method.
  • the competitors used for a particular region are the same length. In other embodiments, the lengths of competitors used for different regions are different.
  • the present invention provides the use of a locked nucleic acid for controlling the amplification efficiency of the competitor in the simultaneous amplification of a target gene and its competitor, so that the amplification efficiency of the competitor is close to or preferably equal to the The amplification efficiency of the target gene.
  • the locked nucleic acid is used to modify the primer pair for amplifying the target gene and its competitor.
  • the present invention provides the use of a locked nucleic acid modified primer for controlling the amplification efficiency of the competitor in the simultaneous amplification of a target gene and its competitor, so that the amplification efficiency of the competitor is close to or It is preferably equal to the amplification efficiency of the gene of interest.
  • Locked nucleic acid is a synthetic antisense oligonucleotide, which is a special bicyclic nucleotide derivative in which the ribose ring ( ⁇ -D- The 2'-oxygen and 4'-carbon of ribofuranose) form a methylene connection by shrinkage, and the structure contains one or more 2'-O-4'-C-methylene- ⁇ -D-ribofuranose nucleic acid Monomer, the 2'-O and 4'-C positions of ribose form oxymethylene bridges, thiomethylene bridges or amine methylene bridges through different shrinkage effects, and connect them into a ring, this ring bridge locks The N configuration of furanose C3'-endotype reduces the flexibility of the ribose structure and increases the stability of the local structure of the phosphate backbone.
  • LNA and DNA/RNA have the same phosphate backbone in structure, they have good recognition ability and strong affinity for DNA and RNA.
  • Locked nucleic acids are generally incorporated into PCR probes, which are soluble in water and standard buffers and follow the Watson-Crick base pairing rules, which offer several advantages over natural DNA bases, These include: higher thermal stability and hybridization specificity, more accurate gene quantification and allele identification, and easier and more flexible probe design for problematic target sequences.
  • the present invention uses a pair of primers modified by locked nucleic acid, and preferably, the primers have the same sequence end corresponding to the target gene to be amplified, and the primers have different sequence ends corresponding to the target gene to be amplified. There is a mismatch at the end of the sequence corresponding to the competitor of the increased target gene. And in the specific embodiment of the present invention, one or both of the primer pairs used to amplify the target gene and the competitor of the target gene are modified with locked nucleic acid, which can significantly reduce the artificial synthesis of low amplification steric hindrance. Amplification efficiency of competitors.
  • amplification primer pair is selected from the sequences shown in Table 1 or a combination thereof.
  • the present invention provides an amplification method, comprising using a pair of locked nucleic acid modified amplification primers to simultaneously amplify a target gene and its competitor.
  • it is used to control the amplification efficiency of the competitor, so that the amplification efficiency of the competitor is close to or preferably equal to the amplification efficiency of the target gene.
  • one or both primers in the pair of amplification primers are modified with a locked nucleic acid; preferably wherein bases at one or more positions of one or both primers in the pair of amplification primers bases are modified with locked nucleic acids; more preferably wherein the bases at the 3' ends of one or both primers in the pair of amplification primers are modified with locked nucleic acids.
  • the gene of interest is selected from the following group of genes or a combination thereof: SMN1, SMN2, ALK, RET, ROS1, NTRK1, NTRK2, NTRK3.
  • the amplification primer pair is selected from the sequences set forth in SEQ ID NOs: 1-72 or a combination thereof.
  • the competitor comprises a nucleotide sequence with different bases compared with the target gene sequence
  • the number of the different bases is 1, 2, 3, 4, 5 , 6, 7, 8, 9 or 10.
  • at least one of the different bases is located in the nucleotide sequence of the competitor corresponding to the amplification primer pair; preferably, at least one of the different bases is located in the competitor corresponding to the nucleotide sequence The nucleotide site of the base that is modified by the locked nucleic acid in the amplification primer pair.
  • the bases at the 3' ends of one or both primers in the pair of amplification primers are modified by locked nucleic acid, and the bases modified by the locked nucleic acid in the pair of amplification primers correspond to The bases of the target gene are different from those of its competitors.
  • the competitor is an artificially synthesized single-stranded or double-stranded nucleic acid molecule; preferably an artificially synthesized single-stranded or double-stranded DNA molecule; more preferably an artificially synthesized plasmid.
  • the competitor is selected from the sequence set forth in SEQ ID NO: 113-145 or a combination thereof.
  • the final concentration of the competitor is 0.05-250ng/ ⁇ l, 0.1-200ng/ ⁇ l, 0.1-150ng/ ⁇ l, 0.1-100ng/ ⁇ l, 0.1-90ng/ ⁇ l, 0.1-80ng/ ⁇ l ⁇ l, 0.1-70ng/ ⁇ l, 0.1-60ng/ ⁇ l, 0.1-50ng/ ⁇ l, 0.1-40ng/ ⁇ l, 0.1-30ng/ ⁇ l, 0.1-20ng/ ⁇ l, 0.1-10ng/ ⁇ l, 0.5-100ng/ ⁇ l, 0.5-90ng/ ⁇ l, 0.5-80ng/ ⁇ l, 0.5-70ng/ ⁇ l, 0.5-60ng/ ⁇ l, 0.5-50ng/ ⁇ l, 0.5-40ng/ ⁇ l, 0.5-30ng/ ⁇ l, 0.5-20ng/ ⁇ l, 0.5- 10ng/ ⁇ l, 1-100ng/ ⁇ l, 1-90ng/ ⁇ l, 1-80ng/ ⁇ l, 1-70ng/ ⁇ l, 1-60ng/ ⁇ l, 1-50ng/ ⁇ l, 1
  • the loading amount of the competitor is 5-80ng, 10-70ng, 15-60ng, 20-50ng, 20-40ng or 20-30ng, preferably 20-40ng.
  • the amplification method described above can be used in techniques such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Taqman-PCR, next-generation sequencing, capillary electrophoresis analysis, or digital PCR.
  • the present invention provides a kit comprising a pair of locked nucleic acid modified amplification primers and a competitor of a target gene.
  • the kit further comprises amplification reagents selected from the group consisting of dNTPs, DNA polymerase, MgCl2 , and combinations thereof.
  • one or both primers in the pair of amplification primers are modified with a locked nucleic acid; preferably wherein bases at one or more positions of one or both primers in the pair of amplification primers bases are modified with locked nucleic acids; more preferably wherein the bases at the 3' ends of one or both primers in the pair of amplification primers are modified with locked nucleic acids.
  • amplification primer pair is selected from the sequences set forth in SEQ ID NOs: 1-72 or a combination thereof.
  • the competitor comprises a nucleotide sequence with different bases compared with the target gene sequence, and the number of the different bases is 1, 2, 3, 4, 5 , 6, 7, 8, 9 or 10.
  • At least one of the different bases is located in the nucleotide sequence of the competitor corresponding to the amplification primer pair; preferably, at least one of the different bases is located in the competitor corresponding to the nucleotide sequence of the amplification primer pair; Amplification primer pairs are nucleotide sites of bases modified by locked nucleic acids.
  • the bases at the 3' ends of one or both primers in the pair of amplification primers are modified by locked nucleic acid, and the bases modified by the locked nucleic acid in the pair of amplification primers correspond to The bases of the target gene are different from those of its competitors.
  • the competitor is an artificially synthesized single-stranded or double-stranded nucleic acid molecule; preferably an artificially synthesized single-stranded or double-stranded DNA molecule; more preferably an artificially synthesized plasmid.
  • the competitor is selected from the sequence set forth in SEQ ID NO: 113-145 or a combination thereof.
  • the final concentration of the competitor is 0.05-250ng/ ⁇ l, 0.1-200ng/ ⁇ l, 0.1-150ng/ ⁇ l, 0.1-100ng/ ⁇ l, 0.1-90ng/ ⁇ l, 0.1-80ng/ ⁇ l ⁇ l, 0.1-70ng/ ⁇ l, 0.1-60ng/ ⁇ l, 0.1-50ng/ ⁇ l, 0.1-40ng/ ⁇ l, 0.1-30ng/ ⁇ l, 0.1-20ng/ ⁇ l, 0.1-10ng/ ⁇ l, 0.5-100ng/ ⁇ l, 0.5-90ng/ ⁇ l, 0.5-80ng/ ⁇ l, 0.5-70ng/ ⁇ l, 0.5-60ng/ ⁇ l, 0.5-50ng/ ⁇ l, 0.5-40ng/ ⁇ l, 0.5-30ng/ ⁇ l, 0.5-20ng/ ⁇ l, 0.5- 10ng/ ⁇ l, 1-100ng/ ⁇ l, 1-90ng/ ⁇ l, 1-80ng/ ⁇ l, 1-70ng/ ⁇ l, 1-60ng/ ⁇ l, 1-50ng/ ⁇ l, 1
  • the loading amount of the competitor is 5-80ng, 10-70ng, 15-60ng, 20-50ng, 20-40ng or 20-30ng, preferably 20-40ng.
  • the kit is used to detect Spinal Muscular Atrophy (SMA) (eg caused by SMN1 or SMN2 gene mutations or copy number abnormalities) in a subject.
  • SMA Spinal Muscular Atrophy
  • the kit is used to detect cancer in a subject; for example, cancer formed by fusion mutations of the following genes: ALK (eg, lung cancer, hematological tumors), ROS1 (eg, lung cancer), RET (eg, Lung cancer, thyroid cancer), NTRK (eg, various adult and pediatric tumors), FGFR (cholangiocarcinoma, lung squamous cell carcinoma), and multiple other fusion genes (blood tumors, soft tissue sarcomas), and copy number abnormalities (CNVs) such as HER2 (breast cancer, gastric cancer, esophageal cancer, colorectal cancer), etc.
  • ALK eg, lung cancer, hematological tumors
  • ROS1 eg, lung cancer
  • RET eg, Lung cancer, thyroid cancer
  • NTRK eg, various adult and pediatric tumors
  • FGFR cholangiocarcinoma, lung squamous cell carcinoma
  • CNVs copy number abnormalities
  • the present invention also provides the use of the kit for controlling the amplification efficiency of the competitor in the simultaneous amplification of the target gene and its competitor.
  • amplification primer pair sequences are selected from the sequences set forth in SEQ ID NOs: 1-72.
  • the competitor sequences of the target gene are selected from the sequences shown in SEQ ID NOs: 113-145.
  • the amplification primer pair is selected from the sequence of SEQ ID NO: 1-10 or a combination thereof
  • the competitor is selected from The sequences of SEQ ID NOs: 113-117 or a combination thereof; wherein the kit can be used to detect gene mutation and/or copy number of SMN1 and/or SMN2 genes.
  • an extension primer is also included, and the extension primer is selected from the sequences described in SEQ ID NOs: 73-79 or a combination thereof.
  • kits wherein in the kit, wherein the amplification primer pair is selected from the sequences of SEQ ID NO: 17-44 or a combination thereof, and the competitor is selected from the group of SEQ ID NO: 118- 131 The sequence or a combination thereof; wherein the kit can be used to detect gene fusion mutations in the ALK and/or RET and/or ROS1 genes.
  • an extension primer is also included, and the extension primer is selected from the sequences described in SEQ ID NOs: 85-98 or a combination thereof.
  • kits wherein in the kit, wherein the amplification primer pair is selected from the sequences of SEQ ID NO: 45-72 or a combination thereof, and the competitor is selected from the group of SEQ ID NO: 132- 145 the sequence or a combination thereof; wherein the kit can be used to detect gene fusion mutations of NTRK1 and/or NTRK2 and/or NTRK3 genes.
  • an extension primer is also included, and the extension primer is selected from the sequences described in SEQ ID NOs: 99-112 or a combination thereof.
  • the kit is an amplification kit for exon 5 of SMN1 and SMN2, wherein the sequence of the amplification primer pair is SMN1-2_E5_F (SEQ ID NO: 3), and SMN1 -2_E5_R (SEQ ID NO: 4), and the competitor sequence (5' ⁇ 3') of the target gene is SMN1-2_E5_QC (SEQ ID NO: 114).
  • the kit further comprises an extension primer, and the sequence of the extension primer is SMN1-2_E5_W1_E (SEQ ID NO:74).
  • the kit is an amplification kit for exon 6 of SMN1 and SMN2, wherein the sequence of the amplification primer pair is SMN1-2_E6_F (SEQ ID NO: 5), and SMN1 -2_E6_R (SEQ ID NO: 6), and the competitor sequence (5' ⁇ 3') of the gene of interest is SMN1-2_E6_QC (SEQ ID NO: 115).
  • the kit further comprises an extension primer, and the sequence of the extension primer is SMN1-2_E6_W1_E (SEQ ID NO:75).
  • the kit is an amplification kit for exon 7 of SMN1 and SMN2, wherein the sequence of the amplification primer pair is SMN1-2_E7_TY_TYI_W1_F (SEQ ID NO: 7), and SMN1 -2_E7_TY_TYI_W1_R (SEQ ID NO: 8), and the competitor sequence (5' ⁇ 3') of the target gene is SMN1-2_E7_QC (SEQ ID NO: 116).
  • the kit further comprises an extension primer, and the extension primer sequence is SMN1-2_E7_TY_W1_E (SEQ ID NO:76) or SMN1-2_E7_TYI_W1_E (SEQ ID NO:77).
  • the kit is an amplification kit for the 8th exon of SMN1 and SMN2, wherein the sequence of the amplification primer pair is SMN1-2_E8_TY_TYI_W1_F (SEQ ID NO: 9), and SMN1 -2_E8_TY_TYI_W1_R (SEQ ID NO: 10), and the competitor sequence (5' ⁇ 3') of the target gene is SMN1-2_E8_QC (SEQ ID NO: 117).
  • the kit further comprises an extension primer, and the extension primer sequence is SMN1-2_E8_TY_W1_E (SEQ ID NO:78) or SMN1-2_E8_TYI_W1_E (SEQ ID NO:79).
  • the kit is an amplification kit for the sixth intron of RPP40, wherein the sequence of the amplification primer pair is: RPP40_F (SEQ ID NO: 1), and RPP40_R (SEQ ID NO: 1) NO: 2), and the competitor sequence (5' ⁇ 3') of the target gene is RPP40_QC (SEQ ID NO: 113).
  • the kit further comprises an extension primer, and the sequence of the extension primer is RPP40#2_W1_E (SEQ ID NO:73).
  • the kit is an amplification kit for exons 1 and 2 of ALK, wherein the sequence of the amplification primer pair is ALK_01-02_F (SEQ ID NO: 21), and ALK_01-02_R (SEQ ID NO: 22), and the competitor sequence (5' ⁇ 3') of the target gene is ALK_01-02_QC (SEQ ID NO: 120).
  • the kit further comprises an extension primer, and the sequence of the extension primer is ALK_01-02_E (SEQ ID NO: 87).
  • the kit is an amplification kit for exons 21 and 22 of ALK, wherein the sequence of the amplification primer pair is ALK_21-22_F (SEQ ID NO: 23), and ALK_21-22_R (SEQ ID NO: 24), and the competitor sequence (5' ⁇ 3') of the target gene is ALK_21-22_QC (SEQ ID NO: 121).
  • the kit further comprises an extension primer, and the sequence of the extension primer is ALK_21-22_E (SEQ ID NO: 88).
  • the kit is an amplification kit for exons 22 and 23 of ALK, wherein the sequence of the amplification primer pair is ALK_22-23_F (SEQ ID NO: 25), and ALK_22-23_R (SEQ ID NO: 26), and the competitor sequence (5' ⁇ 3') of the target gene is ALK_22-23_QC (SEQ ID NO: 122).
  • the kit further comprises an extension primer, and the sequence of the extension primer is ALK_22-23_E (SEQ ID NO: 89).
  • the kit is an amplification kit for exons 23 and 24 of ALK, wherein the sequence of the amplification primer pair is ALK_23-24_F (SEQ ID NO: 27), and ALK_23-24_R (SEQ ID NO: 28), and the competitor sequence (5' ⁇ 3') of the target gene is ALK_23-24_QC (SEQ ID NO: 123).
  • the kit further comprises an extension primer, and the sequence of the extension primer is ALK_23-24_E (SEQ ID NO:90).
  • the kit is an amplification kit for exons 2 and 3 of RET, wherein the sequence of the amplification primer pair is RET_02-03_F (SEQ ID NO: 29), and RET_02-03_R (SEQ ID NO:30), and the competitor sequence (5' ⁇ 3') of the target gene is RET_02-03_QC (SEQ ID NO:124).
  • the kit further comprises an extension primer, and the sequence of the extension primer is RET_02-03_E (SEQ ID NO: 91).
  • the kit is an amplification kit for exons 4 and 5 of RET, wherein the sequence of the amplification primer pair is RET_04-05_F (SEQ ID NO: 31), and RET_04-05_R (SEQ ID NO:32), and the competitor sequence (5' ⁇ 3') of the target gene is RET_04-05_QC (SEQ ID NO:125).
  • the kit further comprises an extension primer, and the sequence of the extension primer is RET_04-05_E (SEQ ID NO: 92).
  • the kit is an amplification kit for exons 12 and 13 of RET, wherein the sequence of the amplification primer pair is RET_12-13_F (SEQ ID NO: 33), and RET_12-13_R (SEQ ID NO:34), and the competitor sequence (5' ⁇ 3') of the target gene is RET_12-13_QC (SEQ ID NO:126).
  • the kit further comprises an extension primer, and the sequence of the extension primer is RET_12-13_E (SEQ ID NO:93).
  • the kit is an amplification kit for exons 13 and 14 of RET, wherein the sequence of the amplification primer pair is RET_13-14_F (SEQ ID NO: 35), and RET_13-14_R (SEQ ID NO:36), and the competitor sequence (5' ⁇ 3') of the target gene is RET_13-14_QC (SEQ ID NO:127).
  • the kit further comprises an extension primer, and the extension primer sequence is RET_13-14_E (SEQ ID NO:94).
  • the kit is an amplification kit for exons 1 and 2 of ROS1, wherein the sequence of the amplification primer pair is ROS1_01-02_F (SEQ ID NO:37), and ROS1_01-02_R (SEQ ID NO:38), and the competitor sequence (5' ⁇ 3') of the target gene is ROS1_01-02_QC (SEQ ID NO:128).
  • the kit further comprises an extension primer, and the sequence of the extension primer is ROS1_01-02_E (SEQ ID NO:95).
  • the kit is an amplification kit for exons 4 and 5 of ROS1, wherein the sequence of the amplification primer pair is ROS1_04-05_F (SEQ ID NO:39), and ROS1_04-05_R (SEQ ID NO:40), and the competitor sequence (5' ⁇ 3') of the target gene is ROS1_04-05_QC (SEQ ID NO:129).
  • the kit further comprises an extension primer, and the sequence of the extension primer is ROS1_04-05_E (SEQ ID NO:96).
  • the kit is an amplification kit for exons 35 and 36 of ROS1, wherein the sequence of the amplification primer pair is ROS1_35-36_F (SEQ ID NO: 41), and ROS1_35-36_R (SEQ ID NO:42), and the competitor sequence (5' ⁇ 3') of the target gene is ROS1_35-36_QC (SEQ ID NO:130).
  • the kit further comprises an extension primer, and the sequence of the extension primer is ROS1_35-36_E (SEQ ID NO:97).
  • the kit is an amplification kit for exons 37 and 38 of ROS1, wherein the amplification primer pair sequence is ROS1_37-38_F (SEQ ID NO: 43), and ROS1_37-38_R (SEQ ID NO:44), and the competitor sequence (5' ⁇ 3') of the target gene is ROS1_37-38_QC (SEQ ID NO:131).
  • the kit further comprises an extension primer, and the sequence of the extension primer is ROS1_37-38_E (SEQ ID NO:98).
  • the kit is an amplification kit for exons 11 and 12 of EML4, wherein the sequence of the amplification primer pair is EML4_11-12_F (SEQ ID NO: 17), and EML4_11-12_R (SEQ ID NO: 18), and the competitor sequence (5' ⁇ 3') of the target gene is EML4_11-12_QC (SEQ ID NO: 118).
  • the kit further comprises an extension primer, and the sequence of the extension primer is EML4_11-12_E (SEQ ID NO: 85).
  • the kit is an amplification kit for exons 13 and 14 of EML4, wherein the sequence of the amplification primer pair is EML4_13-14_F (SEQ ID NO: 19), and EML4_13-14_R (SEQ ID NO: 20), and the competitor sequence (5' ⁇ 3') of the target gene is EML4_13-14_QC (SEQ ID NO: 119).
  • the kit further comprises an extension primer, and the sequence of the extension primer is EML4_13-14_E (SEQ ID NO: 86).
  • the kit is an amplification kit for exons 7 and 8 of NTRK1, wherein the sequence of the amplification primer pair is NTRK1_07-08_F (SEQ ID NO:49), and NTRK1_07-08_R (SEQ ID NO:50), and the competitor sequence (5' ⁇ 3') of the target gene is NTRK1_07-08_QC (SEQ ID NO:134).
  • the kit further comprises an extension primer, and the sequence of the extension primer is NTRK1_07-08_E (SEQ ID NO: 101).
  • the kit is an amplification kit for exons 8 and 9 of NTRK1, wherein the sequence of the amplification primer pair is NTRK1_08-09_F (SEQ ID NO:51), and NTRK1_08-09_R (SEQ ID NO:52), and the competitor sequence (5' ⁇ 3') of the target gene is NTRK1_08-09_QC (SEQ ID NO:135).
  • the kit further comprises an extension primer, and the sequence of the extension primer is NTRK1_08-09_E (SEQ ID NO: 102).
  • the kit is an amplification kit for exons 14 and 14 of NTRK1, wherein the sequence of the amplification primer pair is NTRK1_13-14_F (SEQ ID NO:53), and NTRK1_13-14_R (SEQ ID NO:54), and the competitor sequence (5' ⁇ 3') of the target gene is NTRK1_13-14_QC (SEQ ID NO:136).
  • the kit further comprises an extension primer, and the sequence of the extension primer is NTRK1_13-14_E (SEQ ID NO: 103).
  • the kit is an amplification kit for exons 14 and 15 of NTRK1, wherein the sequence of the amplification primer pair is NTRK1_14-15_F (SEQ ID NO:55), and NTRK1_14-15_R (SEQ ID NO:56), and the competitor sequence (5' ⁇ 3') of the gene of interest is NTRK1_14-15_QC (SEQ ID NO:137).
  • the kit further comprises an extension primer, and the sequence of the extension primer is NTRK1_14-15_E (SEQ ID NO: 104).
  • the kit is an amplification kit for exons 9 and 10 of NTRK2, wherein the sequence of the amplification primer pair is NTRK2_09-10_F (SEQ ID NO: 57), and NTRK2_09-10_R (SEQ ID NO:58), and the competitor sequence (5' ⁇ 3') of the target gene is NTRK2_09-10_QC (SEQ ID NO:138).
  • the kit further comprises an extension primer, and the sequence of the extension primer is NTRK2_09-10_E (SEQ ID NO: 105).
  • the kit is an amplification kit for exons 10 and 11 of NTRK2, wherein the sequence of the amplification primer pair is NTRK2_10-11_F (SEQ ID NO: 59), and NTRK2_10-11_R (SEQ ID NO:60), and the competitor sequence (5' ⁇ 3') of the target gene is NTRK2_10-11_QC (SEQ ID NO:139).
  • the kit further comprises an extension primer, and the sequence of the extension primer is NTRK2_10-11_E (SEQ ID NO: 106).
  • the kit is an amplification kit for exons 13 and 14 of NTRK2, wherein the sequence of the amplification primer pair is NTRK2_13-14_F (SEQ ID NO: 61), and NTRK2_13-14_R (SEQ ID NO:62), and the competitor sequence (5' ⁇ 3') of the target gene is NTRK2_13-14_QC (SEQ ID NO:140).
  • the kit further comprises an extension primer, and the sequence of the extension primer is NTRK2_13-14_E (SEQ ID NO: 107).
  • the kit is an amplification kit for exons 14 and 15 of NTRK2, wherein the sequence of the amplification primer pair is NTRK2_14-15_F (SEQ ID NO:63), and NTRK2_14-15_R (SEQ ID NO:64), and the competitor sequence (5' ⁇ 3') of the target gene is NTRK2_14-15_QC (SEQ ID NO:141).
  • the kit further comprises an extension primer, and the sequence of the extension primer is NTRK2_14-15_E (SEQ ID NO: 108).
  • the kit is an amplification kit for exons 9 and 10 of NTRK3, wherein the sequence of the amplification primer pair is NTRK3_09-10_F (SEQ ID NO:65), and NTRK3_09-10_R (SEQ ID NO:66), and the competitor sequence (5' ⁇ 3') of the target gene is NTRK3_09-10_QC (SEQ ID NO:142).
  • the kit further comprises an extension primer, and the sequence of the extension primer is NTRK3_09-10_E (SEQ ID NO: 109).
  • the kit is an amplification kit for exons 10 and 11 of NTRK3, wherein the sequence of the amplification primer pair is NTRK3_10-11_F (SEQ ID NO: 67), and NTRK3_10-11_R (SEQ ID NO:68), and the competitor sequence (5' ⁇ 3') of the target gene is NTRK3_10-11_QC (SEQ ID NO:143).
  • the kit further comprises an extension primer, and the sequence of the extension primer is NTRK3_10-11_E (SEQ ID NO: 110).
  • the kit is an amplification kit for exons 14 and 15 of NTRK3, wherein the sequence of the amplification primer pair is NTRK3_14-15_F (SEQ ID NO:69), and NTRK3_14-15_R (SEQ ID NO:70), and the competitor sequence (5' ⁇ 3') of the target gene is NTRK3_14-15_QC (SEQ ID NO:144).
  • the kit further comprises an extension primer, and the sequence of the extension primer is NTRK3_14-15_E (SEQ ID NO: 111).
  • the kit is an amplification kit for exons 15 and 16 of NTRK3, wherein the sequence of the amplification primer pair is NTRK3_15-16_F (SEQ ID NO:71), and NTRK3_15-16_R (SEQ ID NO:72), and the competitor sequence (5' ⁇ 3') of the target gene is NTRK3_15-16_QC (SEQ ID NO:145).
  • the kit further comprises an extension primer, and the sequence of the extension primer is NTRK3_15-16_E (SEQ ID NO: 112).
  • the kit is an amplification kit for exons 8 and 9 of TPM3, wherein the sequence of the amplification primer pair is TPM3_08-09_F (SEQ ID NO: 45), and TPM3_08-09_R (SEQ ID NO: 46), and the competitor sequence (5' ⁇ 3') of the target gene is TPM3_08-09_QC (SEQ ID NO: 132).
  • the kit further comprises an extension primer, and the extension primer sequence is TPM3_08-09_E (SEQ ID NO: 99).
  • the kit is an amplification kit for exons 10 and 11 of TPM3, wherein the sequence of the amplification primer pair is TPM3_10-11_F (SEQ ID NO: 47), and TPM3_10-11_R (SEQ ID NO:48), and the competitor sequence (5' ⁇ 3') of the target gene is TPM3_10-11_QC (SEQ ID NO:133).
  • the kit further comprises an extension primer, and the sequence of the extension primer is TPM3_10-11_E (SEQ ID NO: 100).
  • the kit can be any combination of the kits in the different embodiments described above.
  • the present invention can be used to detect target gene mutation. First, a competitor of the target gene and an amplification primer pair containing locked nucleic acid modification are designed according to the mutation site of the target gene; then the target gene and its competitor are amplified by the method provided by the present invention; finally, the target gene in the obtained amplification product is detected. Gene mutation.
  • the amplification method provided by the present invention can be combined with common detection techniques, including but not limited to matrix-assisted laser desorption ionization time-of-flight mass spectrometry method, Taqman-PCR method, next-generation sequencing technology, capillary electrophoresis analysis , digital PCR and other gene semi-quantitative/quantitative detection methods.
  • common detection techniques including but not limited to matrix-assisted laser desorption ionization time-of-flight mass spectrometry method, Taqman-PCR method, next-generation sequencing technology, capillary electrophoresis analysis , digital PCR and other gene semi-quantitative/quantitative detection methods.
  • the amplification method provided by the present invention includes the amplification of controllable competitors, it can solve the problem that the difference in detection signal caused by the difference in sample quality easily leads to misjudgment, and the copy number of the target gene can be estimated according to the amplification of the competitors. , therefore can be used to detect the mutation of a variety of target genes, wherein the target gene mutation is selected from: single nucleotide polymorphism SNP, DNA copy number change CNV, gene fusion, pathogen nucleic acid quantification, gene expression changes and its combination, etc.
  • the advantages of the present invention include:
  • the present invention significantly reduces the amplification efficiency of the competitor without changing the amplification efficiency of the gene locus to be tested by optimizing the combination of the competitor and the amplification primer, thereby realizing the close competitor and the gene to be tested.
  • the level of amplification efficiency of the locus is important to reduce.
  • This method uses the synthetic double-stranded DNA modified by locked nucleic acid as the competitor, so that the added amount of the competitor substrate is increased from the original pg level to the ng level, which makes it easier to control the amount used, and avoids negative control experiments. Unnecessary errors caused by uncontrollable factors such as aerosols improve the success rate and operability of mass spectrometry data.
  • the optimized competitor used in this method has the advantages of low freeze-thaw degradation rate and easy storage in addition to the advantages of more controllable usage amount.
  • the amplification method provided by the present invention can be combined with common detection techniques, including but not limited to gene half-mass based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Taqman-PCR, next-generation sequencing technology, capillary electrophoresis analysis, and digital PCR. Quantitative/quantitative detection method with a wide range of applications.
  • Using the method of the present invention to detect SMA has low requirements on samples and is suitable for general screening.
  • the copy number detection and point mutation detection of SMN1 gene and SMN2 gene can be completed in one reaction well, eliminating the interference between reaction wells.
  • single-base extension the difference in molecular weight between different bases is used to distinguish the detection site, which can directly and accurately detect the base type, with strong specificity; no fluorescent probe is used, which avoids the fluorescence interference of similar sites, and the detection result is accurate , reduce costs, and meet the qualitative and commercial testing needs of SMA.
  • ALK, ROS1, RET, NTRK1, NTRK2, and NTRK3 have gene fusion mutations, which can be well detected from fresh tumor tissue, FFPET, pleural effusion, and puncture fluid samples, and is suitable for Auxiliary diagnosis.
  • ALK, ROS1, and RET can be completed in one reaction well
  • NTRK1, NTRK2, and NTRK3 can be completed in one reaction well, which eliminates the interference between reaction wells, and has abundant sample sources and low cost. Conducive to commercial promotion and application.
  • the designed and used amplification primer information is shown in Table 1.
  • [X] indicates that the base is modified by locked nucleic acid, and X can be A, T, C or G.
  • the purpose of PCR is to obtain target DNA.
  • [X] indicates that the base is modified by locked nucleic acid, and X can be A, T, C or G.
  • the extension primer is taken from a part of the PCR amplification sequence.
  • the purpose of MassArray is to detect the mutation of DNA, which can be used to detect the copy number variation of SMA and SNP of disease-related loci, or to detect ALK, ROS1, RET, NTRK1, NTRK2, Are there fusion mutations in six NTRK3 genes?
  • [X] indicates that the base is modified by locked nucleic acid, and X can be A, T, C or G.
  • t indicates that the sequence of the competitor is different from the target gene sequence at the base t position, and the introduced different base is a genotype that does not appear in the human gene.
  • the black and bold are the PCR amplification primer sequences
  • the italics are the extension primer sequences
  • the brackets and italics are the extension primer sequences added to adjust the molecular weight
  • the underlined bold is the detection site or extension base.
  • MassArray was used to detect SMN1 and SMN2 gene mutations in clinical samples.
  • the samples used in this example were obtained from clinically collected anticoagulant specimens or blood spot card specimens of SMA patients, mutant gene carriers and normal individuals, as well as control human genomic DNA (Promega, G1471).
  • Genomic DNA was extracted from fresh or frozen anticoagulated specimens with Yingruicheng magnetic bead method blood genomic DNA extraction kit.
  • Genomic DNA was extracted from blood spot card specimens with Yingruicheng magnetic bead method blood spot genomic DNA extraction kit.
  • the gDNA is a 2-copy control, and the SMN1 gene, SMN2 gene, and RPP40 gene are also 2 copies.
  • the information of the designed and used amplification primers is shown in Table 1; the information of the designed and used extension primers is shown in the following Table 2; the designed and used competitors are shown in Table 3.
  • the synthesized sequence is connected to the vector.
  • the above-mentioned synthetic sequence is connected to the T vector, and the circularization forms a segment that contains (3, 4, 6) different bases compared with the target gene sequence. nucleotide sequence of the plasmid.
  • the amplification primers obtained in step 2.2 are mixed as follows to obtain PCR Primer MIX, wherein the concentration of each PCR primer in the Primer MIX mixture is 0.5-1 ⁇ M.
  • Each assay requires 2 copies of control gDNA and blank control water. Adhere to the film, centrifuge for a while and place it on the gene amplification instrument, and perform amplification according to the following PCR procedures:
  • the obtained PCR amplification reaction products included SMN1-2_E5, SMN1-2_QC_E5, SMN1-2_E6, SMN1-2_QC_E6, RPP40, and RPP40_QC.
  • system of SAP reaction mixture is as follows:
  • extension primer name Concentration of extension primer in mixture RPP40 RPP40#2_W1_E 10.33 ⁇ 20.65 ⁇ M SMN1, SMN2 SMN1-2_E5_W1_E 8.62 ⁇ 17.24 ⁇ M SMN1, SMN2 SMN1-2_E6_W1_E 8.33 ⁇ 16.65 ⁇ M
  • the above extension reaction system was attached to the membrane, centrifuged for a while, and then placed on the gene amplification instrument, and the extension was carried out according to the following extension reaction procedure.
  • Interpretation logic first judge the grouping of the samples to be tested according to the f value of SMN1. Then, the copy number of SMN2 is determined according to the f value of SMN2 in the group.
  • This formula uses the signal-to-noise ratio to calculate the copy number, which is used to exclude errors caused by the external environment (temperature, pressure) and manual operation, so as to make the detection result more accurate).
  • sample number Sample description 11 normal person 17 carrier 20 patient twenty one patient
  • Figures 1 to 5 show the mass spectrometry detection of five target gene products SMN1-2_E5, SMN1-2_E6, SMN1-2_E7, SMN1-2_E8, and RPP40, respectively, using competitors and locked nucleic acid-modified primers in four samples.
  • this method uses a competitor with a loading amount of ng magnitude for amplification, indicating that the amplification of the competitor of the present invention is in a controllable range, and the product can be used in subsequent detection reactions such as mass spectrometry detection. Therefore, this method improves the clinical practicability of mass spectrometry for detecting gene mutations.
  • the present invention can detect the copy numbers of SMN1 and SMN2 and their hot spot SNPs in one reaction.
  • 2/0, 1/2, 1/3, 2/3, 2/4 copy number situation and can refine the test results (for example, distinguish 1/2 or 2/4), the test results are more accurate, meet the SMA Clinical quantitative testing needs.
  • Result As shown in Figure 6, using the locked nucleic acid modified amplification primers proposed in this method, five target gene products of SMN1-2_E5, SMN1-2_E6, SMN1-2_E7, SMN1-2_E8 and RPP40 can be successfully detected. However, the loading amounts of the competitors used by the two primers were significantly different. Taking SMN1-2_E5_QC as an example, the sample volume corresponding to the unlocked nucleic acid modified primer is about 0.05pg. After using the locked nucleic acid modified primer, the corresponding sample volume is increased to about 10ng, an increase of 2*10 5 times; while other bits The loading volume of the spot competitor was also increased by a factor of 10 5 -10 6 .
  • the primer information used in the reaction is as follows:
  • the qPCR amplification system is as follows:
  • the qPCR amplification conditions are as follows:
  • the same competitor mixture is fed and amplified with locked nucleic acid primers and non-locked nucleic acid primers respectively, wherein the sequences of the two types of primers are consistent, and the locked nucleic acid primers are part of the primer bases replaced by locked nucleic acid modified bases, and the unlocked nucleic acid primers All bases are conventional unmodified bases; the average difference between the obtained locked nucleic acid primers and unlocked nucleic acid primers Cp is 7.86 at the lowest, and 20.64 at the highest. The difference in the amount of amplified product. It is proved that the addition of locked nucleic acid modified primers significantly reduces the amplification efficiency of competitors and greatly reduces the amount of products obtained by amplification.
  • the prepared mixture should be frozen at -80°C.
  • the real-time quantitative qPCR method detects the concentration of the competitor to determine whether the amplification efficiency of the competitor is affected by the storage concentration and is unstable.
  • the specific sequence is as follows:
  • the qPCR amplification system is as follows:
  • the qPCR amplification conditions are as follows:
  • the competitor mixtures in the repeated freezing and thawing experiments in this experiment have two concentrations of 1 pg/ ⁇ l and 5 ng/ ⁇ l, respectively.
  • the theoretical feeding amount of detection is 1pg and 5ng, respectively.
  • the 10 freeze-thaw test data of 5ng remain stable, and the data of 0 freeze-thaw test of 5ng is also stable; There is an obvious gap between the data of 0 freeze-thaw and 2.2 to 3.19, and the 10 freeze-thaw test data of 1 pg is close to the test data of 0 pg. It is proved that after 20 repeated freezing and thawing, the 5ng/ ⁇ l mixture can maintain a stable Cp value, and the 1pg/ ⁇ l mixture is severely degraded after freezing and thawing once.
  • the competitor mixture of the experimental error experiment was carried out with two concentrations of about 50pg/ ⁇ l and 5ng/ ⁇ l, and three batches of experiments were carried out.
  • the first batch experiment was 0 freeze-thaw times, and the second and third batches were Batch experiments consisted of at least one freeze-thaw of the competitor mixture at least 24 hours apart.
  • the concentration of 10ng of gDNA in the detection sample, and the concentration of the competitor of the locked nucleic acid primer set in the mixture QC Mix- the concentration of the competitor of the unlocked nucleic acid primer set in the mixture QC Mix-unlocked nucleic acid as follows shown:
  • SNR (SMN1-2_E6_QC) and SNR (SMN E6) ratios are as follows:
  • the rapid amplification efficiency of the competitor will cause many problems, such as the peak area of the amplified product in the subsequent mass spectrometry detection far exceeding the peak area of the analyte, resulting in judgment failure;
  • the error of preparation is large and the deviation is large; or the concentration of the competitor is so low that it is easily degraded after freezing and thawing, so it is not easy to store. Therefore, the present application greatly reduces the operational difficulty of the amplification reaction with competitors, and greatly improves the success rate of the detection method.
  • Threshold setting configure ALK 1%, 5%, 10% yang ginseng and yin ginseng, carry out intra-batch and inter-batch repeatability verification, and verify the correctness of 20 samples, and finally set a threshold of 15.
  • Judgment logic of test results When cDNA/QC calculation is performed, if the QC SNR value is 0, adjust it to a value of 0.1, and perform formula calculation.
  • the first step internal reference quality control, if EML4_11-12 ⁇ 0.5 and EML4_13-14 ⁇ 0.01 appear at the same time, the quality control is not good; the feeding needs to be increased. If EML4_11-12 ⁇ 7.5 and EML4_13-14 ⁇ 3 appear at the same time, the quality control is not good; it is necessary to reduce the feeding.
  • EML4_11-12 and EML4_13-14 are within the applicable scope, the quality control is passed, and the judgment is continued.
  • Step 2 If the ratio of the 3 arm ends is ⁇ 0.05, it can be directly judged as negative
  • Step 3 If the ratio of the 5-arm end is 0, adjust it to a value of 0.01, and perform formula calculation. If the calculated value is greater than or equal to the corresponding threshold, it is positive.
  • PCR cycling conditions are as follows:
  • PCR products were subjected to 2% agarose gel electrophoresis, and the target bands of equal size were cut out, and purified and recovered according to the Medicaid magnetic bead method PCR product purification kit.
  • the sequencing reaction system is as follows:
  • NH 4 AC.EDTA purification After the sequencing reaction, centrifuge the 96-well plate at 3700 rpm for 0.5 min; add 1 ⁇ l NH 4 AC.EDTA solution to each, centrifuge at 3700 rpm for 30 s; place it on a mixer and shake for 30 s, 3700 Centrifuge for 30s.
  • Washing with 75% alcohol add 50 ⁇ l of 75% alcohol, centrifuge at 4500 rpm for 10 min, invert and centrifuge at 400 rpm for 2 min.
  • Denaturation Add 6 ⁇ l Hi-Di, centrifuge at 3700rpm for 1min, and store at -20°C until the sequencer.
  • the Sanger sequencing results are shown in FIG. 17 .
  • the accuracy was compared, and the gold-label method sanger sequencing was used for comparison and verification.
  • the samples used were the reference samples with 10% of each of the six genes in the standard configuration.
  • the experimental conclusion is that the results detected by the MassArray method are completely consistent with the results of sanger sequencing, which proves the correctness of the competitor reaction system established by this method.
  • the results of the detected 6 genes and their fusion mutations are compared as follows:
  • MassaArray technology is used to detect whether gene fusion mutation occurs in the six genes of ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3. It can be well detected from fresh tumor tissue, FFPET, pleural effusion, and puncture fluid specimens, and is suitable for auxiliary diagnosis. Fusion detection of six genes ALK, ROS1, RET, NTRK1, NTRK2, NTRK3, ALK, ROS1, RET can be completed in one reaction well, NTRK1, NTRK2, NTRK3 can be completed in one reaction well, eliminating the need between reaction wells interference and reduce costs.

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Abstract

Procédé d'amplification, comprenant les étapes suivantes : utilisation d'une paire d'amorces d'amplification modifiées par un acide nucléique verrouillé pour amplifier simultanément un gène cible et un concurrent de celui-ci. Le procédé peut être utilisé pour réguler l'efficacité d'amplification du concurrent, de sorte que l'efficacité d'amplification du concurrent soit proche ou de préférence égale à l'efficacité d'amplification du gène cible.
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