WO2022042399A1

WO2022042399A1 - Gene amplification method and application thereof

Info

Publication number: WO2022042399A1
Application number: PCT/CN2021/113340
Authority: WO
Inventors: 何逖; 覃嘉嘉; 田进
Original assignee: 上海吉凯医学检验所有限公司; 上海吉凯基因医学科技股份有限公司
Priority date: 2020-08-31
Filing date: 2021-08-18
Publication date: 2022-03-03
Also published as: CN114107451A

Abstract

An amplification method, comprising: using a pair of locked nucleic acid-modified amplification primers to simultaneously amplify a target gene and a competitor thereof. The method can be used for controlling the amplification efficiency of the competitor, such that the amplification efficiency of the competitor is close to or preferably equal to the amplification efficiency of the target gene.

Description

A kind of gene amplification method and its application

technical field

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.

Background technique

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.

However, in practice, since the competitor used is an artificially synthesized sequence, when the competitor and the genomic DNA are amplified in the same reaction well, the artificially synthesized sequence has high purity and low amplification steric hindrance, which is incompatible with the amplification efficiency of gDNA. Inconsistent, 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. In the prior art, 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. In addition, 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.

Therefore, there is an urgent need in the art for a gene amplification method involving controllable competitors.

SUMMARY OF THE INVENTION

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.

In one aspect, 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.

In a preferred example, 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.

In a preferred example, 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.

In a preferred example, the target gene is selected from the following group of genes or a combination thereof: SMN1, SMN2, ALK, RET, ROS1, NTRK1, NTRK2, NTRK3.

In a preferred embodiment, wherein the amplification primer pair is selected from the sequences described in SEQ ID NOs: 1-72 or different combinations thereof.

In a preferred example, 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.

In a preferred example, 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.

In a preferred example, 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.

In a preferred example, 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.

In a preferred embodiment, wherein the competitor is selected from the sequences described in SEQ ID NOs: 113-145 or different combinations thereof.

In a preferred example, the final concentration of the competitor is 0.05-250ng/μl, 0.1-200ng/μl, 0.1-150ng/μl, 0.1-100ng/μl, 0.1-90ng/μl, 0.1-80ng/μl, 0.1-70ng/μl, 0.1-60ng/μl, 0.1-50ng/μl, 0.1-40ng/μl, 0.1-30ng/μl, 0.1-20ng/μl, 0.1-10ng/μl, 0.5-100ng/μl, 0.5- 90ng/μl, 0.5-80ng/μl, 0.5-70ng/μl, 0.5-60ng/μl, 0.5-50ng/μl, 0.5-40ng/μl, 0.5-30ng/μl, 0.5-20ng/μl, 0.5-10ng/ μl, 1-100ng/μl, 1-90ng/μl, 1-80ng/μl, 1-70ng/μl, 1-60ng/μl, 1-50ng/μl, 1-40ng/μl, 1-30ng/μl, 1-20ng/μl, 1-10ng/μl, 5-100ng/μl, 5-90ng/μl, 5-80ng/μl, 5-70ng/μl, 5-60ng/μl, 5-50ng/μl, 5- 40ng/μl, 5-30ng/μl, 5-20ng/μl, 5-10ng/μl, 10-100ng/μl, 10-90ng/μl, 10-80ng/μl, 10-70ng/μl, 10-60ng/ μl, 10-50ng/μl, 10-40ng/μl, 10-30ng/μl or 10-20ng/μl.

In a preferred example, the sample loading amount of the competitor is 5-80ng, 10-70ng, 15-60ng, 20-50ng, 20-40ng or 20-30ng, preferably 20-40ng.

In a preferred embodiment, 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.

In another aspect, the present invention provides a kit comprising a pair of locked nucleic acid-modified amplification primers and a competitor of the target gene.

In a preferred embodiment, the kit further comprises amplification reagents selected from the group consisting of dNTP, DNA polymerase, MgCl ₂ and combinations thereof.

In a preferred embodiment, the kit is used to detect spinal muscular atrophy in a subject.

In a preferred embodiment, 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.

On the other hand, 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.

In a preferred example, the kit is used to amplify a gene selected from the group consisting of SMN1, SMN2, ALK, RET, ROS1, NTRK1, NTRK2, and NTRK3.

In another aspect, 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.

In a preferred embodiment, the locked nucleic acid is used to modify the primer pair for amplifying the target gene and its competitor.

In a preferred example, the target gene is selected from the following group of genes or a combination thereof: SMN1, SMN2, ALK, RET, RO1, NTRK1, NTRK2, NTRK3.

In a preferred example, wherein the primer pair is selected from the primer sequences described in SEQ ID NO: 1-72.

In another aspect, 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.

In a preferred embodiment, wherein the amplification primer pair is selected from the sequences described in SEQ ID NOs: 1-72 or a combination thereof.

In a preferred example, 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.

In this context, in all technical solutions of the present invention, the pair of amplification primers, competitors and/or extension primers may be further preferably combined, for example:

In a specific preferred example, wherein the amplification primer pair is selected from the sequences described in SEQ ID NO: 1-10 or a combination thereof, and 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. In a further preferred embodiment, 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.

In a specific preferred example, wherein the amplification primer pair is selected from the sequences described in SEQ ID NO: 17-44 or a combination thereof, and 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. In a further preferred embodiment, 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.

In a specific preferred example, wherein the amplification primer pair is selected from the sequences described in SEQ ID NOs: 45-72 or a combination thereof, and 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. In a further preferred embodiment, 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.

In a specific preferred example, 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). Preferably, the extension primer sequence is SMN1-2_E5_W1_E (SEQ ID NO:74).

In a specific preferred example, 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). Preferably, the extension primer sequence is SMN1-2_E6_W1_E (SEQ ID NO:75).

In a specific preferred example, 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). Preferably, 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).

In a specific preferred example, 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). . Preferably, 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).

In a specific preferred example, 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). Preferably, the extension primer sequence is RPP40#2_W1_E (SEQ ID NO:73).

In another specific preferred example, the 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). Preferably, the extension primer sequence is ALK_01-02_E (SEQ ID NO:87).

In another specific preferred example, the 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). Preferably, the extension primer sequence is ALK_21-22_E (SEQ ID NO:88).

In another specific preferred example, wherein 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). Preferably, the extension primer sequence is ALK_22-23_E (SEQ ID NO:89).

In another specific preferred example, the 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). Preferably, the extension primer sequence is ALK_23-24_E (SEQ ID NO:90).

In another specific preferred example, 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). Preferably, the extension primer sequence is RET_02-03_E (SEQ ID NO: 91).

In another specific preferred example, 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). Preferably, the extension primer sequence is RET_04-05_E (SEQ ID NO:92).

In another specific preference, wherein 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). Preferably, the extension primer sequence is RET_12-13_E (SEQ ID NO:93).

In another specific preferred example, wherein 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). Preferably, the extension primer sequence is RET_13-14_E (SEQ ID NO:94).

In another specific preferred example, wherein 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). Preferably, the extension primer sequence is ROS1_01-02_E (SEQ ID NO:95).

In another specific preferred example, wherein 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). Preferably, the extension primer sequence is ROS1_04-05_E (SEQ ID NO:96).

In another specific preferred example, wherein 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). Preferably, the extension primer sequence is ROS1_35-36_E (SEQ ID NO:97).

In another specific preferred example, 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 of the target gene (5'→3') is ROS1_37-38_QC (SEQ ID NO: 131). Preferably, the extension primer sequence is ROS1_37-38_E (SEQ ID NO:98).

In another specific preferred example, the 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). Preferably, the extension primer sequence is EML4_11-12_E (SEQ ID NO:85).

In another specific preferred example, the 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). Preferably, the extension primer sequence is EML4_13-14_E (SEQ ID NO:86).

In yet another specific preferred example, 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 sequence of the competitor of the target gene (5'→3') is NTRK1_07-08_QC (SEQ ID NO: 134). Preferably, the extension primer sequence is NTRK1_07-08_E (SEQ ID NO: 101).

In yet another specific preference, 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 sequence of the competitor of the target gene (5'→3') is NTRK1_08-09_QC (SEQ ID NO: 135). Preferably, the extension primer sequence is NTRK1_08-09_E (SEQ ID NO: 102).

In yet another specific preferred example, 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 sequence of the competitor of the target gene (5'→3') is NTRK1_13-14_QC (SEQ ID NO: 136). Preferably, the extension primer sequence is NTRK1_13-14_E (SEQ ID NO: 103).

In yet another specific preference, wherein 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). Preferably, the extension primer sequence is NTRK1_14-15_E (SEQ ID NO: 104).

In yet another specific preferred example, wherein 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). Preferably, the extension primer sequence is NTRK2_09-10_E (SEQ ID NO: 105).

In yet another specific preference, 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 sequence of the competitor of the target gene (5'→3') is NTRK2_10-11_QC (SEQ ID NO: 139). Preferably, the extension primer sequence is NTRK2_10-11_E (SEQ ID NO: 106).

In yet another specific preferred example, 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 sequence of the competitor of the target gene (5'→3') is NTRK2_13-14_QC (SEQ ID NO: 140). Preferably, the extension primer sequence is NTRK2_13-14_E (SEQ ID NO: 107).

In yet another specific preference, wherein 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). Preferably, the extension primer sequence is NTRK2_14-15_E (SEQ ID NO: 108).

In yet another specific preferred example, 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 sequence of the competitor of the target gene (5'→3') is NTRK3_09-10_QC (SEQ ID NO: 142). Preferably, the extension primer sequence is NTRK3_09-10_E (SEQ ID NO: 109).

In yet another specific preference, 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 sequence of the competitor of the target gene (5'→3') is NTRK3_10-11_QC (SEQ ID NO: 143). Preferably, the extension primer sequence is NTRK3_10-11_E (SEQ ID NO: 110).

In yet another specific preferred example, 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 sequence of the competitor of the target gene (5'→3') is NTRK3_14-15_QC (SEQ ID NO: 144). Preferably, the extension primer sequence is NTRK3_14-15_E (SEQ ID NO: 111).

In yet another specific preferred example, wherein 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). Preferably, the extension primer sequence is NTRK3_15-16_E (SEQ ID NO: 112).

In yet another specific preferred example, wherein 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). Preferably, the extension primer sequence is TPM3_08-09_E (SEQ ID NO:99).

In yet another specific preference, wherein 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). Preferably, the extension primer sequence is TPM3_10-11_E (SEQ ID NO: 100).

In the above preferred example, 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 .

Description of drawings

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. Repeat the experiment, 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.

detailed description

In order to provide a gene amplification method with controllable amplification efficiency of competitors, 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.

In order to further confirm the technical solution of the present invention and use it for the diagnosis and screening of human spinal muscular atrophy, 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.

Compared with existing technologies such as RT-PCR, MLPA, and NGS, MassARRAY technology has certain advantages.

The signal interpretation of 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. When interpreting 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.

However, 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) 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. According to the age of onset and clinical manifestations, it can be divided into 4 types, namely, spinal muscular atrophy type I (SMN1, also known as Werdnig-Hoffmann disease) with onset of less than 6 months, and from 6 to 18 months. Spinal muscular atrophy type II (SMN2), childhood-onset or adolescent-onset spinal muscular atrophy type III (SMA3, also known as Kugelherg-Welander disease), and adult-onset Spinal muscular atrophy type IV (SMA4). The four subtypes of SMA have the same causative gene, which is motor neuron survival gene 1 (SMN1) [OMIM600354]. 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 One of the common pathogenic mutations in ALK gene is gene rearrangement, including translocation and inversion. 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.

competitor

In the context of the present invention, "competitor" 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.

The competitors have unique characteristics. 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.

In some embodiments, the competitors used for a particular region are the same length. In other embodiments, the lengths of competitors used for different regions are different.

locked nucleic acid

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. In a preferred embodiment, 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 (LNA) is a synthetic antisense oligonucleotide, which is a special bicyclic nucleotide derivative in which the ribose ring (β-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-β-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. Because 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.

In a specific embodiment, wherein the amplification primer pair is selected from the sequences shown in Table 1 or a combination thereof.

Nucleic acid amplification method

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. In a preferred example, 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.

In a specific embodiment, wherein 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.

In a specific embodiment, wherein the gene of interest is selected from the following group of genes or a combination thereof: SMN1, SMN2, ALK, RET, ROS1, NTRK1, NTRK2, NTRK3.

In a preferred embodiment, wherein the amplification primer pair is selected from the sequences set forth in SEQ ID NOs: 1-72 or a combination thereof.

In a specific embodiment, wherein 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. In a preferred example, 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.

In a specific embodiment, 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.

In a specific embodiment, 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.

In a specific embodiment, wherein the competitor is selected from the sequence set forth in SEQ ID NO: 113-145 or a combination thereof.

In a specific embodiment, the final concentration of the competitor is 0.05-250ng/μl, 0.1-200ng/μl, 0.1-150ng/μl, 0.1-100ng/μl, 0.1-90ng/μl, 0.1-80ng/μl μl, 0.1-70ng/μl, 0.1-60ng/μl, 0.1-50ng/μl, 0.1-40ng/μl, 0.1-30ng/μl, 0.1-20ng/μl, 0.1-10ng/μl, 0.5-100ng/μl, 0.5-90ng/μl, 0.5-80ng/μl, 0.5-70ng/μl, 0.5-60ng/μl, 0.5-50ng/μl, 0.5-40ng/μl, 0.5-30ng/μl, 0.5-20ng/μl, 0.5- 10ng/μl, 1-100ng/μl, 1-90ng/μl, 1-80ng/μl, 1-70ng/μl, 1-60ng/μl, 1-50ng/μl, 1-40ng/μl, 1-30ng/ μl, 1-20ng/μl, 1-10ng/μl, 5-100ng/μl, 5-90ng/μl, 5-80ng/μl, 5-70ng/μl, 5-60ng/μl, 5-50ng/μl, 5-40ng/μl, 5-30ng/μl, 5-20ng/μl, 5-10ng/μl, 10-100ng/μl, 10-90ng/μl, 10-80ng/μl, 10-70ng/μl, 10- 60ng/μl, 10-50ng/μl, 10-40ng/μl, 10-30ng/μl or 10-20ng/μl.

In a preferred embodiment, the loading amount of the competitor is 5-80ng, 10-70ng, 15-60ng, 20-50ng, 20-40ng or 20-30ng, preferably 20-40ng.

In a preferred embodiment, 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.

Nucleic acid amplification kit

The present invention provides a kit comprising a pair of locked nucleic acid modified amplification primers and a competitor of a target gene. In a preferred embodiment, the kit further comprises amplification reagents selected from the group consisting of dNTPs, DNA polymerase, _MgCl2 , and combinations thereof.

In a specific embodiment, wherein the amplification primer pair is selected from the sequences set forth in SEQ ID NOs: 1-72 or a combination thereof.

In a specific embodiment, wherein 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.

In a preferred embodiment, 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.

In a preferred embodiment, 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.

In a preferred embodiment, 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.

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.

In a preferred embodiment, wherein the amplification primer pair sequences are selected from the sequences set forth in SEQ ID NOs: 1-72.

In a preferred embodiment, wherein the competitor sequences of the target gene are selected from the sequences shown in SEQ ID NOs: 113-145.

In the context of the present invention, in a specific embodiment, wherein in the kit, the amplification primer pair is selected from the sequence of SEQ ID NO: 1-10 or a combination thereof, and 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. In a further embodiment, 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.

In a specific embodiment, 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. In a further embodiment, 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.

In a specific embodiment, 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. In a further embodiment, 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.

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is SMN1-2_E5_W1_E (SEQ ID NO:74).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is SMN1-2_E6_W1_E (SEQ ID NO:75).

In a preferred embodiment, 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). Preferably, 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).

In a preferred embodiment, 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). Preferably, 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).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is RPP40#2_W1_E (SEQ ID NO:73).

In a preferred embodiment, 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).

Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is ALK_01-02_E (SEQ ID NO: 87).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is ALK_21-22_E (SEQ ID NO: 88).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is ALK_22-23_E (SEQ ID NO: 89).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is ALK_23-24_E (SEQ ID NO:90).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is RET_02-03_E (SEQ ID NO: 91).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is RET_04-05_E (SEQ ID NO: 92).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is RET_12-13_E (SEQ ID NO:93).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the extension primer sequence is RET_13-14_E (SEQ ID NO:94).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is ROS1_01-02_E (SEQ ID NO:95).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is ROS1_04-05_E (SEQ ID NO:96).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is ROS1_35-36_E (SEQ ID NO:97).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is ROS1_37-38_E (SEQ ID NO:98).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is EML4_11-12_E (SEQ ID NO: 85).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is EML4_13-14_E (SEQ ID NO: 86).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is NTRK1_07-08_E (SEQ ID NO: 101).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is NTRK1_08-09_E (SEQ ID NO: 102).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is NTRK1_13-14_E (SEQ ID NO: 103).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is NTRK1_14-15_E (SEQ ID NO: 104).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is NTRK2_09-10_E (SEQ ID NO: 105).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is NTRK2_10-11_E (SEQ ID NO: 106).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is NTRK2_13-14_E (SEQ ID NO: 107).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is NTRK2_14-15_E (SEQ ID NO: 108).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is NTRK3_09-10_E (SEQ ID NO: 109).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is NTRK3_10-11_E (SEQ ID NO: 110).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is NTRK3_14-15_E (SEQ ID NO: 111).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is NTRK3_15-16_E (SEQ ID NO: 112).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the extension primer sequence is TPM3_08-09_E (SEQ ID NO: 99).

In a preferred embodiment, 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). Preferably, the kit further comprises an extension primer, and the sequence of the extension primer is TPM3_10-11_E (SEQ ID NO: 100).

In a preferred embodiment, the kit can be any combination of the kits in the different embodiments described above.

Detection of target gene mutations

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.

In order to detect the mutation of the target gene, 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.

Since 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.

Compared with the prior art, the advantages of the present invention include:

1. 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.

2. 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.

3. 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.

4. 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.

5. 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. Through 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.

6. Use the method of the present invention to detect whether the six genes of 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. For the detection of six genes, ALK, ROS1, and RET can be completed in one reaction well, and 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.

Example

The preferred embodiments of the present application will be further described in detail below with reference to the accompanying drawings. The following descriptions are exemplary and are not intended to limit the present application, and any other similar situations also fall within the protection scope of the present application.

Example 1: Design of primers and competitors

In this example, according to the pathological characteristics of SMA disease and the sequence information of SMN1 and SMN2 genes, a series of amplification primers, competitors and extensions were designed for the fragments corresponding to

exons

5, 6, 7 and 8 of SMN1 and SMN2. Primers, as well as the amplification primers, competitors and extension primers of the RPP40 gene as a reference; the detection site sequences of the 5th and 6th exons are the same (SMN1-2E5:c.541 and SMN1-2E6:c.692 ).

In addition, according to the sequence information of the genes ALK, RET, ROS1, NTRK1, NTRK2, and NTRK3, the amplification primers, competitors and extension primers for the above-mentioned six genes, as well as the EML4 and TPM3 genes as reference are involved.

Specifically, the designed and used amplification primer information is shown in Table 1. Wherein, [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.

Table 1: Amplification Primer Information Sheet

Information on the extension primers designed and used is shown in Table 2 below. Wherein, [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?

Table 2: Extension Primer Information Sheet

Information on the competitors designed and used is shown in Table 3 below. Wherein, [X] indicates that the base is modified by locked nucleic acid, and X can be A, T, C or G.

Table 3: Competitor Information Sheet

Wherein, for example, 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 sequence information of the amplified product of the target gene and competitor is shown in Table 4 below:

Among them, 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, and the underlined bold is the detection site or extension base.

Example 2

Using the primers and competitors described in Example 1, MassArray was used to detect SMN1 and SMN2 gene mutations in clinical samples.

2.1 Sample processing

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.

2.2 Design and synthesis of primers and competitors

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.

Then, the synthesized sequence is connected to the vector. In this example, 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.

2.3 Multiplex amplification of target gene fragments

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 μM.

Mix the competitors obtained in step 2.2 as follows to obtain a QC MIX:

竞争底物名称Competing substrate name	锁核酸方法竞争底物投料Locked Nucleic Acid Method Competitive Substrate Dosing	非锁核酸方法竞争底物投料Unlocked Nucleic Acid Methods Competitive Substrate Dosing
SMN1-2_E7_QCSMN1-2_E7_QC	约2～10ngAbout 2～10ng	约0.02～0.09pgAbout 0.02～0.09pg
SMN1-2_E8_QCSMN1-2_E8_QC	约2～10ngAbout 2～10ng	约0.02～0.09pgAbout 0.02～0.09pg
SMN1-2_E5_QCSMN1-2_E5_QC	约2～10ngAbout 2～10ng	约0.02～0.09pgAbout 0.02～0.09pg
SMN1-2_E6_QCSMN1-2_E6_QC	约2～10ngAbout 2～10ng	约0.02～0.09pgAbout 0.02～0.09pg
RPP40_QCRPP40_QC	约2～10ngAbout 2～10ng	约0.02～0.09pgAbout 0.02～0.09pg
ALK_01-02_QCALK_01-02_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
ALK_21-22_QCALK_21-22_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
ALK_22-23_QCALK_22-23_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
ALK_23-24_QCALK_23-24_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
RET_02-03_QCRET_02-03_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
RET_04-05_QCRET_04-05_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
RET_12-13_QCRET_12-13_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg

RET_13-14_QCRET_13-14_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
EML4_11-12_QCEML4_11-12_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
EML4_13-14_QCEML4_13-14_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
ROS1_01-02_QCROS1_01-02_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
ROS1_04-05_QCROS1_04-05_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
ROS1_35-36_QCROS1_35-36_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
ROS1_37-38_QCROS1_37-38_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
NTRK1_07-08_QCNTRK1_07-08_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
NTRK1_08-09_QCNTRK1_08-09_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
NTRK1_13-14_QCNTRK1_13-14_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
NTRK1_14-15_QCNTRK1_14-15_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
NTRK2_09-10_QCNTRK2_09-10_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
NTRK2_10-11_QCNTRK2_10-11_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
NTRK2_13-14_QCNTRK2_13-14_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
NTRK2_14-15_QCNTRK2_14-15_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
NTRK3_09-10_QCNTRK3_09-10_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
NTRK3_10-11_QCNTRK3_10-11_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
NTRK3_14-15_QCNTRK3_14-15_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
NTRK3_15-16_QCNTRK3_15-16_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
TPM3_08-09_QCTPM3_08-09_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg
TPM3_10-11_QCTPM3_10-11_QC	约10～50ngAbout 10～50ng	约10～50pgAbout 10～50pg

Then, prepare the PCR reaction system as follows:

ddH ₂O ddH ₂ O	0.8μl0.8μl
10x PCR Buffer10x PCR Buffer	0.5μl0.5μl
MgCl ₂ MgCl ₂	0.4μl0.4μl
dNTP MixdNTP Mix	0.1μl0.1μl
PCR EnzymePCR Enzyme	0.2μl0.2μl
QC MIXQC MIX	1μl1μl
PCR Primer MIXPCR Primer MIX	1μl1μl
合计total	4μl4μl

Then, dispense the prepared PCR reaction system into the corresponding reaction wells of the 384-well plate, dispense 4 μl per well, and then add 1 μl of the sample to be tested obtained in step 2.1. 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.

2.4 Validation of amplification by a matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based method result

2.4.1 SAP purification

Add 2 μl of SAP reaction mixture to each well of PCR amplification product obtained in step 1.3, stick it close to the membrane, centrifuge a little and place it on the gene amplifier, and purify it according to the following SAP procedure.

37℃37℃	40min40min
85℃85℃		5min5min
8℃8℃	HoldHold

Wherein the system of SAP reaction mixture is as follows:

ddH ₂O ddH ₂ O	1.53μl1.53μl
SAP BufferSAP Buffer	0.17μl0.17μl
SAP EnzymeSAP Enzyme	0.3μl0.3μl
合计total	2μl2μl

2.4.2 Extension reaction

Add 2 μl of the extension reaction mixture to each well of the SAP purification reaction product obtained in step 2.4.1, where the extension reaction mixture system is as follows:

ddH ₂O ddH ₂ O	0.62μl0.62μl
iPLEX Buffer PlusiPLEX Buffer Plus	0.2μl0.2μl
iPLEX Termination MixiPLEX Termination Mix	0.2μl0.2μl
iPLEX Primer MIXiPLEX Primer MIX	0.94μl0.94μl
iPLEX Pro EnzymeiPLEX Pro Enzyme	0.04μl0.04μl
合计total	2μl2μl

The concentrations of each extension primer in iPLEX Primer MIX are as follows:

检测基因名称detection gene name	延伸引物名称extension primer name	延伸引物在混合液中的浓度Concentration of extension primer in mixture
RPP40RPP40	RPP40#2_W1_ERPP40#2_W1_E	10.33～20.65μM10.33～20.65μM
SMN1、SMN2SMN1, SMN2	SMN1-2_E5_W1_ESMN1-2_E5_W1_E	8.62～17.24μM8.62～17.24μM
SMN1、SMN2SMN1, SMN2	SMN1-2_E6_W1_ESMN1-2_E6_W1_E	8.33～16.65μM8.33～16.65μM

SMN1、SMN2SMN1, SMN2	SMN1-2_E7_TY_W1_ESMN1-2_E7_TY_W1_E	8.99～17.98μM8.99～17.98μM
SMN1、SMN2SMN1, SMN2	SMN1-2_E7_TYI_W1_ESMN1-2_E7_TYI_W1_E	11.34～22.68μM11.34～22.68μM
SMN1、SMN2SMN1, SMN2	SMN1-2_E8_TY_W1_ESMN1-2_E8_TY_W1_E	10.72～21.45μM10.72～21.45μM
SMN1、SMN2SMN1, SMN2	SMN1-2_E8_TYI_W1_ESMN1-2_E8_TYI_W1_E	5.32～10.63μM5.32～10.63μM
SMN1SMN1	rs1221447932_W1_Ers1221447932_W1_E	7.16～14.33μM7.16～14.33μM
SMN1SMN1	rs1561500847_W1_Ers1561500847_W1_E	7.43～14.87μM7.43～14.87μM
SMN1SMN1	rs1554081968_W1_Ers1554081968_W1_E	7.82～15.65μM7.82～15.65μM
SMN1SMN1	rs77668214_W1_Ers77668214_W1_E	9.29～18.57μM9.29～18.57μM
SMN1SMN1	rs104893922_W1_Ers104893922_W1_E	9.52～19.03μM9.52～19.03μM
ALK融合ALK fusion	ALK_01-02_EALK_01-02_E	8.12μM8.12μM
ALK融合ALK fusion	ALK_21-22_EALK_21-22_E	5.99μM5.99μM
ALK融合ALK fusion	ALK_22-23_EALK_22-23_E	7.50μM7.50μM
ALK融合ALK fusion	ALK_23-24_EALK_23-24_E	10.36μM10.36μM
RET融合RET fusion	RET_02-03_ERET_02-03_E	9.28μM9.28μM
RET融合RET fusion	RET_04-05_ERET_04-05_E	8.37μM8.37μM
RET融合RET fusion	RET_12-13_ERET_12-13_E	5.65μM5.65μM
RET融合RET fusion	RET_13-14_ERET_13-14_E	6.75μM6.75μM
ROS1融合ROS1 fusion	ROS1_01-02_EROS1_01-02_E	6.41μM6.41 μM
ROS1融合ROS1 fusion	ROS1_04-05_EROS1_04-05_E	9.59μM9.59μM
ROS1融合ROS1 fusion	ROS1_35-36_EROS1_35-36_E	10.55μM10.55μM
ROS1融合ROS1 fusion	ROS1_37-38_EROS1_37-38_E	8.70μM8.70μM
NTRK1融合NTRK1 fusion	NTRK1_07-08_ENTRK1_07-08_E	10.32μM10.32μM
NTRK1融合NTRK1 fusion	NTRK1_08-09_ENTRK1_08-09_E	9.18μM9.18μM
NTRK1融合NTRK1 fusion	NTRK1_13-14_ENTRK1_13-14_E	6.52μM6.52μM
NTRK1融合NTRK1 fusion	NTRK1_14-15_ENTRK1_14-15_E	7.10μM7.10μM
NTRK2融合NTRK2 fusion	NTRK2_09-10_ENTRK2_09-10_E	9.47μM9.47μM
NTRK2融合NTRK2 fusion	NTRK2_10-11_ENTRK2_10-11_E	7.43μM7.43μM
NTRK2融合NTRK2 fusion	NTRK2_13-14_ENTRK2_13-14_E	7.61μM7.61 μM
NTRK2融合NTRK2 fusion	NTRK2_14-15_ENTRK2_14-15_E	8.64μM8.64μM
NTRK3融合NTRK3 fusion	NTRK3_09-10_ENTRK3_09-10_E	6.22μM6.22μM
NTRK3融合NTRK3 fusion	NTRK3_10-11_ENTRK3_10-11_E	8.44μM8.44μM
NTRK3融合NTRK3 fusion	NTRK3_14-15_ENTRK3_14-15_E	9.02μM9.02μM
NTRK3融合NTRK3 fusion	NTRK3_15-16_ENTRK3_15-16_E	5.76μM5.76μM

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.

2.4.3 Desalting treatment and mass spectrometer

At the end of the extension reaction program, centrifuge briefly. Add 16 μL of sterile double-distilled water and 6 mg of clean resin (Resin) to each well. Invert and mix for 15 min, and centrifuge at 3200 x g for 5 min. Sample spotting, score.

2.4.4 Data analysis and interpretation of test results

The original file exported by the instrument supporting software Typer4.0.

Analysis of copy number detection results: Calculated by the signal-to-noise ratio (SNR) of each detection site. The calculation formula is:

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).

The numerical interpretation range is as follows:

Table 5: SMN1 and SMN2 copy number results of 36 samples (comparison of Massarray method and MLPA gold standard method)

Result :

样本编号sample number	样本说明Sample description
1111	正常人normal person
1717	携带者 carrier
2020	患者patient
21twenty one	患者patient

Take the mass spectrometry results of samples 11, 17, 20, and 21 as an example, where the corresponding phenotypes of the samples are shown above. 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.

It was found that 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.

Based on the MassARRAY technology system, the present invention can detect the copy numbers of SMN1 and SMN2 and their hot spot SNPs in one reaction. Compared with the ordinary MassARRAY detection, the competitive substrate of each CNV detection site is added in the amplification process, and the SMN1/SMN2=1/0 can be accurately distinguished by comparing the peak signal-to-noise ratio with the competitive substrate detection. , 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.

Example 3

3.1 In this example, the same sample, competitor, and amplification system were used, but the primer pair with the same primer sequence but not modified by locked nucleic acid and the amplification primer with locked nucleic acid modification proposed in this method were used respectively. As shown below, amplification of each site is performed, followed by mass spectrometry detection, and the effect of the locked nucleic acid-modified primers proposed in this method in controlling the amplification reaction of competitors is compared.

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 .

3.2 In order to further visually verify the use of locked nucleic acid modified primers in this protocol to reduce the amplification efficiency of competitors, real-time quantitative qPCR was used to amplify the competing substrates using locked nucleic acid and non-locked nucleic acid modified primers respectively, and the Cp of each reaction was compared. value. When the sample amount of the competitor as the template is the same, the larger the Cp value is, the lower the amplification efficiency of the competitor is.

The primer information used in the reaction is as follows:

The competitors and their concentrations contained in the mixture are as follows:

Element

QC Mix 1ng ingredient concentration

QC Mix 5ng ingredient concentration

QC Mix 50ng ingredient concentration

SMN1-2_E5_QCSMN1-2_E5_QC	1ng/μl1ng/μl	5ng/μl5ng/μl	50ng/μl50ng/μl
RPP40_QCRPP40_QC	1ng/μl1ng/μl	5ng/μl5ng/μl	50ng/μl50ng/μl

The qPCR amplification system is as follows:

试剂reagent	体积volume
SybrgreenSybrgreen	5μl5μl
Primer Mix(5p)Primer Mix(5p)	0.4μl0.4μl
竞争物混合液competitor mix	1μl1μl
H ₂O H ₂ O	3.6μl3.6μl
TotalTotal	10μl10μl

The qPCR amplification conditions are as follows:

Results : The amplification reaction of the qPCR experiment is shown in Figure 7 and Figure 8, and the statistical data 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.

Example 4

4.1 Simulate the operation of freezing and thawing high-concentration (5ng) and low-concentration (1pg) competitor stock solutions for multiple times in actual operation according to the following operations:

4.1.1 The prepared mixture should be frozen at -80℃.

4.1.2 After it is completely frozen, take it out and thaw it completely at room temperature. Shake and centrifuge, pipette 10ul into new tubes, mark 1 time to thaw, and store them at -80°C. Return the original tube to -80°C to freeze.

4.1.3 After it is completely frozen, take it out and thaw it completely at room temperature. The original tube was then frozen at -80°C.

4.1.4 After it is completely frozen, take it out and thaw it completely at room temperature. Centrifuge with shaking, pipette 10ul into new tubes, mark as thawed 3 times, and store them at -80°C. Return the original tube to -80°C to freeze.

4.1.5 Repeat 3-4 times to obtain competitor mixtures that have been thawed 5, 7, 9, 11, 13, 15, 17, and 19 times and stored at -80°C.

4.1.6 Take out all the competitor mixtures thawed 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 times, and thaw them again to obtain thawed 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 competitor mixtures.

Then, 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:

Results : The results of qPCR of different concentrations of competitors that were not frozen and thawed are shown below:

After 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 freeze-thaw times, the results of qPCR with different concentrations of competitors are as follows:

From the above results, it can be seen that the competitor mixtures in the repeated freezing and thawing experiments in this experiment have two concentrations of 1 pg/μl and 5 ng/μl, 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/μl mixture can maintain a stable Cp value, and the 1pg/μl mixture is severely degraded after freezing and thawing once.

4.2 In this experiment, the competitor mixture of the experimental error experiment was carried out with two concentrations of about 50pg/μl and 5ng/μl, 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. Then calculate the SMN E6 SNR ratio (SNR(SMN1-2_E6_QC) and SNR(SMN E6) ratio), RPP40 SNR ratio (SNR(RPP40_QC) and SNR(RPP40) ratio) obtained each time. The more consistent the SNR ratio results of different batches are, the more stable the system is.

Among them, 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:

成分Element	QC Mix-锁核酸QC Mix-Locked Nucleic Acids	QC Mix-非锁核酸QC Mix-Unlocked Nucleic Acids
SMN1-2_E6_QCSMN1-2_E6_QC	约5ng/μlAbout 5ng/μl	约50pg/μlAbout 50pg/μl
RPP40_QCRPP40_QC	约5ng/μlAbout 5ng/μl	约50pg/μlAbout 50pg/μl

The obtained SNR (SMN1-2_E6_QC) and SNR (SMN E6) ratios, SNR (RPP40_QC) and SNR (RPP40) ratios are as follows:

The results showed that in the locked nucleic acid primer experiment, the SMN E6 SNR ratio and the RPP40 SNR ratio remained basically stable; while in the second batch of unlocked nucleic acid primers, the second batch was significantly reduced, and no competitor could be detected by the third batch. Therefore, it is proved that the reaction system established by the method of unlocked nucleic acid is unstable, while the relative stability of the competitor reaction system established by this method is greatly improved.

Conclusion : In practice, 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.

Example 5

The accuracy of the 10% abundance of ALK, ROS1, RET, NTRK1, NTRK2, NTRK3 gene fusion mutation reference and Sanger comparison.

5.1 MassArray experimental scheme:

5.1.1 Use Thermo Fisher's High-Capacity cDNA Reverse Transcription Kit (Cat. No. 4368814) to perform reverse transcription experiments on 200 ng of RNA to obtain cDNA.

5.1.2 Calculation of fusion detection formula and determination of threshold: Taking ALK gene fusion detection as an example, the calculation formula is as follows:

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.

If any of 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.

5.1.3 The remaining steps are the same as the method in Example 1. The MassArray maps of the 6 genes and the reference genes EML4 and TPM3 are shown in Figures 9-16. The summary MassArray results are as follows:

5.2 Sanger sequencing protocol:

5.2.1 Use Thermo Fisher's High-Capacity cDNA Reverse Transcription Kit (Cat. No. 4368814) to perform reverse transcription experiments on 200 ng of RNA to obtain cDNA.

5.2.2 PCR reaction, the system is as follows:

试剂名称Reagent name	体积(μl)Volume (μl)
Premix Taq Premix Taq	1010
Primer1(10μM)Primer1 (10μM)	11
Primer2(10μM)Primer2 (10μM)	11
模板 template	11
ddH ₂O ddH ₂ O	77
总体积 total capacity	2020

PCR cycling conditions are as follows:

5.2.3 Gel-tipping purification of PCR products

The 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.

5.2.4 Sanger sequencing

The sequencing reaction system is as follows:

试剂名称Reagent name	体积(μl)Volume (μl)
BigDye Mix kit BigDye Mix kit	11
测序Primer(3.2μM)Sequencing Primer (3.2 μM)	22
纯化后PCR产物PCR product after purification	1-31-3
ddH ₂O ddH ₂ O	0-10-1
总体积 total capacity	55

Sequencing reaction conditions:

NH ₄ AC.EDTA purification: After the sequencing reaction, centrifuge the 96-well plate at 3700 rpm for 0.5 min; add 1 μl 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.

100% alcohol precipitation: add 18 μl of 100% ethanol solution, precipitate at -20° C. for 30 min, centrifuge at 4500 rpm for 30 min, and invert at 400 rpm for 2 min.

Washing with 75% alcohol: add 50 μl of 75% alcohol, centrifuge at 4500 rpm for 10 min, invert and centrifuge at 400 rpm for 2 min.

Denaturation: Add 6μl Hi-Di, centrifuge at 3700rpm for 1min, and store at -20°C until the sequencer.

5.2.5 Sequencing: use a 3730xl sequencer for sequencing. The sequencing primers are as follows:

The Sanger sequencing results are shown in FIG. 17 . In this experiment, 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:

In the present invention, 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.

Claims

An amplification method comprises using a pair of locked nucleic acid-modified amplification primers to simultaneously amplify a target gene and its competitors.
The method of claim 1, which 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 gene of interest.
The method of any one of claims 1-2, wherein one or both primers in the pair of amplification primers are modified with locked nucleic acids; preferably wherein one or both primers in the pair of amplification primers The bases at one or more positions are modified with locked nucleic acid; more preferably, the bases at the 3' end of one or both primers in the pair of amplification primers are modified with locked nucleic acid.
The method of any one of claims 1-3, wherein the gene of interest is selected from the group of genes or different combinations thereof: SMN1, SMN2, ALK, RET, ROS1, NTRK1, NTRK2, NTRK3.
The method of claim 4, wherein the amplification primers are selected from the sequences set forth in SEQ ID NOs: 1-72 or different combinations thereof.
The method of any one of claims 1-5, wherein the competitor comprises a nucleotide sequence having 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.
The method of claim 6, wherein at least 1 of the different bases is located in the nucleotide sequence of the competitor corresponding to the amplification primer pair; preferably, at least 1 of the different bases is located in The competitor corresponds to the nucleotide site of the amplification primer pair to the base modified by the locked nucleic acid.
The method of any one of claims 1-7, wherein the base at the 3' end of one or both primers in the pair of amplification primers is modified by a locked nucleic acid, and the pair of amplification primers is locked The base of the nucleic acid modification corresponding to the base of the target gene is different from the base of its competitor.
The method of any one of claims 1-8, wherein 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 method of any one of claims 1-9, wherein the competitor is selected from the sequences set forth in SEQ ID NOs: 113-145 or different combinations thereof.
The method of any one of claims 1-10, wherein the final concentration of the competitor is 0.05-250ng/μl, 0.1-200ng/μl, 0.1-150ng/μl, 0.1-100ng/μl, 0.1-90ng /μl, 0.1-80ng/μl, 0.1-70ng/μl, 0.1-60ng/μl, 0.1-50ng/μl, 0.1-40ng/μl, 0.1-30ng/μl, 0.1-20ng/μl, 0.1-10ng/μl , 0.5-100ng/μl, 0.5-90ng/μl, 0.5-80ng/μl, 0.5-70ng/μl, 0.5-60ng/μl, 0.5-50ng/μl, 0.5-40ng/μl, 0.5-30ng/μl, 0.5 -20ng/μl, 0.5-10ng/μl, 1-100ng/μl, 1-90ng/μl, 1-80ng/μl, 1-70ng/μl, 1-60ng/μl, 1-50ng/μl, 1-40ng /μl, 1-30ng/μl, 1-20ng/μl, 1-10ng/μl, 5-100ng/μl, 5-90ng/μl, 5-80ng/μl, 5-70ng/μl, 5-60ng/μl , 5-50ng/μl, 5-40ng/μl, 5-30ng/μl, 5-20ng/μl, 5-10ng/μl, 10-100ng/μl, 10-90ng/μl, 10-80ng/μl, 10 -70ng/μl, 10-60ng/μl, 10-50ng/μl, 10-40ng/μl, 10-30ng/μl or 10-20ng/μl.
The method according to any one of claims 1-11, wherein the loading amount of the competitor is 5-80ng, 10-70ng, 15-60ng, 20-50ng, 20-40ng or 20-30ng, preferably 20-40ng.
The method of any one of claims 1-12, wherein the method is used in matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Taqman-PCR, next-generation sequencing, capillary electrophoresis analysis, or digital PCR.
A kit comprising a pair of locked nucleic acid modified amplification primers and a competitor of a target gene.
15. The kit of claim 14, wherein the kit further comprises an amplification reagent selected from the group consisting of dNTPs, DNA polymerase, MgCl2 , and combinations thereof.
The kit of claim 14 or 15, wherein one or both primers in the pair of amplification primers are modified by locked nucleic acids; preferably one or more of one or both primers in the pair of amplification primers The bases at the positions 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 kit of any one of claims 14-16, wherein the pair of amplification primers is selected from the sequences set forth in SEQ ID NOs: 1-72 or different combinations thereof.
The kit according to any one of claims 13-16, wherein 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.
The kit of claim 18, wherein 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 corresponds to the nucleotide site of the amplification primer pair to the base modified by the locked nucleic acid.
The kit of any one of claims 14-19, wherein the base at the 3' end of one or both primers in the pair of amplification primers is modified by a locked nucleic acid, and the pair of amplification primers is modified by a locked nucleic acid. The base of the target gene corresponding to the modified base of the locked nucleic acid is different from the base of its competitor.
The kit of any one of claims 14-20, wherein 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 kit of any one of claims 14-21, wherein the competitor is selected from the sequences described in SEQ ID NOs: 113-145 or different combinations thereof.
The kit of any one of claims 14-22 for use in detecting spinal muscular atrophy in a subject.
The kit of any one of claims 14-22, for use in detecting cancer in a subject; preferably detecting lung cancer, blood tumor, thyroid cancer, cholangiocarcinoma, soft tissue sarcoma, breast cancer, stomach cancer, esophageal cancer or Colorectal cancer.
The kit of any one of claims 14-22, wherein the pair of amplification primers is selected from the sequence described in SEQ ID NO: 1-10 or a combination thereof, and the competitor is selected from SEQ ID NO: 113 - Sequence described in 117 or a combination thereof; wherein the kit is used to detect gene mutation and/or copy number of SMN1 and/or SMN2 gene.
The kit of claim 25, further comprising an extension primer selected from the sequence of SEQ ID NOs: 73-79 or a combination thereof.
The kit of any one of claims 14-26, wherein the pair of amplification primers is selected from the sequence described in SEQ ID NO: 17-44 or a combination thereof, and the competitor is selected from the group consisting of SEQ ID NO: 118 - The sequence described in 131 or a combination thereof; wherein the kit is used to detect gene fusion mutations of ALK and/or RET and/or ROS1 genes.
The kit of claim 27, further comprising an extension primer selected from the sequences described in SEQ ID NOs: 85-98 or a combination thereof.
The kit of any one of claims 14-28, wherein the pair of amplification primers is selected from the sequence described in SEQ ID NO: 45-72 or a combination thereof, and the competitor is selected from the group consisting of SEQ ID NO: 132 -145 the sequence or a combination thereof; wherein the kit is used to detect gene fusion mutations of NTRK1 and/or NTRK2 and/or NTRK3 genes.
The kit of claim 29, further comprising an extension primer selected from the sequence of SEQ ID NOs: 99-112 or a combination thereof.
The kit of any one of claims 14-30, wherein the final concentration of the competitor is 0.05-250ng/μl, 0.1-200ng/μl, 0.1-150ng/μl, 0.1-100ng/μl, 0.1- 90ng/μl, 0.1-80ng/μl, 0.1-70ng/μl, 0.1-60ng/μl, 0.1-50ng/μl, 0.1-40ng/μl, 0.1-30ng/μl, 0.1-20ng/μl, 0.1-10ng/ μl, 0.5-100ng/μl, 0.5-90ng/μl, 0.5-80ng/μl, 0.5-70ng/μl, 0.5-60ng/μl, 0.5-50ng/μl, 0.5-40ng/μl, 0.5-30ng/μl, 0.5-20ng/μl, 0.5-10ng/μl, 1-100ng/μl, 1-90ng/μl, 1-80ng/μl, 1-70ng/μl, 1-60ng/μl, 1-50ng/μl, 1- 40ng/μl, 1-30ng/μl, 1-20ng/μl, 1-10ng/μl, 5-100ng/μl, 5-90ng/μl, 5-80ng/μl, 5-70ng/μl, 5-60ng/ μl, 5-50ng/μl, 5-40ng/μl, 5-30ng/μl, 5-20ng/μl, 5-10ng/μl, 10-100ng/μl, 10-90ng/μl, 10-80ng/μl, 10-70ng/μl, 10-60ng/μl, 10-50ng/μl, 10-40ng/μl, 10-30ng/μl or 10-20ng/μl.
The kit of any one of claims 14-31, wherein the loading amount of the competitor is 5-80ng, 10-70ng, 15-60ng, 20-50ng, 20-40ng or 20-30ng, preferably 20-40ng.
Use of the kit according to any one of claims 14 to 32 for controlling the amplification efficiency of a target gene and its competitor in simultaneous amplification of the competitor.
The use of claim 33, wherein the kit is used to amplify a gene selected from the group consisting of SMN1, SMN2, ALK, RET, ROS1, NTRK1, NTRK2, NTRK3.
The use of locked nucleic acid in the simultaneous amplification of a target gene and its competitors to control the amplification efficiency of said competitors.
The use of claim 35, wherein the locked nucleic acid is used to modify primer pairs that amplify the gene of interest and its competitors.
The use according to claim 35 or 36, wherein the gene of interest is selected from the group of genes or a combination thereof: SMN1, SMN2, ALK, RET, RO1, NTRK1, NTRK2, NTRK3.
The use of claim 35 or 36, wherein the primer pair is selected from the sequences described in SEQ ID NOs: 1-72 or different combinations thereof.
Use of a locked nucleic acid-modified primer pair in the simultaneous amplification of a target gene and its competitor to control the amplification efficiency of the competitor.
The use of claim 39, wherein the gene of interest is selected from the group consisting of genes or combinations thereof: SMN1, SMN2, ALK, RET, ROS1, NTRK1, NTRK2, NTRK3.
The use of claim 39 or 40, wherein the primer pair is selected from the sequences described in SEQ ID NOs: 1-72 or different combinations thereof.