Primers, probe and kit for detecting human MET gene 14 exon splicing mutation

A primer-probe and kit technology, applied in the field of biotechnology and clinical molecular diagnosis, can solve the problems of high requirements, complex operation, long detection cycle, etc., to reduce the probability of deviation, reduce the probability of contamination, and shorten the detection time. Effect

Inactive Publication Date: 2016-06-29
BEIJING FUANHUA BIOLOGICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no commercial kit for the detection of MET14 exon splicing mutations. A few research institutions use high-throughput sequencing to scan the entire exon to detect the presence of MET14 exon splicing mutations. This method has high sensitivity and can Simultaneously detect dozens or even hundreds of tumor-related genes, but the operation is complicated, the later data processing is cumbersome, the detection cycle is long and the detection cost is expensive, the requirements for operation and interpretation technology are very high, and there are still many deficiencies in clinical practice. For the above reasons, high-throughput sequencing is not suitable for large-scale screening and diagnosis of MET mutation-positive NSCLC patients at this stage

Method used

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  • Primers, probe and kit for detecting human MET gene 14 exon splicing mutation
  • Primers, probe and kit for detecting human MET gene 14 exon splicing mutation
  • Primers, probe and kit for detecting human MET gene 14 exon splicing mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, detection method establishment

[0078] 1. Design primers and probes

[0079] 1. After a lot of pre-experiments and effect verification, the specific primer pair A and the specific probe A are obtained.

[0080] Specific primer pair A consists of primer 1 and primer 2.

[0081] Primer 1 (sequence 3 of the sequence listing): 5'-CACTGTTATTACTACTTGGGT-3';

[0082] Primer 2 (sequence 4 of the sequence listing): 5'-AGAGGATACTGCACTTGTCG-3';

[0083] The nucleotide sequence of probe A is as follows (sequence 5 of the sequence listing): 5'-AAGAGAAAGCAAATTAAAGATCAGT-3';

[0084] Probe A has a fluorescent reporter group FAM at its 5' end and a fluorescent quencher group BHQ1 at its 3' end.

[0085] 2. Select the human ACTB gene as an internal control gene, and obtain specific primer pair B and probe B after preliminary experiments and effect verification.

[0086] Specific primer pair B consists of primer 3 and primer 4.

[0087] Primer 3 (sequence 6 of the se...

Embodiment 2

[0108] Embodiment 2, detection method verification

[0109] 1. Construction of positive quality control plasmids and internal control plasmids

[0110] Positive quality control plasmid: Insert the double-stranded DNA molecule shown in Sequence 9 of the sequence listing between the BamI and KpnI restriction sites of the PCU57 vector to obtain a recombinant plasmid (named positive quality control plasmid).

[0111] Internal control plasmid: the recombinant plasmid obtained by inserting the double-stranded DNA molecule (partial segment in the human ACTB gene) shown in sequence 10 of the sequence table between the XbaI and KpnI restriction sites of the PCU57 vector (named internal control plasmid) .

[0112] Two, the kit that application embodiment 1 obtains detects

[0113] 1. Mix the positive quality control plasmid and the internal control plasmid according to the ratio of copy number 10:1, then adjust the DNA concentration with Tris-HCl buffer (2.5mM, pH8.5) to obtain a temp...

Embodiment 3

[0119] Embodiment 3, sensitivity, specificity and repeatability verification

[0120] The kit prepared in Example 1 was used for verification of sensitivity, specificity and repeatability.

[0121] 1. Sensitivity

[0122] 1. Dilute the positive quality control plasmid prepared in Example 2 with a 10-fold gradient with Tris-HCl buffer (2.5mM, pH8.5) to obtain each dilution.

[0123] 2. Take the dilutions obtained in step 1 as templates for real-time fluorescent quantitative PCR reaction.

[0124] Real-time fluorescence quantitative PCR reaction system (25 μL): Mix 20 μL of PCR master mix, 1 μL of enzyme mix and 4 μL of diluent.

[0125] Due to the different diluents used, the following different reaction systems are formed:

[0126] In reaction system 1, the initial content of the positive quality control plasmid DNA is: 10,000 copies;

[0127] In reaction system 2, the initial content of positive quality control plasmid DNA is: 1000 copies;

[0128] In reaction system 3, ...

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Abstract

The invention discloses primers, a probe and a kit for detecting human MET gene 14 exon splicing mutation. The invention provides a specific primer pair A. The specific primer pair A is composed of a primer 1 and a primer 2 which are respectively disclosed as Sequence 3 and Sequence 4 in the sequence table. The invention also provides a primer-probe combination A which is composed of the specific primer pair A and a probe A, wherein the nucleotide sequence of the probe A is disclosed as Sequence 5 in the sequence table. The invention also provides a kit B which comprises the primer-probe combination A. The invention also provides application of the primer-probe combination A or the kit B in detecting whether mutation for causing MET gene 14 exon jump deletion occurs in the human genome DNA (deoxyribonucleic acid). The method is simple to operate, has the advantages of short detection time, high sensitivity, high specificity, high safety, no toxicity, low cost and the like, and can satisfy the actual demands for clinical quick detection.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and clinical molecular diagnosis, and relates to a primer, a probe and a kit for detecting the splicing mutation of exon 14 of human MET gene. Background technique [0002] Lung cancer is the most common malignant tumor in the world, and its morbidity and mortality rank first among all cancers. 80%-85% of lung cancer cases are non-small cell lung cancer (NSCLC). Every year, more than 1.3 million patients die of lung cancer worldwide, nearly half of which occur in developing countries. In recent years, the morbidity and mortality of lung cancer in China have shown a significant upward trend. The 5-year survival rate of lung cancer patients in my country is only 13%. The main reason is that most lung cancers lack highly sensitive gene mutation detection and diagnosis techniques. , and the matching scientific treatment plan, so that patients miss the best time for treatment. [0003] At presen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/106C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 余倩王宏伟葛猛
Owner BEIJING FUANHUA BIOLOGICAL TECH CO LTD
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