Primer set and reagent kit for detecting mutation of lung cancer related genes in human circulating tumor DNA and use method of reagent kit

A technology of a kit and a primer set is applied in the field of primer sets for detecting lung cancer-related gene mutations in human circulating tumor DNA, which can solve the problems of low efficiency of clinical cancer treatment, reduce the burden of medical treatment, increase the signal value, and reduce the probability of error. Effect

Active Publication Date: 2019-08-20
中源维康(天津)医学检验所有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The effective rate of cancer clinical treatment is still low, which is closely related to the complex mechanism of cancer and the individual differences of patients

Method used

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  • Primer set and reagent kit for detecting mutation of lung cancer related genes in human circulating tumor DNA and use method of reagent kit
  • Primer set and reagent kit for detecting mutation of lung cancer related genes in human circulating tumor DNA and use method of reagent kit
  • Primer set and reagent kit for detecting mutation of lung cancer related genes in human circulating tumor DNA and use method of reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Detection of lung cancer-related gene mutation sites by the technology of the present invention

[0073] 1. Primer design and synthesis

[0074]针对BRAF_p.D594G、BRAF_p.G469A、BRAF_p.G469V、BRAF_p.V600E、EGFR_p.C797S、EGFR_p.D770_N771insSVD、EGFR_p.D770_N771insG、EGFR_p.E709A、EGFR_p.E709G、EGFR_p.E709K、EGFR_p.E746_A750del、EGFR_p.E746_E749del、EGFR_p .E746_S752>D、EGFR_p.E746_S752>V、EGFR_p.E746_T751del、EGFR_p.G719A、EGFR_p.G719C、EGFR_p.G719S、EGFR_p.H773_V774insNPH、EGFR_p.H773_V774insH、EGFR_p.L747_A750>P、EGFR_p.L747_E749del、EGFR_p.L747_P753>S 、EGFR_p.L858R、EGFR_p.L861Q、EGFR_p.L861R、EGFR_p.S768I、EGFR_p.T790M、EGFR_p.V769_D770insASV、ERBB2_p.A775_G776insYVMA、ERBB2_p.G776>VC、KRAS_p.G12A、KRAS_p.G12V、KRAS_p.G12D、KRAS_p.G12R , KRAS_p.G12C, KRAS_p.G12S, KRAS_p.G13C, KRAS_p.G13D, KRAS_p.Q61H, KRAS_p.Q61K, KRAS_p.Q61E, KRAS_p.Q61P, KRAS_p.Q61L, KRAS_p.Q61R, PIK3CA_p.E542K, PIK .H1047L, PIK3CA_p.H1047R. Wait for 49 gene mutation sites used to assess the specificity and sensitivity of...

Embodiment 2

[0147] Embodiment two: standard product detection

[0148] 9 cases of standard DNA samples (purchased from Horizon Company) with known mutations were selected for detection, numbered A1-A9. Among them, the mutation frequency of each mutation site corresponding to each sample of A1-A8 is 0.1%, and A9 is wild type.

[0149] According to the method of the present invention, according to the steps described in Example 1, 9 cases of standard DNA samples were detected by mass spectrometry. According to the judgment standard described in the embodiment, the results of the 9 standard samples were analyzed, and Table 14 was obtained.

[0150] Table 14

[0151]

[0152]

[0153] As a result of the test, the mutations corresponding to the 8 mutation-type standard samples can be detected normally, which shows that the method of the present invention can detect sites with a mutation frequency as low as 0.1% of the sample amount of 20 ng, and has frontier advantages.

Embodiment 3

[0154] Example 3: Detection of free DNA samples

[0155] According to the method of the present invention, according to the steps described in Example 1, mass spectrometry was performed on the free DNA samples of 30 lung cancer patients, and the sample numbers were B1-B30. The results of the 30 samples were analyzed according to a criterion described in the embodiment, and Table 15 was obtained.

[0156] Table 15

[0157] sample detected mutation sample detected mutation B1 KRAS_p.G12D B16 EGFR_p.S768I B2 KRAS_p.G12C B17 EGFR_p.L858R B3 EGFR_p.G719S, EGFR_p.L861Q B18 ERBB2_p.A775_G776insYVMA B4 EGFR_p.G719S, EGFR_p.L861Q B19 KRAS_p.G12S B5 EGFR_p.T790M B20 EGFR_p.E746_A750del B6 EGFR_p.E746_A750del B21 KRAS_p.G12D B7 BRAF_p.V600E B22 EGFR_p.V769_D770insASV B8 EGFR_p.S768I, EGFR_p.L858R B23 EGFR_p.E746_A750del B9 EGFR_p.L861Q B24 EGFR_p.S768I, EGFR_p.L858R B10 EGFR_p.L858R B2...

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Abstract

The invention discloses a primer set and reagent kit for detecting mutation of lung cancer related genes in human circulating tumor DNA and a use method of the reagent kit. 49 gene variation sites forassessing specificity and sensibility of risk that human suffer from lung cancer, of 5 genes are provided, and the sites are subjected to gene analysis and detection through a MassARRAY nucleic acidmass spectrum technique. When the reagent kit is in a single-basic-group extension, only ddNTPs for extending mutant genes is added, the ddNTPs has biotin marks, magnetic beads having streptavidin marks are used for capturing extension products, and signal interference of impurity peaks is removed, so that the signal value of mutation genes in the extension products is greatly increased, and the sensitivity of the reagent kit is increased. When the method is used for detecting circulating tumor DNA samples, the sensitivity under 20ng of sampling quantity can be as low as 0.1%. Compared with qPCR and a bibasic sequencing platform, the method has the advantages of being flexible in design, high in accuracy, low in cost, simple and convenient to operate and the like.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a primer set, a kit and a method for detecting lung cancer-related gene mutations in human circulating tumor DNA. Multiplex PCR technology, single base extension technology and nucleic acid mass spectrometry technology are used to detect lung cancer-related gene mutations. The 5 genes and 49 loci were tested for genes. Background technique [0002] Tumor is a new growth formed by the disorder of cell cycle through multi-step changes of multiple genes, which make cells grow out of control. Lung cancer is one of the malignant tumors that seriously threaten human health. Among them, non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer, and adenocarcinoma is the most common type of NSCLC. In oncology, genetics and other multidisciplinary studies, it has been found that most NSCLC patients have mutations in proto-oncogenes and tumor suppressor genes such ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6872C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6872C12Q1/6886C12Q2600/156C12Q2537/143
Inventor 李同恩孟庆艳陈兴京杨俊红张振宇朱兵张双华贾鑫哲崔斌刘宇飞
Owner 中源维康(天津)医学检验所有限公司
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