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Primer set, kit, library and application for multi-gene combined detection of gynecological tumors

A combined detection technology for gynecological tumors, applied in the field of biochemical detection, can solve the problems of expensive instruments, difficult to reduce the cost of building a database experiment, and unfavorable clinical promotion, etc., to achieve the effect of simple process, easy experiment automation, and low false positive rate

Inactive Publication Date: 2019-02-12
GUANGZHOU JIAJIAN MEDICAL TESTING CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are two relatively mature methods for targeted sequencing. One is liquid-phase hybridization capture technology, which is mature and stable, but often takes a long time to build a library (about 3 days) and requires more expensive instruments (such as interrupted instruments). etc.), the cost of library construction experiments is difficult to reduce, etc., so it is not conducive to clinical promotion; another method of targeted sequencing is multiplex PCR capture technology, which has the advantages of fast library construction experiments, short cycle (1 day), no additional equipment and The advantage of low detection cost, easy to carry out in the laboratory

Method used

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  • Primer set, kit, library and application for multi-gene combined detection of gynecological tumors
  • Primer set, kit, library and application for multi-gene combined detection of gynecological tumors
  • Primer set, kit, library and application for multi-gene combined detection of gynecological tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] A kit for multi-gene joint detection of gynecological tumors, which specifically includes the following components: a PCR amplification module and a magnetic bead purification module;

[0063] The PCR amplification module includes the following components:

[0064]

[0065] The magnetic bead purification module includes the following components:

[0066] Magnetic beads 90μL / sample;

[0067] ddH 2 O 100μL / sample.

Embodiment 2

[0069] (1) Genomic DNA extraction

[0070] Collect 2 mL of peripheral blood of the subject (the subject is a person who voluntarily undergoes high-end physical examination) and place it in an EDTA anticoagulation tube, and take 300 μL of EDTA anticoagulant blood from the ultra-clean workbench for genomic DNA extraction. According to the salting-out whole blood DNA purification kit (manufacturer: Guangzhou Meiji Biotechnology Co., Ltd., product model: D3311-02) for genomic DNA extraction, and use Nanodrop to determine the DNA concentration, record the corresponding concentration and 260 / 280 and 260 / The ratio of 230. A total of 1 sample was tested in this test.

[0071] (2) The first round of multiple PCR amplification (the PCR amplification module of the kit prepared in Example 1): Using the genomic DNA of the sample to be tested prepared in step (1) as a template, the reagents prepared in Example 1 are used The box is used to prepare multiple PCR amplification reaction system fo...

Embodiment 3

[0086] Collect 2 mL of peripheral blood from the subject (the subject is a person who voluntarily undergoes a high-end physical examination) to extract genomic DNA and perform the first round of multiplex PCR amplification, the second round of PCR amplification, the second round of PCR product purification, quality inspection, and library Quantitative, diluted library, computer-based sequencing, etc.; apply bioinformatics analysis software NextGENe to convert the sequencing data into FASTQ files, and perform data analysis, compare the DNA sequence obtained by sequencing to the reference target gene sequence, and identify 14 carcinogens Genes (AKT1, APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, PIK3CA, PP2R1A, PTEN, TP53, BRCA1, BRCA2) were extracted from SNVs and Indels, and mutations on target genes were obtained by excluding genetic polymorphism sites For information, see Example 2 for specific methods.

[0087] Genetic test results such as Figure 4 As shown in Table 3, the qua...

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Abstract

The invention relates to the field of biochemical detection, in particular to a primer set, a kit, a library and an application for the multi-gene combined detection of gynecological tumors. The primer set for the multi-gene combined detection of gynecological tumors can be used for carrying out specific amplification of 14 oncogenes APC, AKT1, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, PIK3CA, PPP2R1A, PTEN, TP53, BRCA1 and BRCA2 in the same PCR reaction system. The invention also provides a kit comprising the above primer set, and the kit is based on multiplex PCR targeted capture technology, and can easily complete the enrichment of target sequences within 8 h and obtain the high-quality library which can be directly sequenced on the machine. Using the sequencing library constructed by thekit and combining with the second-generation sequencing technology, the mutations of 1% or lower can be detected, and the false positive rate is extremely low.

Description

Technical field [0001] The invention relates to the field of biochemical detection, in particular to a primer set, kit, library and application for multi-gene joint detection of gynecological tumors. Background technique [0002] The incidence of malignant tumors of the female genital tract in China ranks first among female malignancies, and it is one of the main causes of death of women with cancer. The main reason for the high mortality of women with cancer is the lack of early screening methods, especially ovarian cancer. Therefore, seeking an early, simple, specific, and non-invasive screening method is an important problem to be solved urgently to improve the prognosis of patients with gynecological malignant tumors. [0003] The establishment of early screening methods can enable women's cancer patients to be detected, diagnosed, and treated early, so that the survival rate of patients is improved, and the mortality rate is reduced, so as to achieve the goal of promoting the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6869C12N15/11C40B40/06
CPCC12Q1/6869C12Q1/6886C12Q2600/16C40B40/06C12Q2537/143C12Q2535/122C12Q2531/113
Inventor 张巍陆国辉
Owner GUANGZHOU JIAJIAN MEDICAL TESTING CO LTD
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