Method for synchronously detecting gene mutations of 8th, 19th, 20th and 21st exons of ERBB2 gene

A technology for simultaneous detection and detection methods, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of low detection throughput, improve detection throughput, save costs, The effect of reducing inspection costs

Pending Publication Date: 2020-07-10
BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Low detection throughput since each PCR reaction targets only one exon

Method used

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  • Method for synchronously detecting gene mutations of 8th, 19th, 20th and 21st exons of ERBB2 gene
  • Method for synchronously detecting gene mutations of 8th, 19th, 20th and 21st exons of ERBB2 gene
  • Method for synchronously detecting gene mutations of 8th, 19th, 20th and 21st exons of ERBB2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Design and synthesize primer sets, including the following steps:

[0036] Step 1.1: Based on exons 8, 19, 20, and 21 of the ERBB2 gene, design upstream and downstream primers and corresponding sequencing primers for specifically amplifying the mutation regions of exons 8, 19, 20, and 21 of the ERBB2 gene.

[0037] For the design of primers, Primer Quest and Primer Premier 5.0 were used to design primers and analyze dimers and stem-loop mismatches. Primers were designed at both ends containing mutation sites, and the annealing temperatures of the three pairs of primers were basically consistent.

[0038] The primer set provided in this example covers all gene mutations of exons 8, 19, 20, and 21 of the ERBB2 gene. Since small sequence changes will lead to a significant decrease in primer amplification efficiency and poor specificity, multiple PCR primer sets were designed for different sites / exons, and after pre-experimental screening, the length and position of the pro...

Embodiment 2

[0043] Genomic DNA, tumor genomic DNA or tumor cell-free DNA (ctDNA) is extracted from the sample to be tested, comprising the following steps:

[0044] Step 2.1: The samples to be tested are serum / plasma samples separated from fresh peripheral blood containing DNA, tumor tissue samples, oral exfoliated cells collected from oral swabs, or fresh peripheral blood samples.

[0045] In this embodiment, the sources of samples are tumor patients and normal human body.

[0046] Step 2.2: Use Qiagen QIAamp Circulating Nucleic Acid Kit (55114), or, use Tiangen Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304), to extract tumor cell-free DNA (ctDNA), or, tumor For genomic DNA, use Tiangen oral swab genomic DNA extraction kit (DP322), or use blood / cell / tissue genomic DNA extraction kit (DP304) to extract genomic DNA from the sample to be tested, and use NP80-touch ( German IMPLEN) to measure the concentration and purity of DNA, and store the DNA.

Embodiment 3

[0048] A multiplex PCR detection method for synchronously detecting mutations in exons 8, 19, 20, and 21 of the ERBB2 gene, comprising the following steps:

[0049] Step 3.1: Use the DNA obtained in step 2.2 as an amplification template, and use the primer set synthesized in step 1.2 to configure a multiplex PCR reaction system.

[0050] In this example, the DNA polymerase and buffer in the KOD FX enzyme system (article number: KFX-101) of TOYOBO Co., Ltd. were used as the basic raw materials. concentration, buffer concentration, and enzyme dosage to prepare a multiplex PCR amplification system. The specific composition of this reaction system is shown in Table 2 below. Of course, the proportional amplification / reduction of the reaction system is within the protection scope of the embodiment of the present invention; the purpose of amplification can also be achieved by replacing other DNA polymerase systems and adjusting the appropriate ratio.

[0051] Table 2

[0052] ...

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Abstract

The invention provides primer sets and detection method for synchronously detecting gene mutations of 8th, 19th, 20th and 21st exons of an ERBB2 gene, and is beneficial to improvement of detection throughput. The invention provides the primer set for synchronously detecting gene mutations of the 8th, 19th, 20th and 21st exons of the ERBB2 gene. The nucleotide sequence of an upstream primer of thefirst set of primer pairs is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO.2; the nucleotide sequence of an upstream primer of a second set of primer pairs is shown as SEQ ID NO.3, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO.4; and the nucleotide sequence of an upstream primer of a third set of primer pairs is shown as SEQ ID NO.5, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO.6. The primer sets and method can detect the gene mutations of the 8th, 19th, 20th and 21st exons of the ERBB2 genethrough one reaction, so that the detection efficiency is improved, and the costs are saved to a large extent.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a primer set and a detection method for synchronous detection of gene mutations in exons 8, 19, 20, and 21 of the ERBB2 gene. Background technique [0002] Anti-tumor drug resistance has become a major problem in the clinical application of anti-tumor drugs, and the mutation of the target is an important mechanism of resistance to anti-tumor targeted drugs. Therefore, finding sites with functional significance, such as activating mutation sites and drug resistance mutation sites, as drug targets for targeted tumor therapy and molecular markers for tumor diagnosis is an important factor for precision tumor therapy to play a role and overcome drug resistance. key. The epidermal growth factor receptor gene family has always been an important target for cancer treatment, for example, small molecule tyrosine kinase inhibitors such as gefitinib and erlotinib targeting EGFR, mon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6886C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2565/125
Inventor 冯薇赵方圆智慧芳倪君君
Owner BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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