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Nucleic acid test strip method for detecting polymorphism of 118th codon of ERCC1 (excision repair cross complement)

A technology of nucleic acid test strips and site polymorphism, which is applied in the direction of measuring devices, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of large instruments and equipment, long detection time, expensive prices, etc., and achieve High reaction specificity and sensitivity, simple operation, and low-cost effect

Inactive Publication Date: 2014-05-28
SUZHOU YOUBEIWO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems that most of the ERCC1 site polymorphism detection methods are complex in operation, expensive in price, long in detection time, and require large-scale instruments and equipment, the present invention utilizes amplification technology (PCR&AS-PCR) and nucleic acid thin film chromatography rapid detection method and Its test strip technology (patent number CN1234567890) is used for the rapid detection of polymorphism at the 118th codon of ERCC1

Method used

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  • Nucleic acid test strip method for detecting polymorphism of 118th codon of ERCC1 (excision repair cross complement)
  • Nucleic acid test strip method for detecting polymorphism of 118th codon of ERCC1 (excision repair cross complement)
  • Nucleic acid test strip method for detecting polymorphism of 118th codon of ERCC1 (excision repair cross complement)

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Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1. Detection of polymorphism in codon 118 of ERCC1 in a single human

[0040] a sampling

[0041] 200ul of peripheral venous anticoagulant blood (frozen storage) of the subject was used for nucleic acid extraction using the micro DNA rapid extraction kit produced by Hangzhou Ustar Biotechnology Co., Ltd. for gene amplification.

[0042] b: firstly amplify the target template containing the 118th codon polymorphism site of ERCC1 by PCR, and at the same time, the primers for PCR amplification are not marked;

[0043] It is characterized by:

[0044] c: On the basis of step b, use AS-PCR to perform specific amplification according to the base type of the ERCC1 codon 118 polymorphic genotype,

[0045] In a sterilized 0.2ml PCR tube, in a 20ul reaction system, add different PCR amplification primers and probes for different genotypes;

[0046] PCR & AS-PCR nucleic acid test strip method to detect ERCC1 codon 118 polymorphism reaction system:

[0047] Template...

Embodiment 2

[0064] 2B: Corresponding sequencing results, the bases underlined in red are the sites to be detected: 1. TT homozygous; 2. CC homozygous. Example 2, Detection of Polymorphism in Codon 118 of Multiple Persons ERCC1

[0065] a sampling

[0066] 200ul of peripheral venous anticoagulant blood (frozen storage) of the subject was used for nucleic acid extraction using the micro DNA rapid extraction kit produced by Hangzhou Ustar Biotechnology Co., Ltd. for gene amplification.

[0067] b: firstly amplify the target template containing the 118th codon polymorphism site of ERCC1 by PCR, and at the same time, the primers for PCR amplification are not marked;

[0068] It is characterized by:

[0069] c: On the basis of step b, use AS-PCR to perform specific amplification according to the base type of the ERCC1 codon 118 polymorphic genotype,

[0070] In a sterilized 0.2ml PCR tube, in a 20ul reaction system, add different PCR amplification primers and probes for different genotypes; ...

Embodiment 3

[0089] Example 3, to further define Example 1 or 2, in step c, PCR & AS-PCR nucleic acid test strip method to detect ERCC1 118th codon polymorphism reaction system, PCR primers: 0.2 each of 181PF and 181PR μM, labeled primer: 0.1 μM each of 181D5F-C / 181D5G-A, specific probe: 0.1 μM of 181D3B, MgCl 2 2.5 mM, 2 units of Taq DNA polymerase, PCR reaction program: 95°C, 5 minutes; 94°C, 20 seconds; 60°C, 20 seconds; 72°C, 20 seconds; 40 cycles; 94°C, 20 seconds; 45 cycles ℃, 20 seconds; 5 cycles.

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Abstract

The invention is a technology for performing allele specific amplification by using a labeled primer on the basis of PCR (polymerase chain reaction) amplification of a target template, and eventually detecting the 118th codon of an ERCC1 (excision repair cross complement) by a nucleic acid test strip method. The main detection principle is as follows: according to immunological specific binding of an antigen and a specific antibody thereof, a nucleic acid fragment is fixed on the nucleic acid detection test strip, and then according to color development of visual colorimetric latex particles on the test strip, a result is detected. In the technology for detecting polymorphic genotypes of the 118th codon of the ERCC1 by the PCR & AS-PCR nucleic acid test strip method, a specific PCR amplification primer for PCR amplification, an allele-specific primer with a biotin label at 5' end, and a specific probe with an FITC (fluorescein isothiocyanate) label at 3' end are mainly used.

Description

technical field [0001] The invention relates to a method for detecting the polymorphism of the 118th codon of excision repair cross-complementing gene 1 (excision repair cross-complementing gene 1, ERCC1) by using a nucleic acid test strip method. More specifically, the rapid detection of the polymorphism of the 118th codon of ERCC1 is carried out by using the amplification technology (PCR&AS-PCR), the rapid detection method of nucleic acid thin film chromatography and the test strip technology. Background technique [0002] The main mechanism of action of platinum-based drugs is to combine with guanine, adenine, and cytosine on DNA to form platinum-DNA adducts, resulting in inter-strand cross-linking or intra-strand cross-linking of DNA, causing DNA damage, resulting in cell death . Differences in DNA repair ability will directly lead to individual differences in the susceptibility of tumor cells to DNA-related cytotoxic drugs. ERCC The 118th codon Asn-Asn of the 1 gene h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/558
CPCC12Q1/6804C12Q1/6858C12Q2531/113C12Q2563/131C12Q2565/137
Inventor 胡琳江建辉侯建华徐高连孙悦
Owner SUZHOU YOUBEIWO BIOTECH
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