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Kit for detection of K-ras gene mutation and application thereof

A kit and gene technology, applied in the field of kits for detecting K-ras gene mutations, can solve the problems of difficult screening of homozygous mutations, harsh PCR conditions, and inability to type, so as to improve detection sensitivity and resolution, and promote Nucleic acid hybridization, the effect of shortening the operation time

Inactive Publication Date: 2016-02-24
杨华卫 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is difficult to screen for homozygous mutations, and the false positives and false negatives are high. The PCR conditions are harsh and cannot be typed, and the detection results need to be confirmed by sequencing.
Although gene chips and liquid-phase chips can accurately distinguish specific mutation types, they require high equipment and are difficult to promote on a large scale. They are mostly used in scientific research institutions
Fluorescent quantitative PCR method has the disadvantages of high false positives, and it is difficult to be applied in large-scale clinical diagnosis

Method used

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  • Kit for detection of K-ras gene mutation and application thereof
  • Kit for detection of K-ras gene mutation and application thereof
  • Kit for detection of K-ras gene mutation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Main raw materials and reagents

[0087] Cycloolefin copolymer plastic sheet and 99.99% silver wire;

[0088] 20×SSC buffer solution, the preparation process is as follows: Weigh 88.2g of trisodium citrate (purchased from Shanghai Sangong) and 175.3g of NaCl in 800ml of pure water, mix well, adjust the pH of the solution to 7.0± with concentrated HCl 0.1, just add pure water to 1000ml;

[0089] A primer pair for amplifying the K-ras gene, wherein the upstream primer sequence for amplifying the K-ras gene is shown in SEQ ID NO: 1, and the 5' end biotin is labeled; the downstream primer sequence for amplifying the K-ras gene is as shown in Shown in SEQ ID NO: 2. Entrust Shanghai Sangong to synthesize, and add water to the dry powder of the primers to prepare the concentration of 20μM;

[0090] A primer pair for amplifying the human Actin gene, wherein the nucleotide sequence of the upstream primer is as shown in SEQ ID NO: 5, the nucleotide sequence of the downstream p...

Embodiment 2

[0164] With embodiment 1, difference is that the pretreatment liquid composition in embodiment 1 is adjusted to: 5 parts of cystamine, 3 parts of C 17 Fatty alcohol polyoxyethylene ether and 2 parts of C 11 Isomerized alcohol, the balance is water; the composition of the hybridization solution is adjusted to: 3×SSC, 0.5μg / ml SA-AP, 5mM ZnCl 2 , 5mMMgCl 2 , 0.05% TritonX-100, 0.05% PLL and 2% polyethylene glycol 8000, the balance is water; the post-treatment liquid is adjusted to: PH9.0, 0.1mol / LTris-HCl, 0.1mol / LNaCl and 1% n-octyl glucoside, the balance is water. Other conditions remain unchanged.

Embodiment 3

[0166] With embodiment 1, difference is that the solid phase support among the embodiment 1 is replaced by slide glass, and the metal film of solid phase support surface load becomes the aluminum film of thickness 60nm, purity 99.9%; Pretreatment solution The composition is adjusted to: 10 parts of cystamine, 12 parts of C 18 Fatty alcohol polyoxyethylene ether and 8 parts of C 12 Isomerized alcohol, the balance is water; the composition of the hybridization solution is adjusted to: 3×SSC, 1.2μg / ml SA-AP, 50mM ZnCl 2, 50mMMgCl 2 , 1% Tween-20, 0.2% CPAM and 4% polyethylene glycol 8000, the balance is water; the post-treatment liquid is adjusted to: PH10.0, 0.1mol / LTris-HCl, 0.1mol / LNaCl and 4% n-decane glucoside, the balance is water. Other conditions remain unchanged.

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Abstract

The invention discloses a kit for detection of K-ras gene mutation. The kit comprises a PCR reaction reagent, a solid phase support coated with a metallic film and a hybridization solution containing alkaline phosphatase labeled-streptavidin, wherein the PCR reaction reagent comprises biotin-labeled primers used for amplifying target nucleic acids; and a nucleic acid probe used for detecting K-ras gene mutation is fixed on the surface of the solid phase support. The kit is a rapid highly-sensitive reverse hybridization technique product developed on the basis of the solid phase support coated with the metallic film. The kit has advantages of few operating step, simple process and rapid detection, can be used for highly-sensitively and highly-specifically detecting base mutation of K-ras gene in nucleic acid of a clinic colorectal cancer tissue sample, and is beneficial to guide clinical rectal cancer treatment.

Description

technical field [0001] The invention relates to the detection of gene mutations, in particular to a kit for detecting K-ras gene mutations. Background technique [0002] Colorectal cancer is one of the most common clinical malignant tumors that seriously endanger human life and health. Its incidence rate ranks third in the world and ranks fourth in China. Finding effective treatment methods for colorectal cancer has always been an important task. Research directions in the oncology community. In recent years, a new treatment method - targeted therapy, has made individualized treatment possible and significantly improved the curative effect of tumor treatment. In the targeted therapy of colorectal cancer, two anti-EGFR monoclonal antibodies Cetuximab (Cetuximab, trade name Erbitux) and Panitumumab (Panitumumab, trade name Vectibix) can specifically inhibit the The growth of colorectal cancer tissue cells with type K-ras gene improves the prognosis of patients. Therefore, t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 杨华卫曾冀
Owner 杨华卫
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