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Composition for detecting instability of microsatellite and application of composition

An unstable, microsatellite technology, applied in recombinant DNA technology, microbial assay/test, biochemical equipment and methods, etc., can solve problems such as low sensitivity and specificity, achieve accurate results, avoid cross-contamination, less demanding effects

Active Publication Date: 2017-10-27
北京鑫诺美迪基因检测技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Further through the adjustment of the above system, the sensitivity and specificity of the whole kit can be improved, and the sensitivity and specificity can reach 95% and 100% respectively, which solves the problem of low sensitivity and specificity in the detection of microsatellite instability in the past. question

Method used

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  • Composition for detecting instability of microsatellite and application of composition
  • Composition for detecting instability of microsatellite and application of composition
  • Composition for detecting instability of microsatellite and application of composition

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Embodiment 1: On the basis of multiplex fluorescent PCR, the present invention is formulated into a premixed primer composition for detecting 12 detection sites. The extracted sample can be tested directly, which makes the detection step easier.

[0053] Specific steps are as follows:

[0054] 1) Extract genomic DNA from control samples (such as whole blood samples) and paraffin-embedded tumor tissue samples (5-10 slices, about 5um) (using commercial peripheral blood genomic DNA and paraffin-embedded tissue genomic DNA extraction kits).

[0055] 2) Measure the concentration of the extracted genomic DNA, and dilute the concentration to 10-20 ng / μL for subsequent PCR reaction process.

[0056] 3) Composition of the reaction system: dNTPs, Mg 2+ , DNA polymerase buffer, reverse transcription buffer, primer pairs for 12 genes, DNA polymerase, etc.

[0057] 4) PCR reaction conditions: UDG enzyme reaction at 37°C for 2min; pre-denaturation at 95°C for 3min; denaturation at...

Embodiment 2

[0086]Collect 400 random colorectal cancer samples and 400 corresponding peripheral blood control samples from Wuhan Tongji Hospital, and use the above-mentioned finished kit for detection. The specific operation steps are as described in Example 1. Among the 400 random samples of colorectal cancer, 60 samples of MSI-H type were detected, 13 samples of MSI-L type were detected, and the rest samples were of MSS type. According to the above results, it can be concluded that the proportion of microsatellite instability in colorectal cancer accounts for about 18%, which is consistent with the corresponding literature reports.

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Abstract

The invention discloses a composition for detecting the instability of a microsatellite and application of the composition. The composition comprises primer pairs respectively used for detecting specific target genes relevant with the instability of the microsatellite in a to-be-detected sample. A kit for detecting the instability of the microsatellite is used for detecting microsatellite distribution conditions of nine loci of Bat 26, Bat 25, NR-21, NR-24, NR-27, Mono-27, D5S346, D2S123 and D17S250 and three loci of Penta C, Penta D and Amel by virtue of a specific multi-fluorescence PCR fragment through an analysis capillary electrophoresis method and detecting whether the microsatellite is in a stable or instable state. By adjusting the proportion of 12 pairs of primers, the use amounts of Taq enzyme and magnesium ions, a PCR system and the like, and the sensitivity and specificity of the whole kit are improved.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a technology for detecting microsatellite instability. Background technique [0002] The genetic factors of colorectal cancer (Colorectal Cancer, CRC) include chromosome instability (Chromosome Instability, CIN) and microsatellite instability (Microsatellite Instability, MSI). About 80-85% of CRC is caused by CIN, including familial adenomatous polyposis (FAP) (germline mutation of APC gene) and sporadic CRC (mutation of APC, P53, DCC, KRAS and other genes); 15-20% of CRC is mainly caused by MSI, including hereditary nonpolyposis colorectal cancer (HNPCC, also known as Lynch syndrome) (mismatch repair gene germline mutation) and sporadic MSI ( +) CRC (mismatch repair gene MLH1 gene promoter methylation). [0003] Microsatellite instability (Microsatellite Instability, MSI) is the same microsatellite locus between different individuals and between normal tissu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2565/125C12Q2563/107
Inventor 刘沛王林海
Owner 北京鑫诺美迪基因检测技术有限公司
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