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Multiplex amplification detecting system of microsatellite instable state and application thereof

A microsatellite instability and compound amplification technology is applied in the field of compound amplification detection systems in the unstable state of microsatellites, and can solve the problems of lack of clear judgment criteria, the need for improvement in specificity and sensitivity, and uneven detection results.

Active Publication Date: 2018-11-06
QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the final detection results of the detection system provided in the above-mentioned prior art are uneven, the specificity and sensitivity need to be improved, and there is no clear judgment standard, which needs to be optimized and improved urgently

Method used

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  • Multiplex amplification detecting system of microsatellite instable state and application thereof
  • Multiplex amplification detecting system of microsatellite instable state and application thereof
  • Multiplex amplification detecting system of microsatellite instable state and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Design and Establishment of Microsatellite Instability Compound Amplification System

[0074] 1. Design of primer combinations

[0075] Firstly, microsatellite sites with high sensitivity and specificity were designed and selected through complex experiments, including 5 single nucleotide sites and 4 long single nucleotide (LMR) repeat sites, including: NR-21 , NR-24, BAT-25, BAT-26, MONO-27, BAT-52, BAT-56, BAT-59, and BAT-60, as well as 2 sample internal reference sites. We refer to the GeneBank sequence numbers of the genes where the 11 sites are reported in the literature, and find the gene sequences corresponding to each site from the NCBI nucleic acid database. The GeneBank numbers of each site are shown in Table 4, and the primer pair sequences corresponding to each site are listed in Primer Sequence Listing 4;

[0076] Table 4. MSI site information

[0077] MSI site

GeneBank number

major repeat sequence

NR-21

XM_033393.1 ...

Embodiment 2

[0087] The assembly of embodiment 2 kit

[0088] The PCR buffer, 10× primer mix, hot start Taq enzyme, reagent internal reference template, template DNA, deionized water and instructions are assembled into a kit.

Embodiment 3

[0089] Example 3 Tumor tissue sample detection

[0090] Selected 3 patients with colorectal cancer (the patient's blood sample was used as a control sample), and the complex amplification system of the present invention was used to detect the microsatellite instability state of its tumor cells. The specific operation steps are as follows:

[0091] 1. DNA extraction from FFPE samples

[0092] Use the QIAamp DNA FFPE Tissue Kit from QIAGEN to extract DNA from tumor and paracancerous FFPE samples of colorectal cancer patients, and then use Nanodrop to detect DNA concentration and quality. The DNA extracted from FFPE samples is best quantified again with Qubit, and finally diluted into a concentration of 5ng / μL.

[0093] 2. PCR amplification

[0094] 2.1 Preparation of 10×MSI primer mixture

[0095] Firstly, dissolve 13 pairs of fluorescently labeled primers into 100 μM stock solution with TE Buffer, and then prepare 10× primer mixture according to the requirements in Table 3; ...

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Abstract

The invention provides a multiplex amplification detecting system of a microsatellite instable state and application thereof. The multiplex amplification detecting system comprises 13 pairs of primerswhich are capable of amplifying 13 sites comprising 9 mononucleotide repeat sites (NR-21, NR-24, BAT-25, BAT-26, MONO-27, BAT-52, BAT-56, BAT-59 and BAT-60), 2 pentanucleotide repeat sites (Penta C and Penta D) and 2 reagent internal reference sites (QS90 and QS330), and optimizing the proportioning of the detecting system and the concentrations of the primers. A large amount of experiments verify that 13 sites can be finally and simultaneously amplified in one-tube reaction, and higher specificity and higher sensitivity are obtained; meanwhile, sample internal reference sites and reagent internal reference sites are added, so that interference caused by factors of sample mixing, reagent invalidation and the like during a whole detecting process can be avoided, and higher application value is obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a composite amplification detection system for microsatellite instability and its application. Background technique [0002] Microsatellite sequences are short tandem repeats of 1–6 bases that are highly variable and ubiquitous in eukaryotic genomes. Because of their high variability, microsatellites tend to vary in populations and are therefore widely used for the purposes of molecular markers, linkage mapping, pedigree mapping, and genotyping. About 3% of the human genome contains microsatellite sequences, which have the characteristics of single-nucleotide repeat sequences, among which poly(A / T) fragments are the most abundant. The mutation rate of microsatellites varies greatly among loci, and the variation range of each locus is between 10-6 and 10-2. At the same time, the tendency of microsatellite mutation becomes larger with the increase of repeat number. [0003] Microsate...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6886C12Q2600/166C12Q2600/156C12Q2525/151
Inventor 徐梅波吴子侠张亚飞李强朱爱思
Owner QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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