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Kit for detecting microsatellite instability

A microsatellite instability and kit technology, applied in the field of nucleic acid detection, can solve the problem of short time

Inactive Publication Date: 2022-03-01
艾普拜生物科技(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to overcome the defects of the existing MSI detection method and provide a MSI detection kit with simple operation, short time, ultra-sensitive and good detection repeatability

Method used

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  • Kit for detecting microsatellite instability
  • Kit for detecting microsatellite instability
  • Kit for detecting microsatellite instability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: A kind of test kit for detecting microsatellite instability

[0037] 1.1 Check the gene number of the MSI site to be tested in the Gene Bank, and determine the main repeat sequence of each MSI site and the base sequence of about 400 bp upstream and downstream of the main repeat sequence. The relevant information of the micro-MSI site is shown in Table 2 Show.

[0038] Table 2: MSI site information

[0039] Microsatellite loci Gene gene number major repeat sequence BAT-25 c-KIT L04143 (A)25 BAT-26 hMSH2 U41210 (A)26 NR-21 SLC7A8 XM_033393 (A)21 NR-24 Zinc finger2 X60152 (A)24 MONO-27 MAP4K3 Ac007684 (A)27

[0040] 1.2 According to the target gene sequence, design multiple primers and multiple specific probes for each MSI site. The 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quencher group. After multiple screening and detection , pick out the p...

Embodiment 2

[0053] Embodiment 2: A method of using a kit for detecting microsatellite instability

[0054] 2.1 Instrument: Naica TM Stilla digital PCR instrument (French STILLA TECHNOLOGE company).

[0055] 2.2 Extraction of DNA in the sample: extract the DNA in the sample, and dilute the extracted DNA to 50 ng / μL as a template for digital PCR amplification.

[0056] 2.3 Preparation of digital PCR amplification reagents:

[0057] Eight components of the test kit are taken out from a test kit for detecting microsatellite instability according to the present invention, namely digital PCR reaction solution 1, digital PCR reaction solution 2, digital PCR reaction solution 3, and digital PCR premix solution 1 , digital PCR master mix 2, negative quality control product, positive quality control product, nuclease-free water, after completely melting on ice, shake evenly, briefly centrifuge for a few seconds, prepare a digital PCR reaction system according to Table 5, Table 6, and Table 7 Mi...

Embodiment 3

[0078] Embodiment 3: Determination of the sensitivity of a kit for detecting microsatellite instability

[0079] In order to verify the detection sensitivity of the kit in the present invention, we mixed the genomic DNA solutions of the MSI highly unstable cell line HCT-116 and the stable cell line K562 according to the ratio, and obtained the proportions of HCT-116 as 10%, 1% and 1% respectively. %, 0.1% and 0.05% of the four DNA mixtures.

[0080] Using the kit described in Example 1 and the method of using the kit described in Example 2, the above four DNA mixtures of 10%, 1%, 0.1% and 0.05% were tested and calculated after three parallel repetitions average value. The results show that the kit of the present invention is close to the detection limit when the MSI highly unstable ratio is 0.1%, and when the MSI highly unstable ratio is 0.0.5%, only one MSI site can be detected. Therefore, the detection sensitivity of the kit of the present invention is 0.1%, and the detect...

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Abstract

Based on the digital PCR technology, the invention provides a novel microsatellite instability detection kit which is simple to operate, short in time consumption, ultra-sensitive and good in detection repeatability. The kit does not need a normal sample as a contrast, does not need complex analysis software, overcomes the situation that low frequency and heterozygosity deficiency are difficult to detect in an unstable state of the microsatellite, and has the detection sensitivity of 0.1%.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a kit for detecting microsatellite instability. Background technique [0002] Microsatellite instability (MSI) is a phenomenon in which the number of repetitions of microsatellite site repeat units (usually tandem repeat DNA sequences with 1 to 6 nucleotides as the unit) fluctuates, that is, the insertion or delete. The stability of different MSI sites is different. The size of the microsatellite repeat unit, the base composition of the repeat unit, the structure of the repeat sequence and the number of repeats will all affect the stability of the site to a certain extent. According to the degree of MSI, MSI can be divided into Microsatellite Instability High (MSI-H), Microsatellite Instability Low (MSI-L) and Microsatellite Stable (MSS). Since MSI is a relatively special type of mutation with multiple additions or deletions of the same base, the dete...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2563/159C12Q2525/151
Inventor 龚建郑祖亮于祥春冯晓燕林挺
Owner 艾普拜生物科技(苏州)有限公司