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Microsatellite unstable state detection kit and detection method thereof

A technology of microsatellite instability and detection kits, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problems of unreliable judgment results, inability to detect other polymorphic sites, Incomplete detection and other problems, to achieve accurate and reliable detection results, avoid missed judgments, and ensure sensitivity

Pending Publication Date: 2020-03-24
WUHAN BIOTECH GENE ENG
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Problems solved by technology

[0005] At present, PCR technology is mainly used to study the changes of microsatellite instability. The most commonly used method is to detect the instability of the selected 6 single-base repeat sites, and other polymorphic sites of the selected genes cannot be detected. , the detection is not comprehensive, and the judgment result is not reliable

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  • Microsatellite unstable state detection kit and detection method thereof
  • Microsatellite unstable state detection kit and detection method thereof
  • Microsatellite unstable state detection kit and detection method thereof

Examples

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Embodiment 1

[0047] This embodiment provides a primer set for detecting microsatellite instability.

[0048] (1) This example is aimed at 5 mononucleotide repeat sites (NR21, NR24, BAT25, MONO27 and BAT26), 3 dinucleotide repeat sites (D5S346, D17S250 and D2S123) and control pentanucleotide repeats Primers were designed for the site (Penta D) and the control tetranucleotide repeat site (TPOX). The details of the 10 detection sites are as follows:

[0049]

[0050]

[0051] (2) Design of multiple fluorescently labeled primers

[0052] NR21-F1: 5'-VIC-TAAATGTATGTCCCCTGG-3' (SEQ ID NO.1)

[0053] NR21-R1: 5'-TTACATACAGAGGGGACCTG-3' (SEQ ID NO.2)

[0054] D5S346-F2: 5'-FAM-AATGAGTGAGATCACTATTTAGCCC-3' (SEQ ID NO.3)

[0055] D5S346-R3: 5'-TCGTCTATTCTGTCATAATGATCAA-3' (SEQ ID NO.4)

[0056] NR24-F2: 5'-NED-GACTTAAAATGGAGGACTGAGGTTT-3' (SEQ ID NO.5)

[0057] NR24-R1: 5'-AACACGGTAACGTAAGGTTGGACCC-3' (SEQ ID NO.6)

[0058] BAT25-F1: 5'-VIC-CGCCTCCAAGAATGTAATGT-3' (SEQ ID NO.7)

[0059]...

Embodiment 2

[0077] For the detection method of microsatellite instability provided in this embodiment, the sample to be tested can be any form of sample that can extract nucleic acid, including but not limited to whole blood, serum, plasma and tissue samples; tissue samples include but not limited to paraffin-coated Buried tissue, fresh tissue, and frozen sections.

[0078] S1. Sample genomic DNA extraction

[0079] Two patients with colorectal cancer were taken, one of which was detected as MSI-H by MMR immunohistochemistry, and the other was detected as MSS by MMR immunohistochemistry, and the cancerous tissue samples and normal tissue samples (peripheral blood or cancerous tissue 3cm around), make paraffin-embedded tissue sections with a thickness of 5-20μm (make sure the section contains 9mm 2 Tissue blocks of the above size), use the paraffin-embedded tissue DNA extraction kit from TIANGEN Company to extract genomic DNA from the paraffin-embedded tissue sections of the two samples, ...

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Abstract

The invention provides a detection kit for an unstable state of a microsatellite. The kit comprises a primer group for detecting the unstable state of the microsatellite. The primer group is characterized in that upstream and downstream primers are designed for 10 microsatellite sites, namely NR21, D5S346, NR24, BAT25, D17S250, MONO27, BAT26, D2S123, Penta D and TPOX; base sequences of the primersare shown as SEQ ID NO.1 to 20; and the primers are high in specificity and wide in coverage range. The invention further provides a detection method of the unstable state of the microsatellite. Thedetection method is based on a multiplex fluorescence PCR technology and a capillary electrophoresis technology. The kit provided by the invention is used for detecting the unstable state of multiplemicrosatellite sites, and has the advantages of convenient and rapid operation, high specificity and sensitivity, good reproducibility, and accurate and reliable results.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a detection kit for microsatellite instability and a detection method thereof. Background technique [0002] Microsatellite sequences refer to short tandem repeat structures with a core sequence of 1 to 6 bases, which are widely distributed throughout the human genome. When the function of DNA mismatch repair is impaired, the length of microsatellite repeat sequence can be changed, which is called microsatellite instability (Micro-Satellite Instability, MSI). MSI is one of the characteristics of DNA repair-deficient tumor cells, and about 15% of colorectal cancers have MSI. MSI detection is recommended by the National Comprehensive Cancer Network (NCCN) colorectal cancer clinical practice guidelines and the Chinese Anti-Cancer Association Clinical Oncology Collaborative Professional Committee (CSCO) colorectal cancer diagnosis and treatment guidelines for all colorectal cancer pati...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107C12Q2565/125
Inventor 熊慧陶慧清谢立群吴琼肖樊周丹丹高峰英
Owner WUHAN BIOTECH GENE ENG
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