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Diagnostic kit and method for detecting V561D mutation of human PDGFRalpha gene

A technology of α gene and kit, which is applied in the field of diagnostic kits for detecting the V561D mutation of human PDGFRα gene, can solve the problems of high cost and unfavorable clinical detection, and achieve low cost, fast detection and good clinical application prospects

Inactive Publication Date: 2015-09-23
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, at present, the PDGFRα gene V561D mutation is detected by sequencing, but the cost of sequencing is too high, which is not conducive to ordinary clinical detection

Method used

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  • Diagnostic kit and method for detecting V561D mutation of human PDGFRalpha gene
  • Diagnostic kit and method for detecting V561D mutation of human PDGFRalpha gene
  • Diagnostic kit and method for detecting V561D mutation of human PDGFRalpha gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 The present invention detects the kit and detection method of V561D mutation

[0040] PCR amplification reagent (50 servings):

[0041] components

1 serving

50 servings

MgCl2

5-10mmol

500mmol

dUTP PLUS

0.5μl

25μl

Various primers

0.1-1μl

50μl

Each probe

0.5-1.5μmol

75μmol

UNG

0.2μl

19μl

Taq

0.3μl

15μl

overall system

20μl

1000μl

[0042] Amplification primers and detection probes for PDGFRα gene V561D mutation:

[0043] Primers:

[0044] 12F 5'-tat gaa att cgc tgg agg ga-3' Forward (SEQ ID NO.1)

[0045] 12R 5'-AGTTGTGTGCAAGGGAAA-3' Reverse (SEQ ID NO.2)

[0046] Probe:

[0047] 12P FAM-5'-CCCATGGAACTTACCAAAGCACTAGTCC-3'-BQ1 Forward (SEQ ID NO.3)

[0048] Quality control primers and probes:

[0049] Primers:

[0050] 5'-cctcaagatcatcagcaatgcctc-3' Forward in CtlF (SEQ ID NO.4)

[0051] 5'-GTG GTC ATG AGT CCT TCC ACG ATA-3' Reve...

experiment example 1

[0102] Experimental example 1 The detection of the accuracy of kit and method of the present invention

[0103] 1. Sample to be tested

[0104] One clinical GIST paraffin tissue sample with known mutation type V561D and one known wild-type clinical GIST paraffin tissue sample were selected to detect human PDGFRα gene mutation results.

[0105] 2. Detection method

[0106] The method of the present invention: adopt the kit of embodiment 1, detect according to the method of embodiment 1.

[0107] Sample DNA extraction method: extract with Qiagen kit:

[0108] 1. Use a scalpel to remove the useless paraffin at the tissue border;

[0109] 2. Cut the paraffin-embedded tissue into 4 μm thick slices;

[0110] 3. Quickly use sterilized tweezers to take 2-6 pieces and put them into DNase / RNase Free EP tubes (each spreading area is 500 (maximum) mm) 2 , which is the size of a square with a side length of 1.6 cm;

[0111] 4. Add 800μl xylene, shake on the shaker for 10s, and centri...

experiment example 2

[0131] Experimental example 2 The specificity of kit and method of the present invention

[0132] 1. Sample to be tested

[0133] Randomly select 200 cases of clinical GIST paraffin tissue samples to detect human PDGFRα gene mutation results.

[0134] 2. Detection method

[0135] Sample DNA extraction method: extract with Qiagen kit:

[0136] ●Using a scalpel to remove useless paraffin at the tissue border;

[0137]●Slice the paraffin-embedded tissue into 4μm thick slices;

[0138] Quickly take 2-6 pieces with sterilized tweezers and put them into DNase / RNase Free EP tubes (the area of ​​each sheet is 500 (maximum) mm) 2 , which is the size of a square with a side length of 1.6 cm;

[0139] ●Add 800μl xylene, shake on the shaker for 10s, and centrifuge at the maximum speed for 3 minutes;

[0140] ●Add 800μl xylene, shake on the shaker for 10s, and centrifuge at the maximum speed for 3 minutes;

[0141] Discard the xylene, add 800 μl absolute ethanol, and centrifuge at t...

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PUM

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Abstract

The invention discloses a diagnostic kit for detecting V561D mutation of human PDGFRalpha gene. The diagnostic kit comprises probes shown in SEQ ID NO.1-2 and SEQ ID NO.3. The invention also discloses primer pairs shown in SEQ ID NO.1-2 and the probe shown in SEQ ID NO.3. The kit provided by the invention can accurately detect the V561D mutation of human gene PDGFRalpha, has the advantages of rapid detection, simpleness and low cost, can be popularized for usage by a large scale and shows good prospects for clinical application.

Description

technical field [0001] The invention relates to a diagnostic kit for detecting V561D mutation of human PDGFRα gene. Background technique [0002] Gastrointestinal stromal tumors (Gastrointestinal Stromal Tumors, GIST) are a type of tumor originating from the mesenchymal tissue of the gastrointestinal tract, accounting for most of the gastrointestinal mesenchymal tumors. Gastrointestinal stromal tumors account for 1-3% of gastrointestinal malignant tumors, and the estimated annual incidence is about 10-20 / 1 million. They mostly occur in middle-aged and elderly patients, and are rare in patients under 40 years old. There is no significant difference in incidence between men and women. GIST mostly occurs in the stomach (50-70%) and small intestine (20-30%), the colorectum accounts for about 10-20%, the esophagus accounts for 0-6%, and the mesentery, omentum and retroperitoneal are rare. 20-30% of GIST patients are malignant, and about 11-47% have metastasized at the first visi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/106C12Q2600/156
Inventor 叶丰陈卉娇张红英步宏
Owner WEST CHINA HOSPITAL SICHUAN UNIV