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A qPCR composition and kit for detecting human c-kit gene mutation

A technology for detecting human and kits, which is applied in the field of QPCR compositions and kits for detecting human c-kit gene mutations, can solve the problems of time-consuming direct sequencing methods, cannot be widely promoted, and difficult to clinical detection, etc. High speed, improved detection sensitivity and specificity, and strong specificity

Active Publication Date: 2018-09-28
武汉海吉力生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The direct sequencing method is time-consuming and has low sensitivity. It is usually only used in scientific research and is difficult to use in clinical detection.
The instruments used in the high-resolution melting curve method are relatively special, and it is difficult to popularize them in hospitals
Beijing Baikangan Company has developed a human c-kit gene D816V mutation detection kit based on fluorescent PCR method, which can detect the D816V site mutation in vitro, but due to unknown reasons, it is limited to scientific research and cannot be widely promoted

Method used

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  • A qPCR composition and kit for detecting human c-kit gene mutation
  • A qPCR composition and kit for detecting human c-kit gene mutation
  • A qPCR composition and kit for detecting human c-kit gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: Extraction of clinical sample DNA

[0052] In this example, DNA is extracted from paraffin-embedded tissue sections of the lungs of patients with non-small cell lung cancer and quantified as a template for PCR detection. The QIAamp paraffin-embedded tissue extraction kit from Qiagen was used, and the specific details are as follows:

[0053] 1. Preparation before experiment

[0054] (1) Adjust the water bath to 56°C.

[0055] (2) Place Buffer ATL and Buffer AL in the QIAamp kit at 56°C until the precipitate dissolves.

[0056] 2. Sample processing

[0057] (1) Cut off the excess paraffin on the paraffin section, cut the section into a suitable size, and install it on a paraffin microtome.

[0058] (2) Adjust the caliper to 10 μm, and then cut the wax block until a uniform tissue can be clearly seen on the cut section.

[0059] (3) Remove the first 5 slices, start from the 6th slice, put them into 1.5mL EP tubes, 4 slices per tube, and then number them...

Embodiment 2

[0078] Example 2: Real-time fluorescent PCR method to amplify clinical sample DNA

[0079] In this example, the primers and probes provided by the present invention are used to prepare the mutation detection reaction solution and the external control detection reaction solution according to the formula in Table 1. After the reaction solution is prepared and mixed, it is divided into 8-section PCR reaction strips, the odd-numbered wells are divided into the mutation detection reaction solution, and the even-numbered wells are divided into the external control detection reaction solution. The DNA sample extracted in Example 1 was used as the reaction sample.

[0080] Table 1 Preparation of PCR detection reaction solution

[0081]

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Abstract

The invention discloses a QPCR composition used for detecting c-kit gene mutation of people and a reagent kit. The composition comprises an internal control detection primer pair, an internal control detection probe, a point mutation detection primer pair and a point mutation detection probe. The internal control detection primer pair is shown in SEQ ID NO:9-10. The internal control detection probe is a sequence shown in SEQ ID NO:11, wherein in the sequence, the end 5' is connected with FAM, and the end 3' is connected with BHQ1. The point mutation detection primer pair is shown in SEQ ID NO:1-2 or SEQ ID NO:3-4. The point mutation detection probe is a sequence shown in SEQ ID NO:5, wherein in the sequence, the end 5' is connected with FAM, and the end 3' is connected with BHQ1. The QPCR composition further comprises a peptide nucleotide sequence and an external control detection composition. By means of the detection primer pairs and the probes, D816V point mutation on the number 4 chromosome exon 17 of people can be rapidly, sensitively and effectively detected.

Description

technical field [0001] The invention belongs to the technical field of gene mutation detection, in particular to a QPCR composition and kit for detecting human c-kit gene mutation. Background technique [0002] The c-kit gene is located on human chromosome 4q12-13, and its encoded product is stem cell growth factor receptor, which belongs to type III tyrosine kinase, and its extracellular region has 5 or 3 immunoglobulin domains, which can bind The combination of body SCF makes kit dimerization. After dimerization, the kinase domain in kit cells is autophosphorylated to activate downstream signal transduction, thereby regulating gene expression and controlling cell growth, proliferation and differentiation. Studies have found that gastrointestinal stromal tumors (GIST) are mainly formed due to the continuous activation of tyrosine kinases caused by mutations in the proto-oncogene c-kit, resulting in uncontrolled proliferation of mutant cells. [0003] The drug small molecul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2600/166C12Q2561/101C12Q2531/113C12Q2525/107
Inventor 郭骁段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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