PIK3CA gene mutation fluorescence quantitative PCR genotype detection kit and detection method

A detection kit and fluorescence quantitative technology, applied in the field of molecular biology, can solve the problems of complex operation process, insufficient detection sensitivity and time-consuming

Inactive Publication Date: 2013-01-02
广州达健生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 1. The detection cycle is long, and it takes about 48-72 hours at the shortest from the time the specimen is submitted for inspection to the result;
[0008] 2. The operation process is complicated and involves the operation after PCR amplification, so it is easy to be contaminated, resulting in unsatisfactory results (see item F for details);
[0009] 3. The interpretation of sequencing results is complex and time-consuming (this is especially prominent when the sample size is large, see item F for details);
[0010] 4. The detection sensitivity is not high

Method used

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  • PIK3CA gene mutation fluorescence quantitative PCR genotype detection kit and detection method
  • PIK3CA gene mutation fluorescence quantitative PCR genotype detection kit and detection method
  • PIK3CA gene mutation fluorescence quantitative PCR genotype detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Detection of base substitution mutations at positions 542 (G→A mutation) and 545 (G→A mutation) of exon 9 of PIK3CA gene

[0071] Sample Description: Collect tumor tissues from 50 patients with clinical pathological diagnosis of intestinal cancer (adenocarcinoma), all of whom have not received radiotherapy and chemotherapy before operation. Genomic DNA is extracted from the tumor tissues of case specimens for the following experimental applications. Prior to this, the tumor tissue content of the test specimen must be evaluated first, and the cases with tumor cell components greater than 30% are selected for the implementation test of the kit of the present invention.

[0072] Design a fluorescent probe and a pair of common primers that can specifically detect base substitution mutations at the 542nd (G→A mutation) and 545th positions (G→A mutation) of exon 9 of the PIK3CA gene, and one pair of common primers, plus one for simultaneous coverage The wild-type f...

Embodiment 2

[0085] Example 2: Detection of base substitution mutation at position 1047 (A→G) of exon 20 of PIK3CA gene

[0086] Design a fluorescent probe and a pair of common primers that can specifically detect the base substitution mutation at the 1047th (A→G) site of exon 20 of the PIK3CA gene, plus a wild-type fluorescent probe sequence to detect this site.

[0087] Among them, the PCR primers are:

[0088] Exon20F: 5'-TAC ATT CGA AAG ACC CTA GCC T-3' (SEQ ID NO: 6);

[0089] Exon20R: 5'-ATC CAT TTT TGT TGT CCA GCC-3' (SEQ ID NO: 7)

[0090] Fluorescent probes are:

[0091] Exon20-1047MT: 5'-FAM-TGA TGC ACG TCA TGG T-BHQ 1-3' (SEQ ID NO:8);

[0092] Exon20-1047WT: 5'-HEX-TGA TGC ACG TCA TGG T-BHQ1-3' (SEQ ID NO: 9).

[0093] Wherein, SEQ ID NO: B3 is a probe for detecting the base corresponding to the 1047 site of the 20th exon of the mutant PIK3CA gene, and SEQ ID NO: B4 is a wild-type fluorescent probe covering the 1047 site of the 20th exon of the PIK3CA gene. The needle sequ...

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Abstract

The invention discloses a PIK3CA gene mutation fluorescence quantitative PCR genotype detection kit comprising a PCR mixed reaction liquid, a positive control, and a fluorescent probe used for detecting PIK3CA gene mutant genotypes. The PCR mixed reaction liquid comprises PCR primers used for amplifying PIK3CA gene segments where the mutation sites exist. The invention also discloses a PIK3CA gene mutation fluorescence quantitative PCR genotype detection method. According to the method, PIK3CA gene mutant genotypes are subjected to fluorescence quantitative PCR detections by using the kit provided by the invention. With the technical scheme provided by the invention, detections can be carried out upon tissue PIK3CA gene mutations, and especially rapid, accurate, and high-sensitively detections upon PIK3CA gene 542,545 and 1047 nucleotide mutations can be realized.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a fluorescent quantitative PCR kit for detecting gene mutations and a detection method thereof, in particular to a fluorescent quantitative PCR typing detection kit for PIK3CA gene mutations and a detection method thereof. Background technique [0002] PIK3CA (phosphatidylinositol 3-kinase catalytic alpha) was detected by Volinia et al. in 1994 using in situ hybridization. It is a newly discovered oncogene with somatic mutations, which may be regulated by participating in the PIK3 / AKT cell signal transduction pathway. Cell growth and apoptosis. Studies have shown that PIK3CA is related to a variety of malignant tumors, such as colorectal cancer, cervical cancer, breast cancer, lung cancer and so on. At present, the relationship between oncogene PIK3CA and tumors and its mechanism of tumorigenesis have become a hot spot in tumor research. The occurrence of human tumors is a mult...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 邵琦
Owner 广州达健生物科技有限公司
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