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PIK3CA gene mutation detection method and kit

A kit and mutant technology, applied in the field of biotechnology and tumor diagnosis, can solve the problems of long cycle, low sensitivity, high cost, etc.

Pending Publication Date: 2020-08-07
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that the cycle is long, the cost is high, and cross-contamination is prone to occur, and the sensitivity is not high
[0008] (2) High-throughput sequencing, high-throughput sequencing technology is a revolutionary change to next-generation sequencing, which can sequence hundreds of thousands or even millions of DNA molecules at a time, with high throughput and high accuracy , high sensitivity, etc., but its cost is high, it is not suitable for single gene mutation detection
Fluorescence PCR has poor specificity and poor detection sensitivity when detecting mutant gene mutations, and is not suitable for the detection of plasma free nucleic acids

Method used

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  • PIK3CA gene mutation detection method and kit
  • PIK3CA gene mutation detection method and kit

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Experimental program
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Effect test

preparation example Construction

[0170] Step 2, preparation of dPCR system

[0171] Preparations before dPCR system preparation: Take out the primer-probe mixture A1-A11, PCR reaction master mix B, RNase-free water, etc. in the kit, melt at room temperature, vortex and oscillate to mix, and then centrifuge for 10 seconds to prepare the dPCR system; dPCR The system composition is shown in Table 6:

[0172] Table 6. dPCR system

[0173]

[0174]

[0175] The nucleotide sequence information of the primer probes are respectively as above;

[0176] Step 3, add sample

[0177] Take 5 μL of the sample DNA template prepared in step 1 and 5 μL of each control sample in the kit, and add the sample to the eight-tube tube of the dPCR reaction system prepared in step 2, so that the total volume of each tube of dPCR reaction solution is 15 μL; cap tightly The eight-tube tube cap, mixed well, and centrifuged at high speed for 10 seconds, is used to prepare micro-reactions; the control samples of the kit are shown i...

Embodiment 1

[0197] Embodiment 1: Kit

[0198] The composition, packaging and quantity (20 reactions / box) of a breast cancer plasma free nucleic acid PIK3CA gene mutation detection kit provided by the present embodiment are shown in Table 8,

[0199] Table 8. The composition, packaging and quantity of the kit

[0200]

[0201]

Embodiment 2

[0202] Embodiment 2: Sensitivity detection and minimum detection rate experiment

[0203] The wild-type nucleic acid control sample is the fragmented PIK3CA gene wild-type nucleic acid derived from Caco2 cell line DNA; the sensitivity reference product is composed of 11 kinds of PIK3CA mutant gene template DNA and Caco2 cell line DNA respectively mixed in a certain proportion, and the mutation rate of the mixture is (%) were 1%, 0.1%, and 0.05%, respectively; wherein, 11 kinds of PIK3CA mutant gene template DNAs were derived from artificially synthesized DNA fragments 1-11; the negative control was RNase-free water;

[0204] Take 5 μL each of the negative control, wild-type nucleic acid control, and sensitivity reference substance, and add the sample to the eight-tube tube of the dPCR reaction system prepared in step 2, so that the total volume of each tube of dPCR reaction solution is 15 μL; tightly cover the tube cap of the eight-tube tube, Mix well and centrifuge at high sp...

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Abstract

The invention provides a PIK3CA gene mutation detection method and kit, and concretely discloses a primer and a probe for detecting PIK3CA gene mutation, and a kit comprising a primer and probe mixedsolution. 11 mutation types of the PIK3CA gene can be detected at the same time, and the method and the kit are suitable for dPCR detection, and have the advantages of multiple detectable mutation types, high sensitivity, small sample dependence and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology and tumor diagnosis, in particular, the invention relates to a PIK3CA gene mutation detection method and a kit thereof. Background technique [0002] Phosphatidylinositol-3-kinase catalytic subunit alpha gene (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha, PIK3CA) is located on chromosome 3q26.32, 92kb in length, composed of 23 exons, encoding IA The catalytic subunit p110α of phosphatidylinositol 3-kinases (PI3Ks), its main function is to catalyze 4,5-PIP2 into 3,4,5-PIP3 as a second messenger to bind and activate multicellular target protein. [0003] PIK3CA is an oncogene. When the gene is mutated, it can continuously stimulate downstream AKT activation through the PI3K / AKT pathway, making fibroblasts and breast epithelial cells continue to proliferate and inhibit cell apoptosis. Therefore, it is closely related to the occurrence and development of tumors. [0004] Breast...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/156C12Q2563/159
Inventor 蒋析文朱小亚潘翠园徐小解黄志文
Owner DAAN GENE CO LTD
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