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PIK3CA gene mutation detection probe, detection liquid phase chip and detection method thereof

一种液相芯片、探针的技术，应用在分子生物学领域，能够解决实时荧光定量PCR检测技术假阳性率高、特异性差等问题，达到避免假阳性率高、反应条件均一、提高特异性和准确率的效果

Active Publication Date: 2011-09-14
GUANGZHOU SUREXAM THERANOSIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, real-time fluorescence quantitative PCR detection technology itself has the problem of high false positive rate, that is, poor specificity, which is also criticized by clinical diagnosis.

Method used

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  • PIK3CA gene mutation detection probe, detection liquid phase chip and detection method thereof
  • PIK3CA gene mutation detection probe, detection liquid phase chip and detection method thereof
  • PIK3CA gene mutation detection probe, detection liquid phase chip and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Preparation of liquid-phase chip for detection of PIK3CA gene mutation

[0055] 1. Probe sequence design and microsphere coating

[0056] Specific oligonucleotide probes were designed for the wild-type and mutant sequences of exons 9 and 20 of the PIK3CA gene. The 5' end of the probe is an amino group followed by a 10 T spacer. The probe was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Probes were coupled with different color-coded microspheres (purchased from Luminex) through covalent bonding (coating process).

[0057] The specific steps of each microsphere coating are as follows:

[0058] (1) Take the microsphere mother solution (purchased from Luminex Company) and vibrate on a vortex instrument to form a microsphere suspension;

[0059] (2) Take out 8ul microsphere mother solution, containing 0.8×10 5 -1.2×10 5 microspheres into a 0.5ml centrifuge tube;

[0060] (3) Centrifuge at 10,000rpm for 2min, discard the supernatant;

[0...

Embodiment 2

[0078] Using the PIK3CA gene mutation detection liquid chip in Example 1 to detect breast cancer tissue samples

[0079] 1. Preparation of samples to be tested

[0080] Extraction of DNA from breast cancer tissue samples: take 5-50 mg of tissue samples after breast cancer surgery, grind them, and wash them twice with PBS solution of pH 7.4; the washed tissue samples are resuspended in 1ml of digestive solution (50mmol / L Tris, 1mmo / L Na 2 EDTA, 0.5% Tween-20, 200ug / ml proteinase K 200, pH 8.5), digested in 55°C water bath for 1 hour, inactivated proteinase K in 99°C water bath for 15 minutes; centrifuged at 12000 rpm for 10 minutes; took the supernatant and passed through phenol - Chloroform-isoamyl alcohol extraction and ethanol precipitation to obtain DNA samples for PCR reactions. DNA can also be extracted by micro spin column method;

[0081] 2. PCR amplification and enzyme digestion enrichment of the samples to be tested:

[0082] (1) PCR amplification and enzyme dige...

Embodiment 3

[0189] Example 3, PIK3CA gene mutation detection liquid chip detection sensitivity experiment

[0190] In order to test the detection sensitivity of this liquid-phase chip, the ability to detect mutant DNA in PIK3CA wild-type DNA was designed, that is, to investigate the minimum ratio of mutant DNA that this liquid-phase chip system can detect. The copy numbers of wild-type DNA and mutant DNA in each group of samples in this experiment are shown in Table 2. Each set of experiments was repeated. The steps of the first round of PCR, enzyme digestion reaction, second round of PCR reaction and luminex detection of the samples in this example are the same as those described in Example 2.

[0191] Table 3 PIK3CA gene mutation detection liquid-phase chip sensitivity detection results

[0192] wild-type DNA

copy number

mutant DNA

copy number

mutant DNA

The proportion

E542K-P

(MFI)

E545K-P

(MFI)

E545D-P

(MFI)

H1047R-P

...

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Abstract

The invention discloses a PIK3CA gene mutation site detection probe, a detection liquid phase chip and a detection method thereof. The PIK3CA gene mutation detection liquid phase chip mainly comprises a microball covered with a probe and a primer for amplifying a target sequence carrying a 9th exon and / or a 20th exon. The PIK3CA gene mutation detection liquid phase chip and the detection method can help simultaneously detect the sites with relatively higher frequency of the PIK3CA gene mutation, and can separately detect the 9th exon and the 20th exon or detect the 9th exon and the 20th exon at the same time, and the detection has uniform reaction condition, good specificity of detection results, high sensitivity, good accuracy up to 90% and short detection time.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a detection probe for PIK3CA gene mutation, a liquid phase chip and a detection method thereof. technical background [0002] The PIK3CA gene is the intracellular homologue of the retroviral v-p3k oncogene, encoding I A p110α catalytic subunit of phosphatidylinositol 3-kinases (PI3Ks). Class I PI3Ks are heterodimers composed of a p110 catalytic subunit and a p85 regulatory subunit, which can be regulated by receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and insulin receptor activation. Activated PI3Ks phosphorylate phosphatidylinositol 4,5-bisphosphate [phosphatidylinositol 4,5-bisphosphate, PI(4,5)P2] to generate an important second messenger in cells - phosphatidylinositol (3, 4,5) Triphosphate (phosphatidyli-3,4,5-triphosphate, PIP3). PIP3 can activate the AKT pathway to produce a wide range of biological effects, including regulation...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q2600/156C12Q1/6837C12Q1/6886
Inventor 许嘉森郭元杰
Owner GUANGZHOU SUREXAM THERANOSIS
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