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Reagent kit used for testing PIK3CA (phosphatidylinositol-4,5-bisphosphate3-kinase, catalytic subunit alpha) gene mutations

A kit and reagent technology, applied in biotechnology and medical fields, can solve the problem of low sensitivity, achieve high sensitivity, simple and effective detection method, and excellent performance

Inactive Publication Date: 2015-02-25
WUHAN YZY MEDICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The object of the present invention is to provide a PIK3CA gene mutation detection kit and detection method thereof, aiming at solving the problem of low sensitivity in the prior art

Method used

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  • Reagent kit used for testing PIK3CA (phosphatidylinositol-4,5-bisphosphate3-kinase, catalytic subunit alpha) gene mutations
  • Reagent kit used for testing PIK3CA (phosphatidylinositol-4,5-bisphosphate3-kinase, catalytic subunit alpha) gene mutations
  • Reagent kit used for testing PIK3CA (phosphatidylinositol-4,5-bisphosphate3-kinase, catalytic subunit alpha) gene mutations

Examples

Experimental program
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Effect test

Embodiment 1

[0107] Prepare a PIK3CA gene mutation detection kit, specifically comprising the following steps:

[0108] 1. Synthetic primers and probe sequences

[0109] Synthetic primer sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10-SEQ ID NO:13 ; Synthetic specific probe sequences SEQ ID NO:3, SEQ ID NO:9 and SEQ ID NO:14, wherein SEQ ID NO:3 and SEQ ID NO:9 label the FAM fluorescent group at the 5' end, and the 3' end Label the quenching group NFQ-MGB modification group, SEQ ID NO: 14 is labeled with the VIC fluorescent group at the 5' end, and the quenching group TAMRA is labeled at the 3' end.

[0110]The above primer sequences were respectively prepared into 100 μM mother solution for storage, and the above probe sequences were respectively prepared into 100 μM mother solution for storage.

[0111] 2. Synthetic Blocking Nucleic Acid Sequence

[0112] Synthetic MGB blocking nucleic acid sequence SEQ ID NO:6, and carry out MGB-NF...

Embodiment 2

[0120] Use the PIK3CA gene mutation detection kit prepared in Example 1 to detect the samples to be tested.

[0121] In this embodiment, 50 cases of paraffin-embedded tissue sections of colorectal cancer patients diagnosed by clinicopathology were collected, and genomic DNA was extracted therefrom, and the PIK3CA gene mutation detection kit obtained in Example 1 was used to detect whether there was PIK3CA gene in the samples to be tested E542K, E545K, E545D, H1047R, and H1047L mutations are verified by traditional sequencing methods. The specific steps are as follows:

[0122] 1. Extraction of Genomic DNA from Samples

[0123] Genomic DNA was extracted from the tissue samples of the above lung cancer patients using a DNA extraction kit (QIAamp DNA FFPE Tissue Kit, Cat No. 56404). The DNA extraction method was carried out according to the instruction manual, and the operation steps were briefly described as follows: Place the clinically collected paraffin-embedded tissue (≤25m...

Embodiment 3

[0140] Prepare a PIK3CA gene mutation detection kit, specifically comprising the following steps:

[0141] 1. synthetic primer and probe sequence (with embodiment 1)

[0142] 2. synthetic blocking nucleic acid sequence (same as embodiment 1)

[0143] 3. Preparation of fluorescent quantitative PCR reaction system

[0144] Prepare the mutation detection reaction system containing No. 1-5 reagent, the quality control detection reaction system containing No. 6 reagent, the No. 7 reagent containing enzyme mixture, weak positive control and blank control, and the components are as follows in Table 3 Shown:

[0145] Components of No. 1-9 reagents in Table 3

[0146]

[0147]

[0148] The above PCR buffer contains dNTPs, magnesium ions and other necessary components for PCR reaction.

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Abstract

The invention is applicable to biotechnology and the medical field and relates to a reagent kit used for testing PIK3CA (phosphatidylinositol-4,5-bisphosphate3-kinase, catalytic subunit alpha) gene mutations. A first reagent, a second reagent, a third reagent, a fourth reagent and a fifth reagent are sequentially targeted to E542K, E545K, E545D, H1047R and H1047L mutations of a PIK3CA gene, and a sixth reagent is targeted to a conserved region of the PIK3CA gene and used for quality control. A testing method is carried out by the testing reagent kit by means of fluorescence quantitative PCR (polymerase chain reaction). The testing reagent kit is high in sensitivity and good in specificity and is a high-performance human PIK3CA gene mutation testing reagent kit. The fluorescence quantitative PCR method is adopted for performing specific amplification testing on sequences in PIK3CA gene mutation regions, and specific binding of the MGB (minor groove binder) blocked nucleic acid and a wild type DNA (deoxyribo nucleic acid) template in a sample to be tested is utilized to inhibit binding of the wild type DNA template and a primer probe. Forward primers are designed by methods of lowering the primer Tm value, tailing the 5' end and adding and inserting bases among the primers and the like, specificity of the reagent kit can be improved, then more than 45 cycle numbers are introduced, and accordingly effect of improving sensitivity of the regent kit is achieved.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and in particular relates to a human PIK3CA gene mutation detection kit and detection method. Background technique [0002] PI3K (Phosphatidylinositol-3-Kinase, phosphatidylinositol 3-kinase) is an intracellular phosphatidylinositol kinase, which consists of a 110k Da catalytic subunit p110 and a 85k Da regulatory subunit p85. Among them, PIK3CA (phosphatidylinositol-4,5-bisphosphate3-kinase, catalytic subunit alpha, phosphatidylinositol 3'-kinase catalytic subunit alpha) is the p110α catalytic subunit of PI3K family type IA PI3K. PI3K is expressed in normal human brain, lung, breast, gastrointestinal, cervical and ovarian tissues, and has important physiological functions such as regulating cell proliferation, survival, death and differentiation in the body. Under normal circumstances, PI3K can produce phosphatidylinositol 3,4-diphosphate (PIP2) and phosphatidylinositol 3,4,5-triphosp...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/156
Inventor 蔡从利董瑞华彭盼张喆周鹏飞
Owner WUHAN YZY MEDICAL SCI & TECH
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