Kit utilizing stem loop primers to detect PIK3CA gene mutation sites

A stem-loop primer and mutation site technology, applied in the biological field, can solve the problems of cumbersome second-generation sequencing operations, limited detection capability, and high cost, and achieve the effects of no cross-reaction, high accuracy and sensitivity, and high precision.

Active Publication Date: 2019-07-19
上海伯豪生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The kit is a high-sensitivity and high-specificity kit based on optimized amplification reaction stem-loop primers, MGB fluorescent probes and ddPCR system, which can overcome the problems of low throughput, time-con

Method used

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  • Kit utilizing stem loop primers to detect PIK3CA gene mutation sites
  • Kit utilizing stem loop primers to detect PIK3CA gene mutation sites
  • Kit utilizing stem loop primers to detect PIK3CA gene mutation sites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Use the plasmid template containing the H1047R mutation site of the human PIK3CA gene (corresponding to each site to be tested), and use the primers and probes in Table 1 to perform qPCR to optimize and select the best stem-loop primers and MGB probes.

[0072] Wherein, the wild-type plasmids and mutant-type plasmids involved in the following tables can be prepared according to conventional plasmid construction methods and PCR cloning methods.

[0073] 1) Plasmid treatment and extraction:

[0074] Plasmid extraction was carried out using TIANGEN (HighPure Plasmid Kit, DP116) plasmid extraction kit, and the specific operation steps are detailed in the product manual. The extracted DNA was dissolved in Tris-HCl (10 mmol / L, pH 8.0), and the quality of the sample was detected and the concentration was determined by an ultraviolet spectrophotometer. Then, dilute the sample to 1000 copies / uL. Take 1 µL for ddPCR reaction.

[0075] 2) Prepare a PCR reaction system according...

Embodiment 2

[0092] Using the present invention to detect mutant cell line DNA samples, culturing cells containing the H1047R mutation site of the human PIK3CA gene, extracting DNA, using the specific stem-loop primers and MGB probes of the present invention to detect it, and at the same time, the minimum detection limit of mutation detection Determination.

[0093] Proceed as follows:

[0094] 1) Sample processing and DNA extraction:

[0095] Use a commercial DNA extraction kit to extract sample DNA, and refer to the kit instructions for specific operations.

[0096] Samples were diluted to 10ng / µL.

[0097] 2) Carry out PCR amplification according to the following amplification system (total volume 20 μL)

[0098]2 × ddPCR supermix for Probes 10μl

[0099] Each primer 0.005~1.0μmol

[0100] Probe 0.001~1.0μmol

[0101] 10ng / μL DNA template 0.1~1.0μL

[0102] Make up to 20μL with water;

[0103] Wherein, the primers in the above PCR amplification are the primers shown in SEQ ID NO...

Embodiment 3

[0118] Using the stem-loop primers and common primers (Table 5) of the present invention to detect plasmid samples and mutant cell line DNA samples respectively, the sample acquisition and specific implementation steps are as in Example 1 and Example 2, and the obtained data are shown in Table 6.

[0119] Table 5 Common primer sequences

[0120]

[0121] Table 6 Data comparison between stem-loop primers and common primers

[0122]

[0123] The results show that the specificity and accuracy of the stem-loop primers of the present invention are better than those of common primers. As shown in Table 6, the positive detection rate of stem-loop primers used in the present invention can reach 100%, far exceeding the positive detection rate of common primers. The output rate has achieved unexpected technical effects. Stem-loop primers are designed so that bases in their stems are complementary to each other, and form a stem-loop structure by themselves without binding to the t...

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Abstract

The invention discloses a kit utilizing stem loop primers to detect PIK3CA gene mutation sites. The kit utilizing the stem loop primers to detect the PIK3CA gene mutation sites comprises a set of stemloop primers, and two MGC fluorescent probes; sequence of upstream primers of the set of the stem loop primers is shown as SEQ ID NO. 1 while sequence of downstream primers of the set of the stem loop primers is shown as SEQ ID NO. 8; and the two MGC fluorescent probes are shown as SEQ ID NO. 9-10. The kit utilizing the stem loop primers to detect the PIK3CA gene mutation sites is capable of overcoming defect of low throughput of existing first-generation sequencing technologies, long time consumption of existing second-generation sequencing technologies, limited detection capacity of fluorescent quantitative PCR and the like; moreover, the kit has the advantages of being high in sensitivity, good in specificity, rapid, high in throughput and so on.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for detecting mutations in PIK3CA gene (H1047R), in particular to a kit for detecting mutations in PIK3CA genes using stem-loop primers. This kit is used for the qualitative detection of the H1047R site mutation of the human PIK3CA gene in paraffin-embedded pathological tissue samples. Background technique [0002] The PIK3CA gene is expressed in normal human brain, lung, breast, gastrointestinal, cervix, ovary and other tissues. It has important physiological functions such as regulating cell proliferation, survival, death, and differentiation in the body. It mostly exists in an inactive state under physiological conditions. , are usually not easily detected. However, PIK3CA gene mutations can be detected in a variety of tumor tissues. The mutated PIK3CA abnormally activates the PI3K-AKT signaling pathway, resulting in abnormal cell cycle, decreased cell adhesion,...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/156C12Q2563/159
Inventor 徐晓晶赵莹贺华陆凌佳
Owner 上海伯豪生物技术有限公司
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