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Kit for detecting multiple mutation sites of KRAS gene

A technology for detecting kits and mutation sites, which is applied in the fields of molecular biotechnology and genetic testing, can solve the problems of wasting treatment costs, KRAS mutant patients cannot benefit, and delay treatment, so as to reduce complexity and reduce primers and probes Interaction and the effect of reducing the cost of detection reagents

Active Publication Date: 2018-12-14
广州市宝创生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As early as June 2008, the clinical research results were released at the annual meeting of the American Society of Clinical Oncology (ASCO), that is, patients with KRAS mutations do not benefit from anti-EGFR therapy, but instead delay the disease and waste treatment costs; while patients with wild-type KRAS are likely to benefit from this type of drug treatment

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  • Kit for detecting multiple mutation sites of KRAS gene
  • Kit for detecting multiple mutation sites of KRAS gene
  • Kit for detecting multiple mutation sites of KRAS gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The detection principle of embodiment 1 Kras gene multiple mutation detection kit (see figure 1 )

[0032] A pair of PCR primers are designed to amplify the target region of the KRAS gene mutation site to amplify the target region to be detected. Targeted design of a shared auxiliary probe, design of detection probes for each hot spot mutation, and labeling of fluorescent groups and quenching groups. At the same time, in order to further increase the specificity, the specific probe also introduces LNA bases to increase the binding specificity of the probe and increase the difference with the wild probe. In the reaction, the auxiliary probe and the detection probe can anneal and hybridize with the amplified target template to form a single-base overlapping intrusion structure, which can be specifically recognized by the Afu enzyme, thereby cutting the first base complementary to the template of the detection probe base chemical bonds, resulting in signal molecules. Si...

Embodiment 2

[0049] The sensitivity of embodiment 2 Kras gene multiple mutation detection kit

[0050] In this example, KRAS gene mutation DNA templates with different concentrations and gene mutation percentages were detected using the Kras gene multiple mutation detection kit, which was used to verify the sensitivity of the kit of the present invention. The detected hot spot mutation c.34G> A is used as an example for sensitivity verification.

[0051] Reaction conditions:

[0052] 40μL amplification system includes: 4μL 10×buffer, 200-500μmol / L dNTP, 200-500nmol / LPCR upstream and downstream primers and probes (SEQ ID NO.1 to SEQ ID NO.11), 3U Taq DNA polymerase, 60U Afu enzyme and template DNA. Taking the detected hotspot mutation c.34G>A as an example, the added template is the kit reference product whose concentration is determined by a UV spectrophotometer, and the mutation percentage is determined by mixing the corresponding mutant plasmid and the reference copy number ratio of hu...

Embodiment 3

[0054] Example 3 Validation Results of Different Combination Probe Systems of Kras Gene Multiple Mutation Detection Kit

[0055] This example is to verify that the Kras gene multiple mutation detection kit can be flexibly combined to detect specific Kras gene mutation sites. The combination of the hot spot mutation sites shown in Table 2 is used as an example to verify the results under the conditions of the quadruple system. Single detection to determine the detection accuracy of the kit of the present invention.

[0056] In order to better verify the flexibility and accuracy of the detection kit of the present invention, taking the hot spot mutation site shown in Table 2 as an example, the quadruple mutation probe system detects the corresponding four target templates respectively, and simultaneously prepares each hot spot mutation The detection system detects the corresponding single target template.

[0057] Reaction conditions:

[0058] 40μL amplification system include...

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Abstract

The invention discloses a kit for detecting multiple mutation sites of the KRAS gene. The kit comprises primers and probes directed at a plurality of hot spot mutations of the KRAS gene. The probes are sequences as shown in SEQ ID No. 3 and at least one selected from the group consisting of SEQ ID No. 4-11, or are reverse complementary sequences of the above sequences. The primers are sequences asshown in SEQ ID No. 1 and SEQ ID No. 2. According to the invention, the primers and the auxiliary probes are designed to be common sequences, so the complexity of such a detection system can be reduced, the defects of optimization of a reaction system in a later phase and mutual influence of the primers and the probes can be reduced, the practicality of a detection method is improved, and cost for a detection reagent is lowered.

Description

technical field [0001] The invention belongs to the field of molecular biotechnology and gene detection, in particular to a KRAS gene multiple mutation site detection kit. Background technique [0002] KRAS gene is a recognized oncogene, which is involved in EGFR signal transduction process. As early as June 2008, the results of clinical research were released at the annual meeting of the American Society of Clinical Oncology (ASCO), that is, patients with KRAS mutations do not benefit from anti-EGFR therapy, but instead delay the disease and waste treatment costs; while patients with wild-type KRAS are likely to benefit from such drug therapy. At the same time, the "NCCN Clinical Practice Guidelines for Colorectal Cancer" clearly pointed out two points: first, all patients with metastatic colorectal cancer should be tested for KRAS gene status, and second, only KRAS wild-type patients are recommended to receive EGFR inhibitors (such as cetuximab Anti-and panitumumab) trea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156C12Q2600/16
Inventor 王建平潘腾飞
Owner 广州市宝创生物技术有限公司