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KRAS gene mutation detection reagent kit

A kit and probe technology, applied in the field of molecular biology, can solve the problem of no KRAS gene mutation detection, and achieve the effect of reducing the possibility of later contamination, rapid detection, and good stability

Inactive Publication Date: 2019-03-19
WUHAN CMLABS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This patent uses the combination of RNase H2 and PCR technology to develop a new type of detection for KRAS gene mutation. At present, there is no related technology that applies this method to the detection of KRAS gene mutation.

Method used

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  • KRAS gene mutation detection reagent kit
  • KRAS gene mutation detection reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] In this example, primers and probe sequences for the detection of KRAS gene mutations were designed, the primer sequences SEQ ID NO.1 to SEQ ID NO.8 were designed for the detection of KRAS gene mutations, and the probe sequence SEQ ID for KRAS gene mutations was designed. NO.9, the specific sequence is as follows:

[0037] 5'-GTGGTAGTTGGAGCTGaTGG-x, the sequence is shown in SEQ ID NO.1;

[0038] 5'-GTGGTAGTTGGAGCTGcTGG-x, the sequence is shown in SEQ ID NO.2;

[0039] 5'-GTGGTAGTTGGAGCTGtTGG-x, the sequence is shown in SEQ ID NO.3;

[0040] 5'-GTGGTAGTTGGAGCTaGTGG-x, the sequence is shown in SEQ ID NO.4;

[0041] 5'-GTGGTAGTTGGAGCTcGTG-x, the sequence is shown in SEQ ID NO.5;

[0042] 5'-GTGGTAGTTGGAGCTtGTGG-x, the sequence is shown in SEQ ID NO.6;

[0043] 5'-GTAGTTGGAGCTGGTGaCGTA-x, the sequence is shown in SEQ ID NO.7;

[0044] 5'-ATCAAAGAATGGTCCTGCAC-3', the sequence is shown in SEQ ID NO.8;

[0045] FAM-TAGCTGTATCGTCAAGGCACTC-BHQ1, the sequence is shown in SE...

Embodiment 2

[0048] In this example, a kit for detecting KRAS gene mutations was prepared. In order to ensure the reliability of the detection system, the patent of the present invention also introduced positive and negative quality control products.

[0049] The specific design of the kit is as follows:

[0050] (1) PCR reaction master mix: components include PCR buffer, 4 kinds of dNTPs, probe SEQ ID NO.9 and general human internal standard probe, primers from SEQ ID NO.1 to SEQ ID NO.8 and general Internal standard primers are mixed;

[0051] (2) Enzyme mixture: the components include RNase H2 and Taq DNA polymerase, the enzyme activity concentration ratio of RNase H2 and Taq DNA polymerase is 1:50;

[0052] (3) Negative quality control: the constructed KRAS gene mutation is a negative plasmid template;

[0053] (4) Positive quality control product: the constructed KRAS gene mutation is a positive plasmid template;

[0054] More preferably, in the enzyme mixture, the concentration ra...

Embodiment 3

[0057] In this example, a kit for detecting KRAS gene mutations was prepared. In order to ensure the reliability of the detection system, the patent of the present invention also introduced positive and negative quality control products.

[0058] The specific design of the kit is as follows:

[0059] (1) PCR reaction master mix: components include PCR buffer, 4 kinds of dNTPs, probe SEQ ID NO.9 and general human internal standard probe, primers from SEQ ID NO.1 to SEQ ID NO.8 and general Internal standard primers are mixed;

[0060] (2) Enzyme mixture: the components include RNase H2 and Taq DNA polymerase, and the enzyme activity concentration ratio of RNase H2 and Taq DNA polymerase is 1:300;

[0061] (3) Negative quality control: the constructed KRAS gene mutation is a negative plasmid template;

[0062] (4) Positive quality control product: the constructed KRAS gene mutation is a positive plasmid template;

[0063] The above four reagents are packaged into kits of diffe...

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Abstract

The invention provides a novel KRAS gene mutation detection reagent kit. The novel KRAS gene mutation detection reagent kit has the advantages that diphase enzyme systems RNase H2 and DNA (deoxyribonucleic acid) polymerase technologies are applied, diphase system amplification processes are adopted, accordingly, the KRAS gene mutation detection reagent kit is changed to a certain extent on the basis of common primers, in other words, spacer arms x are connected with 3' ends of primer sequences SEQ ID NO.1-SEQ ID NO.7, and primers can be specifically bound with template sequences and can be specifically amplified after RNase H2 is shorn; primer probes for KRAS gene mutation sites are designed, fluorescence PCR (polymerase chain reaction) closed detection is adopted, and accordingly the possibility of pollution in later periods can be reduced; the KRAS gene mutation detection reagent kit is high in sensitivity and detection speed and good in stability, and the possibility of pollution inthe later periods can be reduced by means of closed detection.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a kit for detecting KRAS gene mutation. Background technique [0002] KRAS is one of the main members of the Ras gene family and is located downstream of the EGFR signaling pathway. KRAS encodes the P21 protein, plays a role in the MAPK signaling pathway, is an oncogene that can bind to GDP / GTP and promote GTPase activity. When KRAS is mutated, it cannot be hydrolyzed and inactivated by hydrolytic enzymes, and is in a continuously activated state, which can cause the upregulation of RAF / MAPK and transmit a variety of survival pathway signals, thereby causing excessive cell growth and proliferation, and inhibiting the effects of EGFR-TKIs. KRAS gene mutation exists in 17% to 25% of human tumors, and its mutation can lead to a variety of malignant tumors, including pancreatic (95%), thyroid (55%), colorectal cancer (35%), lung cancer (35%) and so on. The most common way...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2521/101C12Q2521/327C12Q2563/107C12Q2531/113
Inventor 徐志勇秦伟
Owner WUHAN CMLABS CO LTD
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