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KRAS gene mutation detection kit and detection method

A detection kit and mutation detection technology, applied in the fields of biotechnology and medicine, can solve the problems of long detection period, low sensitivity, and inaccurate detection results, and achieve the effects of improving specificity, high sensitivity, and simple and fast operation.

Inactive Publication Date: 2013-01-23
WUHAN YZY BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the embodiments of the present invention is to provide a human KRAS gene mutation detection kit and detection method, aiming to solve the problems of long detection cycle, low sensitivity and inaccurate detection results in the prior art

Method used

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  • KRAS gene mutation detection kit and detection method
  • KRAS gene mutation detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Prepare the KRAS gene mutation detection kit of the present invention, comprising the following steps:

[0075]1. Primer and probe synthesis: Design and synthesize 7 pairs of specific primers for the seven mutations of human KRAS gene, the sequences are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, the base mutations corresponding to the 7 pairs of specific primers are shown in Table 1; design and synthesize 1 pair of quality control primers, the sequence of the quality control primer pair is SEQ ID NO :15, SEQ ID NO:16; design and synthesize a pair of specific probes, and modify it, the modified specific probes are:

[0076] FAM-5'-TAGGCAAGAGTGCCTTGA-3'-NFQ-MGB SEQ ID NO: 17;

[0077] Design and synthesize a locked nucleic acid probe whose sequence is:

[0078] 5'-T+AG+T+TGG+AGC+TGGTGGC-3'-PO4 SEQ ID NO:18.

[0079] The above...

Embodiment 2

[0086] Prepare the KRAS gene mutation detection kit of the present invention, comprising the following steps:

[0087] 1. Synthesis of primers and probes: The synthesis method was the same as in Example 1. After synthesis, the above-mentioned pairs of primers were respectively prepared into 100 μM mother solutions for storage, and the above two probes were respectively prepared into 100 μM mother solutions for storage.

[0088] 2. Prepare the internal standard system: the preparation method is the same as in Example 1. After synthesis, the internal standard primers are prepared into 100 μM mother solution for storage, the internal standard probe is prepared into 100 μM mother solution for storage, and the internal standard template is configured as 5×10 4 copies / ml of the stock solution for storage. .

[0089] 3. prepare positive control solution and blank control solution, contain 7 kinds of plasmid DNAs in this positive control solution, contain respectively 7 kinds of diff...

Embodiment 3

[0093] Use the kit of the present invention to detect the KRAS gene mutation. In this embodiment, the colon cancer tissues of 47 patients diagnosed as colon cancer were collected, and paraffin embedding was carried out to obtain tissue samples. Genomic DNA was extracted, obtained in Example 1. The kit detects 7 common mutations of the KRAS gene, and at the same time uses the traditional pyrosequencing method for verification. The specific steps are as follows:

[0094] (1) Sample processing: DNA extraction kit (QIAamp DNA FFPE Tissue Kit, Cat No.56404) was used to extract genomic DNA from colon cancer tissue specimens. The extraction method was carried out according to the instructions. The specific steps were to place the clinically collected paraffin-embedded tissue (≤25mg) in a 1.5ml centrifuge tube, add 1200μl xylene, vortex vigorously for 10s, and centrifuge at 12000rpm for 5min at room temperature. Discard the supernatant, taking care not to pour off the pellet. Add 120...

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Abstract

The invention belongs to the field of biotechnology and medical science, and provides a KRAS gene mutation detection kit. The kit comprises a polymerase chain reaction (PCR) buffer solution, 7 pairs of specificity primers, one pair of quality control primers, a specificity probe, a locked nucleic acid probe, an interior label system, an enzyme solution and a reference dye. The invention also provides a human KRAS gene seven-mutation detection method. The detection kit has the advantages that the sensitivity is high, the specificity is strong, the pollution is reduced, the result is genuine and believable, and the operation is simple and rapid.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, in particular to a human KRAS gene mutation detection kit and detection method. Background technique [0002] Ras is a proto-oncogene involved in the occurrence and development of human tumors, which was originally cloned from rat sarcoma virus. Since Weinberg et al. discovered in 1982 that there is an activated H-Ras gene in human bladder cancer cells, the Ras gene has attracted great attention. The protein encoded by the Ras gene is P21 protein, with a molecular weight of 21KD and composed of 188-189 amino acids, also known as P21 highly related protein. [0003] Among the Ras gene family, KRAS has the greatest impact on human cancer. KRAS protein is a key downstream regulator in the EGFR signaling pathway. KRAS mutants encode abnormal proteins that stimulate the growth and spread of malignant tumor cells and are not affected by EGFR signaling. In the process of studying the relati...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 蔡从利张喆周鹏飞
Owner WUHAN YZY BIOPHARMA CO LTD
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