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

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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 detecti

Method used

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

Examples

Experimental program
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Example Embodiment

[0073] Example 1

[0074] The preparation of the KRAS gene mutation detection kit of the present invention includes the following steps:

[0075] 1. Synthesis of primers and probes: 7 pairs of specific primers were designed and synthesized 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; a pair of quality control primers was designed and synthesized, and the sequence of the quality control primer pair is SEQ ID NO :15, SEQ ID NO:16; Designed and synthesized a pair of specific probes and modified them. 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. The sequence of the locked nucleic...

Example Embodiment

[0085] Example 2

[0086] The preparation of the KRAS gene mutation detection kit of the present invention includes the following steps:

[0087] 1. Synthesis of primers and probes: The synthesis method is the same as in Example 1. After synthesis, the above-mentioned primers are prepared as 100 μM mother liquor for storage, and the above two probes are prepared as 100 μM mother liquor for storage.

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

[0089] 3. Prepare a positive control solution and a blank control solution. The positive control solution contains 7 kinds of plasmid DNAs, and these plasmid DNAs respectively contain 7 different mutations of ...

Example Embodiment

[0092] Example 3

[0093] The kit of the present invention is used to detect KRAS gene mutations. In this example, colon cancer tissues of 47 patients diagnosed as colon cancer were collected by clinical pathology, and paraffin embedding was performed to obtain tissue specimens, and genomic DNA was extracted, which was obtained in Example 1. The kit detects 7 common mutations in the KRAS gene and uses traditional pyrosequencing methods for verification. The specific steps are:

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

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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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