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Probes, primers and kits for detecting seven mutations in human nras gene

A technology for detecting probes and kits, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., which can solve the problems of easy contamination of samples, low detection efficiency, and poor accuracy

Active Publication Date: 2020-08-25
武汉海吉力生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of the existing NRAS gene mutation detection methods, such as low detection efficiency, easy sample contamination, long detection time, poor accuracy, and difficulty in discrimination, the present invention provides primers and probes for detecting seven kinds of mutations in human NRAS genes

Method used

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  • Probes, primers and kits for detecting seven mutations in human nras gene
  • Probes, primers and kits for detecting seven mutations in human nras gene
  • Probes, primers and kits for detecting seven mutations in human nras gene

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Embodiment 1

[0089] 1. Primers and probes of the detection kit for detecting 7 mutations of human NRAS gene:

[0090] According to the NRAS gene (NCBI Reference Sequence: NM_002524.4) wild-type sequence published by the NCBI database, the mutation sites of exon 2 G12C (NM1) and G12D (NM2), exon 3 Q61K (NM3), Q61L (NM4) ), Q61R (NM5), Q61H (NM6) and Q61H (NM7) mutation sites as reference, design specific NM1 mutation primers and probes (see Table 2), specific NM2 mutation primers and probes (see Table 3) , Specific NM3 mutation primers and probes (see Table 4), specific NM4 mutation primers and probes (see Table 5), specific NM5 mutation primers and probes (see Table 6), specific NM6 mutation primers and probes Needle (see Table 7) and specific NM7 mutant primers and probes (see Table 8). Using mutations and wild-type plasmids constructed by genetic engineering as templates, a real-time fluorescent PCR mutation detection system was established to achieve high sensitivity and high specificity ...

Embodiment 2

[0177] The seven human NRAS gene mutation detection kits of the present invention are used to detect clinically collected tissue samples.

[0178] In this embodiment, tissue samples of patients diagnosed as colorectal cancer or melanoma were collected clinically and pathologically, and genomic DNA was extracted therefrom. The NRAS gene mutation detection kit is used to detect the NRAS gene mutation status in the sample to be tested. Specific steps are as follows:

[0179] (1) Extraction of sample genomic DNA

[0180] The extraction of paraffin-embedded tissue samples uses QIAamp DNAFFPE Tissue Kit (CatNo.56404) to extract the genomic DNA of the sample to be tested according to the instructions. The extracted DNA sample is used to detect the concentration and DNA quality with an ultraviolet spectrophotometer, and the concentration of DNA is required ≥10ng / μL, DNA quality OD260 / 280 is between 1.8~2.0. The genomic DNA that meets the requirements is diluted with TE (10mmol / L, PH8.0) t...

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Abstract

The invention discloses probes, primers and kits for detecting seven kinds of mutations in human NRAS genes, wherein there are seven sets of primers and probes from NM1 to NM7, and the mutation primers in each group can combine with the conserved sequence of the NRAS gene, and the mutation ARMS primers It can specifically bind to the corresponding mutant sequence and amplify the mutant sequence; the detection probe can bind to the amplified fragment, and the blocking probe can specifically bind to the wild-type sequence corresponding to the mutation site, inhibiting the non-specific amplification of the wild-type . The present invention adopts specific mutation primer and probe blocking technology, can accurately detect 7 kinds of mutation types of NRAS gene; the kit of the real-time fluorescent PCR amplification reaction system established is convenient for rapid detection of NRAS gene mutation, and the operation is simple and the result is easy Read; the detection method has high sensitivity, and 50 copies of mutations can be detected stably; the specificity is good, 20ng of wild-type genomic DNA has no non-specific amplification, and the detection ability is as high as 1%.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a probe, primer and kit for detecting 7 mutations of human NRAS gene. Background technique [0002] There are three genes related to human tumors in the ras gene family-HRAS, KRAS and NRAS, which are located on chromosomes 11, 12 and 1, respectively. Functionally, the Ras protein functions as a molecular switch, and under normal expression, it can regulate cell growth. Abnormal conditions such as point mutations, overexpression, or gene translocation can lead to abnormal cell proliferation, and ultimately lead to tumor formation. More than 30% of human tumors have Ras mutations. As a member of the ras family, NRAS has many similarities with KRAS in structure and function. With the deepening of research in recent years, it has gradually become another molecular indicator that provides important basis for clinical evaluation and treatment after KRAS. [0003] Among colorectal cancer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2531/113C12Q2535/137C12Q2537/163C12Q2561/101C12Q2561/113
Inventor 朱峰杨晶晶段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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