Probe, primer and kit for detecting seven kinds of mutations of human NRAS genes

A technology for detecting probes and kits, which is applied in DNA/RNA fragments, recombinant DNA technology, and microbial measurement/inspection, etc., and can solve problems such as low detection efficiency, easy contamination of samples, and poor accuracy

Active Publication Date: 2016-07-20
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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  • Probe, primer and kit for detecting seven kinds of mutations of human NRAS genes
  • Probe, primer and kit for detecting seven kinds of mutations of human NRAS genes
  • Probe, primer and kit for detecting seven kinds of mutations of human NRAS genes

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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 wild-type sequence of the NRAS gene (NCBIReferenceSequence: NM_002524.4) published in the NCBI database, the mutation sites of exon 2 G12C (NM1) and G12D (NM2), exon 3 Q61K (NM3), Q61L (NM4), The Q61R (NM5), Q61H (NM6) and Q61H (NM7) mutation sites were used as a reference to design specific NM1 mutation primers and probes (see Table 2), specific NM2 mutation primers and probes (see Table 3), specific 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 ( See Table 7) and specific NM7 mutation primers and probes (see Table 8). Using the mutant and wild-type plasmids constructed by genetic engineering as templates, a real-time fluorescent PCR mutation detection system was established to achieve high sens...

Embodiment 2

[0178] The human NRAS gene mutation detection kit of the present invention is used to detect clinically collected tissue samples.

[0179] In this example, tissue samples from patients diagnosed as colorectal cancer or melanoma were collected and genomic DNA was extracted from them. The NRAS gene mutation status in the samples to be tested was detected with the 7 kinds of NRAS gene mutation detection kit. Specific steps are as follows:

[0180] (1) Sample genomic DNA extraction

[0181] Extract the paraffin-embedded tissue samples using QIAampDNAFFPETissueKit (CatNo.56404), extract the genomic DNA of the sample to be tested according to the instructions, and use the UV spectrophotometer to detect the concentration and DNA quality of the extracted DNA samples. The concentration of DNA is required to be ≥ 10ng / μL, the quality OD260 / 280 of DNA is between 1.8 and 2.0. Genomic DNA that meets the requirements was diluted to 2-10 ng / μL with TE (10 mmol / L, pH 8.0) as a PCR templat...

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Abstract

The invention discloses a probe, a primer and a kit for detecting seven kinds of mutations of human NRAS genes, wherein in seven groups of primers and probes from NM1 to NM7, the mutation primer in each group can be combined with a conserved sequence of the NRAS genes; the mutation ARMS primers can realize the specific binding with the corresponding mutation sequences; the mutation sequences are amplified; the detection probe can be combined with an amplification segment; the specific binding of the probe with the wild sequence corresponding to the mutation site is blocked; and the wild nonspecific amplification is inhibited. The specific mutation primer and probe blocking technology is used; the seven kinds of mutation types of the NRAS genes can be accurately detected; the built kit for a real-time fluorescent PCR (Polymerase Chain Reaction) amplification reaction system can be used for conveniently and fast detecting the mutations of the NRAS genes; the operation is simple; the result can be easily read; the sensitivity of a detection method is high; 50 copy mutations can be stably detected; the specificity is good; the 20ng wild genome DNA (Deoxyribose Nucleic Acid) does not have specific amplification; and the detection capability is as high as 1 percent.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a probe, primer and kit for detecting seven mutations of human NRAS gene. Background technique [0002] The ras gene family has three genes related to human tumors—HRAS, KRAS and NRAS, which are located on chromosome 11, 12 and 1, respectively. Functionally, Ras protein acts as a molecular switch, and under normal expression, it can regulate cell growth. Abnormal conditions such as point mutation, overexpression or gene translocation can lead to abnormal proliferation of cells, and eventually lead to tumor formation, and 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, and with the deepening of research in recent years, it has gradually become another molecular indicator that provides important basis for clinical disease assessment and treatment after KRAS. [0003] NRAS gene mutations a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
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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