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Primers and probes and kit for detecting seven mutation types of human RET gene

A technology for detecting probes and kits, which is applied in the biological field and can solve problems such as poor accuracy, long detection time, and low detection efficiency

Active Publication Date: 2016-04-06
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In order to solve the problems of the existing RET 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 RET gene

Method used

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  • Primers and probes and kit for detecting seven mutation types of human RET gene
  • Primers and probes and kit for detecting seven mutation types of human RET gene
  • Primers and probes and kit for detecting seven mutation types of human RET gene

Examples

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Effect test

Embodiment 1

[0095] 1. Primers and probes of the detection kit for detecting 7 mutations of human RET gene:

[0096] According to the wild-type sequence of the RET gene (NCBIReferenceSequence: NM_020975.4) published in the NCBI database, the mutation site of exon 16 M918T (RM1), exon 11 C634R (RM2), C634G (RM3), C634S (RM4), The C634F (RM5), C634Y (RM6) and C634W (RM7) mutation sites were used as references to design specific M918T mutation primers and probes (see Table 2), C634R mutation primers and probes (see Table 3), and C634G mutation primers and probes (see Table 4), C634S mutation primers and probes (see Table 5), C634F mutation primers and probes (see Table 6), C634Y mutation primers and probes (see Table 7) and C634W mutation primers and probes needle (see Table 8). Using the mutant plasmid and wild-type plasmid constructed by genetic engineering as templates, a real-time fluorescent PCR mutation (Mutation) detection system was established to achieve high sensitivity and high sp...

Embodiment 2

[0184] The human RET gene mutation detection kit of the present invention is used to detect clinically collected tissue samples.

[0185] In this example, the blood samples of patients diagnosed as medullary thyroid carcinoma and multiple endocrine neoplasia type 2 were collected clinically and pathologically, and genomic DNA was extracted therefrom. Use the RET gene mutation detection kit to detect the RET gene mutation status in the sample to be tested. Specific steps are as follows:

[0186] (1) Sample genomic DNA extraction

[0187] The blood sample was extracted using Ningbo Youcheng Blood Genomic DNA Extraction Kit (KR008F), and the genomic DNA of the sample to be tested was extracted according to the instructions. The extracted DNA sample was tested for concentration and DNA quality with a UV spectrophotometer, and the DNA concentration requirements ≥10ng / μL, the quality of DNA OD260 / 280 is between 1.8 and 2.0. Genomic DNA that meets the requirements was diluted to 2...

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Abstract

The invention discloses primers and probes and a kit for detecting seven mutation types of a human RET gene. According to each group of the seven groups (RM1-RM7) of the primers and probes, the mutation upstream ARMS primer can be specially combined with a corresponding mutation sequence of the mutation upstream ARMS primer to amplify the mutation sequence, the mutation downstream primer can be combined with a conserved sequence of the RET gene, the detection probe can be combined with an amplified fragment, and the blocking probe can be specially combined with a wild type sequence corresponding to a mutation locus to inhibit wild type nonspecific amplification. Accordingly, by adopting a specific mutation primer and probe blocking technology, the seven mutation types of the RET gene can be accurately detected; through the established kit of a real-time fluorescence PCR amplification reaction system, rapid detection on the RET gene mutation is facilitated, operation is easy, and a result is easy to read; the detection method is high in sensitivity, and 50-copy mutation can be stably detected; the specificity is good, no nonspecific amplification is generated in 20-ng wild type genome DNA, and the detectability reaches up to 1%.

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 RET gene. Background technique [0002] Medullary thyroid carcinoma (medμLlarythyroidcarcinoma) is actually not a thyroid cancer. It is derived from the thyroid parafollicular cells (also known as C cells) that secrete calcitonin. It is a neuroendocrine cell and has nothing to do with thyroid follicular cells. It was first proposed by Hazand et al. in 1959 as an independent clinicopathological type. It only accounts for a small part of thyroid tumors (about 3-12%), and its pathogenesis, diagnosis and treatment have unique characteristics. According to whether it is hereditary, MTC can be divided into two categories: sporadic and hereditary: 1. Sporadic MTC: the most common clinically, accounting for about 75-80% of MTC, most of them are middle-aged and elderly, and women are slightly more common; 2. Hereditary MTC (MEN2)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2531/113C12Q2535/137C12Q2537/163C12Q2561/101C12Q2561/113C12Q2565/1015
Inventor 朱峰裴冰雪段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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