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Primers, kit and pcr method for detecting m918t site mutation of ret gene

A site mutation and kit technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc. High price and other problems, to achieve the effect of avoiding site mismatch, fast detection speed, and high detection sensitivity

Active Publication Date: 2018-04-27
沈阳优吉诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] In view of this, the present invention provides a primer for detecting the M918T site mutation of the RET gene, a kit and a PCR method thereof, to at least solve the complex interpretation of the results, the high price of the detection instrument, the difficulty in operation, and the existence of certain false positives in the previous kits. One or more problems such as negative and false positive, high testing cost, low clinical popularity, inability to test clinical specimens on a large scale at the same time

Method used

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  • Primers, kit and pcr method for detecting m918t site mutation of ret gene
  • Primers, kit and pcr method for detecting m918t site mutation of ret gene
  • Primers, kit and pcr method for detecting m918t site mutation of ret gene

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Effect test

Embodiment 1

[0116] Example 1: Preparation of wild-type and mutant-type positive plasmids for the mutation of the RET gene M918T site

[0117] Multiple endocrine neoplasia type 2 (MEN2) is caused by mutations in the proto-oncogene RET, which is located on chromosome 10q11. Certain mutations in the RET gene activate RET kinase activity, leading to tumorigenesis or transformation. Clinical evidence shows that the occurrence of more than 90% of multiple endocrine neoplasia type 2 is closely related to the M918T site mutation of the RET gene. Through RET gene mutation screening, carriers of the diseased genotype can be detected early, and effective intervention can be carried out, thereby changing the clinical course of the disease and improving the prognosis.

[0118] First, we called out the gene sequence before and after the M918T mutation site of the RET gene from the gene bank, and marked the polymorphic site with a double underline, at the appropriate position upstream and downstream of...

Embodiment 2

[0135] Example 2: Design and specificity screening of allele-specific primers (ASP)

[0136] For RET-M918T, wild-type and a series of mutant-specific primers were designed as follows:

[0137] RET-M918T-WT-R: caaaaagggattcaattggca (SEQ No. 8)

[0138] RET-M918T-mut-R: caaaaagggattcaattggcg (SEQ No. 9)

[0139] RET-M918T-mut-R1: aaaaagggattcaattggcg (SEQ No. 10)

[0140] RET-M918T-mut-R2: aaaagggattcaattggcg (SEQ No. 11)

[0141] RET-M918T-mut-R3: caaaaagggattcaattgcgg (SEQ No. 12)

[0142] RET-M918T-mut-R4: aaaaagggattcaattgcgg (SEQ No. 13)

[0143] RET-M918T-mut-R5: aaaagggattcaattgcgg (SEQ No. 14)

[0144] RET-M918T-mut-R6: aaagggattcaattgcgg (SEQ No. 15)

[0145] Simultaneously design and synthesize Taqman-specific probes:

[0146] SEQ No. 16: FAM-ccctccttcctagagagttagag-BHQ1.

[0147] Relevant primers and probes were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

[0148] Then use the above 8 primers to pair with the common downstream primer RET-M918T-F...

Embodiment 3

[0150] Embodiment 3: ASP sensitivity screening

[0151] Then use the No. 8 mutant primer to pair with the common downstream primer SEQ No.17 of the RET gene M918T mutation site, and use the mutant recombinant plasmid according to 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10, 0 for serial dilution, plus Taqman-specific probes, sensitivity verification was performed on a fluorescent quantitative PCR instrument. The No. 8 mutation-specific primer can detect 100 copies of the mutant, so this primer is the best primer for detecting the RET gene M918T mutation site screened according to our method, as shown in Table 3.

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Abstract

The invention discloses a primer and a kit for detecting mutation of a locus M918T of a RET gene and a PCR (Polymerase Chain Reaction) method of the primer and the kit. The primer comprises a wild type specific forward primer, a mutant type specific forward primer and a reverse primer, wherein the reverse primer is shared by the wild type specific forward primer and the mutant type specific forward primer; the wild type specific forward primer has a sequence shown in SEQ No.18; the mutant type forward primer has a sequence shown in SEQ No.15; the shared reverse primer has a sequence shown in SEQ No.17. The kit has the advantages of simplicity, rapidness, accuracy, low cost and the like when used for detection; a powerful tool is provided for the scientific research and clinical analysis of the locus M918T of the RET gene.

Description

technical field [0001] The invention relates to the field of molecular biological gene detection, and in particular provides a primer, a kit and a PCR method for detecting the mutation of the M918T site of the RET gene, which are used for the rapid detection of the mutation of the M918T site of the RET gene. Background technique [0002] Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant genetic disease characterized by hyperplasia or tumors of neuroendocrine cells in the thyroid, adrenal medulla, and parathyroid glands. Divided into three subtypes, MEN2A, MEN2B, and familial medullary thyroid carcinoma. MEN2A mainly involves thyroid C cells, adrenal medulla, and parathyroid glands, manifesting as medullary thyroid carcinoma, pheochromocytoma, and hyperparathyroidism, respectively. ; MEN2B is mainly manifested as medullary thyroid carcinoma, pheochromocytoma and lip tongue and gastrointestinal neuroma and Marfan syndrome body type. [0003] MEN2 is caused b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2531/113C12Q2535/125C12Q2561/101
Inventor 高劲松张英杰李星颐隋欣魏潇魏奇
Owner 沈阳优吉诺生物科技有限公司
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