Specific primer combination for identifying RET gene mutation of medullary thyroid carcinoma as well as kit and application of specific primer combination
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A primer combination, thyroid technology, applied in the biological field, can solve the problems of long detection cycle, huge workload, high detection price, etc., and achieve the effect of saving resources, reducing operation steps, and improving efficiency
Active Publication Date: 2019-03-08
SHANGHAI PERSONAL BIOTECH
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The number of mutation sites that can be detected by PCR quantification is small each time, and the workload is huge when m
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Embodiment 1
[0070] Detection of RET gene mutation in tissue samples of medullary thyroid tumor using the primers of the present invention
[0071] 1 Materials and methods
[0072] 1.1 Sample source
[0073] Through cooperation, 25 tissue samples of medullary thyroid tumor in the Head and Neck Surgery Department of Cancer Hospital Affiliated to Fudan University were collected from January 1, 2017 to June 30, 2018, and tested.
[0074] 1.2 Specimen extraction
[0075] Thyroid medullary tumor tissue samples were frozen in liquid nitrogen, stored at -80°C, transported to the laboratory at 4°C, and stored at -80°C for later use. The DNA in the sample was extracted with Tiangen Blood / Cell / Tissue Extraction Kit (DP304-02), and stored at -20°C for future use.
[0076] 1.3 Synthesis of PCR amplification primers
[0077] Using bioinformatics knowledge and related bioinformatics software such as DNAstar, the gene sequence comparison and analysis of the RET gene nucleic acid sequences that can be...
Embodiment 2
[0156] Contain following reagent and material in the kit of the present invention:
[0157] 1. Primer solution (100 μmol / L): composed of primer pairs SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9 -10, SEQ ID NO.11-12, SEQ ID NO.13-14
[0158] 2. 2×qPCR SYBR Green Master Mix
[0159] The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the method of the present invention, some improvements and supplements can also be made, and these improvements and supplements should also be considered Be the protection scope of the present invention.
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Abstract
The invention discloses a specific primer combination for identifying RET gene mutation of medullary thyroid carcinoma. The specific primer combination is characterized in that the combination is usedfor amplifying a sequence shown in SEQ ID NO.1 in RET gene mutation of medullary thyroid carcinoma. The primer combination comprises a combination of a primer RET8, a primer RET10, a primer RET11, aprimer RET13, a primer RET14, a primer RET15 and a primer RET16. The invention further discloses a kit of the primer combination and an application of the primer combination. The primer combination ishigh in specificity, covers targeted area fragments accurately and detects various known mutant sites. By adopting a mixed solution of the specific primers and marks with fluorescent edges to carry out PCR amplification on batched samples and sanger sequencing analysis, a lot of samples can be analyzed at one time, and various known and unknown mutation can be detected, so that the operating steps are reduced, the resources are saved and the efficiency is improved. A kit provided by the invention can be widely applied to assisted diagnostic detection of clinical medullary thyroid carcinoma and has a wide application prospect.
Description
technical field [0001] The invention belongs to the field of biotechnology, and relates to a combination of specific primers for identifying RET gene mutations in medullary thyroid cancer, a kit and applications thereof. Background technique [0002] Medullary thyroid carcinoma (MTC) is one of the malignant tumors of the thyroid gland, accounting for about 2% of thyroid cancers. MTC includes hereditary MTC and sporadic MTC. Sporadic MTC usually occurs between the ages of forty and sixty, and the clinical manifestations are not stable. Prediction, age at diagnosis and disease stage were significantly associated with prognosis. Hereditary MTC is mainly MEN2, and MEN2 has two syndromes, MEN2A and MEN2B. MEN2A accounts for 95% of MEN2 cases and has four variants: classic MEN2A (emerging from the uniform presence of MTC and less commonly from pheochromocytoma (PHEO) or hyperparathyroidism (HPTH) or both), MEN2A with cutaneous Lichen amyloidosis (CLA), MEN2A and Hirschsprung dis...
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