A thyroid multi-gene combined detection kit

Abstract

The invention belongs to the technical field of molecular biology, and in particular relates to a thyroid multi-gene combined detection kit. The kit of the invention comprises: a special adapter for DNA library construction; a specific composite primer for detecting fusion genes and point mutations; and a library amplification composite primer. The kit of the present invention jointly extracts the DNA and RNA of the samples submitted for inspection, reverse transcribes the RNA into cDNA, constructs a high-throughput sequencing library together with the DNA, and performs high-throughput sequencing, which can detect CCDC6- RET / NCOA4-RET fusion and PAX8 / PPARγ fusion, BRAF(V600E), HRAS(61), NRAS(61), KRAS(12,13), KRAS(61), TERTp and other point mutations, the operation is simplified and time-consuming Short, the sensitivity can reach 0.1%.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a thyroid polygene combined detection kit. Background technique [0002] Thyroid nodule is a common thyroid disease in the population, and it is a lesion caused by abnormal growth of thyroid cells in the local area. In most cases, thyroid nodules are benign and can be treated conservatively, but 5%-10% of thyroid nodules are malignant, that is, thyroid cancer, which requires early surgical treatment to obtain a good prognosis. At present, obtaining thyroid cells by fine needle aspiration (FNA) for cytological examination is the gold standard for assessing whether thyroid nodules are malignant tumors. However, 20%-30% of thyroid nodules cannot be definitively diagnosed in this way. [0003] With the development of molecular biology techniques, molecular diagnosis has gradually attracted people's attention in the differential diagnosis of benign and malignant thyroid no...

AI Technical Summary

Problems solved by technology

[0006] Chinese patent CN111349694A discloses a detection method and kit for thyroid cancer-related gene fusion mutations. Although it can detect the three thyroid cancer-related gene fusion mutations, RET / PTC1 fusion, RET / PTC3 fusion, and PAX8-PPARG rearrangement, Unable to detect point mutations such as BRAF, KRAS, NRAS, HRAS, TERTp

[0007] Another example is Chinese patent CN110241215B which discloses a primer and a kit for detecting benign and malignant gene mutations related to thyroid nodules, which can simultaneously detect 15 site mutations of 6 genes and 3 fusion gene mutations, including the BRAF gene , KRAS gene, HRAS gene, NRAS gene, TERT gene, EIF1AX gene and RET / PTC1 fusion, RET / PTC3 fusion, PAX8 / PPARγ fusion, but this technical solution has the following disadvantages: the detection range of the fusion gene partner gene is small, only It can detect two fusion types, PTC1 / RET and PTC3 / RET; the constructed DNA sequencing library has low complexity and is prone to false positives; the operation is complex and requires independent construction of RNA and DNA libraries

Disadvantages are: high cost of liquid phase hybridization, and complicated operation process

[0009] However, the traditional fluorescent PCR method has defects such as only one site can be detected at a time, and a large amount of samples is required; the detection cost is high; the number of experimental operations is large, and the detection procedure takes a long time.

[0010] In summary, the above-mentioned methods can only detect known gene fusions or point mutations, and are limited by the detection throughput, and cannot realize one-time full detection of fusion genes and point mutations that cause thyroid cancer.

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