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A primer and kit for detecting gene variation related to prognosis of thyroid cancer

A technology for thyroid cancer and gene mutation, applied in the field of molecular biology, can solve the problems of undetectable low-level mutations, large impact on results, and many operating procedures, so as to improve treatment effects and quality of life, reduce detection costs, and improve The effect of detection efficiency

Active Publication Date: 2022-04-05
润安医学科技(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Sanger sequencing method is the "gold standard" of gene detection. It can directly detect the mutation signals of different bases, and the result analysis is convenient. However, the detection sensitivity of this method is low, and low-level mutations cannot be detected, and the operation steps are many and time-consuming.
For the detection of multiple genes and multiple loci, the ARMS-PCR method often requires multiple tube reactions, which is time-consuming and labor-intensive.
The dPCR method is suitable for the detection of low-copy samples and low-frequency mutation samples, but there are many operating procedures, which have a great impact on the results, and the cost of consumables is high and the throughput is small.

Method used

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  • A primer and kit for detecting gene variation related to prognosis of thyroid cancer
  • A primer and kit for detecting gene variation related to prognosis of thyroid cancer
  • A primer and kit for detecting gene variation related to prognosis of thyroid cancer

Examples

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Effect test

Embodiment 1

[0037]Example 1 A combination of primers used to detect gene mutations related to the prognosis of thyroid cancer.

[0038] The thyroid cancer prognosis-related genes and loci involved in this example were selected from the COSMIC (Catalogue of Somatic Mutations in Cancer) database, and mutation hotspot primers were designed based on relevant gene sequences, and the design scope included mutation hotspots in thyroid cancer prognosis-related genes.

[0039] As shown in Table 2, a total of 12 specific primers were designed for hotspot mutations in genes related to thyroid cancer prognosis in this example. 280bp, has the advantages of wide coverage, multiple detection sites, balanced GC content, stable product structure, and less dimer structure. The specific primers of this example exhibit good specificity, stability and uniformity during multiplex PCR amplification, and can ensure the amplification efficiency while ensuring the specificity of PCR amplification.

Embodiment 2

[0040] Example 2 A kit for detecting gene mutations related to prognosis of thyroid cancer.

[0041] The kits used in the detection of thyroid cancer prognosis-related gene mutations described in this example mainly include:

[0042] (1) PCR-specific primers: used to amplify multiple target regions on the target gene of the sample to be tested. The amplification range covers the hotspot mutation region of the target gene. The sequences are shown in Table 2. SEQ ID NO.1 to SEQ ID NO. 12 shown. Preferably, multiple amplification primer pairs are mixed together to form a primer pool;

[0043] (2) Universal primers: used to re-amplify the amplification products of the target region amplified by specific primers during the library construction process, to mark the sequencing libraries of different samples to be tested, and then to distinguish different samples. The sequence is shown in the table Shown in SEQ ID NO.13 and SEQ ID NO.14 in 2;

[0044] (3) NGS library construction r...

Embodiment 3

[0047] Example 3 A detection method of a kit for detection of gene mutations related to prognosis of thyroid cancer.

[0048] The detection method of the present embodiment comprises the following steps ( figure 2 ):

[0049] (1) Sample DNA extraction: Refer to the kit instructions, use the AllPrep DNA Mini Kit kit to extract DNA, use NanoDrop to measure the concentration and purity, and use nuclease-free water to dilute the nucleic acid sample to 10 -50ng / μL was used as the initial nucleic acid concentration for amplification and library construction.

[0050] (2) DNA library construction:

[0051] (2a) DNA target region amplification:

[0052] Carry out the first round of PCR amplification according to the following reaction system and amplification conditions:

[0053] Reagent volume High Fidelity DNA Enzyme Mix 12.5μL DNA-specific primer mix pool 4μL DNA template 2μL nuclease free water Make up to 25 μL

[0054] DNA multipl...

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Abstract

The invention provides a primer and a kit for detecting the variation of genes related to the prognosis of thyroid cancer. Based on high-throughput sequencing technology, this method can quickly, comprehensively and accurately detect BRAF gene T1799A, A1801G mutation, TERT gene C228T, C250T mutation, RET gene T2753C mutation, EIF1AX gene G25C / A / T, G370T, G371T mutation and TP53 Gene G818A mutation. The above-mentioned mutations of these genes are closely related to the prognosis of thyroid cancer. Therefore, using the present invention to detect patient samples can assist doctors in evaluating and judging the prognosis of thyroid cancer patients, and provide valuable decision-making basis and guidance for doctors to choose appropriate surgical methods and formulate postoperative follow-up plans in a targeted manner. Reference information is helpful to improve the treatment effect and quality of life of patients with thyroid cancer, and has very important clinical value.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer and a kit for detecting gene variation related to the prognosis of thyroid cancer. Background technique [0002] Thyroid cancer is the most common endocrine malignancy. According to the data of GLOBOCAN2018, the incidence of thyroid cancer in the world accounts for 3.1% of all malignant tumors and 5.1% of female malignant tumors. Since the 1970s, a rapid increase in the incidence of thyroid cancer and a relatively stable mortality rate have been reported in most parts of the world, including the United States, Canada, Europe, Australia, Asia, and parts of South America. According to statistics from the China Cancer Center, the incidence of thyroid cancer among women in my country ranks fourth among all malignant tumors in women. [0003] Thyroid cancer can be divided into papillary carcinoma, follicular carcinoma, medullary carcinoma, and undifferentiated car...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156C12Q2600/118
Inventor 张宇清宣涛王宇
Owner 润安医学科技(苏州)有限公司
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