Biomarker for thyroid nodule benign and malignant discrimination, multi-gene joint detection kit and application

A technology of thyroid nodules and biomarkers, which is applied in the field of biomedicine, can solve the problems of complex experimental operation process, low sensitivity, and long detection cycle, and achieve the effect of low template input, stable reaction system, and short detection cycle

Pending Publication Date: 2022-07-29
上海睿璟生物科技有限公司
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conventional gene detection method is Sanger sequencing method, which has the disadvantages of long detection cycle and low sensitivity
The main advantages of NGS sequencing, which is developing rapidly, are high detection throughput and can detect unknown mutations, but the disadvantages are that the detection cycle is long, the experimental operation process is complicated, the data analysis is difficult, and the cost of reagent equipment is expensive, so it is difficu

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Biomarker for thyroid nodule benign and malignant discrimination, multi-gene joint detection kit and application
  • Biomarker for thyroid nodule benign and malignant discrimination, multi-gene joint detection kit and application
  • Biomarker for thyroid nodule benign and malignant discrimination, multi-gene joint detection kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0205] Composition of multi-gene combined detection kit for judging benign and malignant thyroid nodules

[0206] The kit includes detection of target genes (BRAF gene V600E, TERT gene C228T / C250T, KRAS gene G12C / G12V / Q61R, NRAS gene Q61R, HRAS gene Q61R a total of 8 DNA single base mutations, CCDC6-RET, NCOA4-RET, PAX8 -PPARG, ETV6-NTRK3, a total of 4 gene fusions) and internal reference genes ACTB, NONO detection reaction solution, PCRMix, RT mixed enzyme, positive control.

[0207] 1. Primer probe combination

[0208] The detection reaction solution includes a total of 8 DNA single-base mutations, CCDC6-RET, NCOA4- RET, PAX8-PPARG, ETV6-NTRK3 (a total of 4 gene fusions) and primer probe compositions of the internal reference genes ACTB and NONO.

[0209] In this example, BRAF(NG_007873.3), TERT(NG_009265.1), KRAS(NG_007524.2), NRAS(NG_007572.1), HRAS(NG_007666.1) and ACTB(NG_007992.1) gDNA sequences are used as templates respectively , designed the respective specific pr...

Embodiment 2

[0276] Application method of multi-gene combined detection kit for judging benign and malignant thyroid nodules

[0277] The use of the multi-gene combined detection kit for judging benign and malignant thyroid nodules includes the following steps:

[0278] 1. Collect fine needle aspiration samples of thyroid nodules from patients to be tested, extract genomic DNA and total RNA, and store them at -20°C for later use. It is recommended to use the tissue cell DNA / RNA extraction kit (spin column method) (Shanghai Ruijing Biotechnology Co., Ltd., China) to extract nucleic acids according to the instructions.

[0279] 2. Prepare a PCR reaction system (the composition is shown in Table 14) with the DNA, RNA, positive control substance of the kit and blank control substance obtained above as templates respectively, and then carry out PCR reaction after fully mixing.

[0280] Table 14 qPCR reaction system

[0281]

[0282] PCR reaction conditions: the first stage: 50°C for 15min,...

Embodiment 3

[0293] Detection limit test of multi-gene combined detection kit for the discrimination of benign and malignant thyroid nodules

[0294] Select 12 locus positive samples or standards in the kit, of which 8 DNA mutation locus positive samples are diluted to 1% mutation abundance, and the concentrations are: 10ng / μL, 5ng / μL, 2.5ng / μL, 1ng / μL . The four fusion-positive samples were diluted to different copy number concentration gradients, 666 copies / μL, 333 copies / μL, 166 copies / μL, 83 copies / μL (corresponding to the total input amount of 2000 copies, 1000 copies, 500 copies, 250 copies) copy). Use the kit to dilute the template for detection, and repeat each gradient three times to confirm the detection sensitivity of the kit. The detection template gradient and detection results of each site are as follows:

[0295] For BRAF V600E

[0296]

[0297] The detection limit gradient test results of BRAF V600E are as follows Figure 13A shown.

[0298] For TERT C228T:

[0299...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention belongs to the field of biological medicine, and discloses a biomarker for judging benign and malignant thyroid nodules, a multi-gene joint detection kit and application. The biomarker group comprises at least one of eight DNA single base mutations and four gene fusions; the eight kinds of DNA single base mutations are selected from a BRAF gene V600E, a TERT gene C228T/C250T, a KRAS gene G12C/G12V/Q61R, an NRAS gene Q61R and an HRAS gene Q61R, and the four kinds of gene fusion are selected from CCDC6-RET (Exon1-Exon12), NCOA4-RET (Exon8-Exon12), PAX8-PPARG (Exon10-Exon2) and ETV6-NTRK3 (Exon4-Exon14). The method and the kit are high in detection sensitivity, high in specificity and wide in application scene.

Description

technical field [0001] The invention belongs to the field of biomedicine and discloses a high-performance multi-gene combined detection kit for judging benign and malignant thyroid nodules. Background technique [0002] Thyroid nodule is a common thyroid disease in the population, which is caused by the 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, and require early surgical treatment to obtain a good prognosis. At present, thyroid cells obtained by fine needle aspiration (FNA) and cytology are the gold standard for evaluating whether thyroid nodules are malignant tumors. However, 20%-30% of thyroid nodules cannot be definitively diagnosed this way. [0003] With the development of molecular biology technology, molecular diagnosis has gradually attracted people's attention in the differential diagnosis of benign and malign...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/6883C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6883C12Q1/6886C12Q1/6851C12Q2600/156C12Q2600/166C12Q2535/137C12Q2525/186C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 龚伟李雯石涵杨峰洪跟东
Owner 上海睿璟生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap