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Extraction-free detection kit and detection method for oncogene mutation in human thyroid glands

A detection kit and technology for thyroid cancer, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of poor prognosis and high tumor invasiveness

Inactive Publication Date: 2020-11-24
上海睿璟生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If this mutation occurs at the same time as BRAF mutation, RAS mutation and other oncogenic mutations, it indicates that the tumor is highly aggressive and has a poor prognosis

Method used

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  • Extraction-free detection kit and detection method for oncogene mutation in human thyroid glands
  • Extraction-free detection kit and detection method for oncogene mutation in human thyroid glands
  • Extraction-free detection kit and detection method for oncogene mutation in human thyroid glands

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 The specific primer sequence for amplifying the mutation site of thyroid cancer-related genes was synthesized by Shanghai Sangong.

[0032] Primer sequence pairs for BRAF:

[0033] BRAF-F

[0034] 5'-GAACGACATGGCTACGATCCGACTTCAGTGGAAAAATAGCCTCAATTCTTACCA-3'

[0035] BRAF-R

[0036] 5'-TCCTAAGACCGCTTGGCCTCCGACTTCTTCATGAAGACCTCACAGTAAAAATAGGT-3'

[0037] Primer sequence pairs for TERT:

[0038] TERT_F 5'-GAACGACATGGCTACGATCCGACTTAGCGCTGCCTGAAACTCG-3'

[0039] TERT_R 5'-TCCTAAGACCGCTTGGCCTCCGACTTCCTTCACCTTCCAGCTCCG-3'

[0040] Primer sequence pairs for NRAS:

[0041] NRAS-F

[0042] 5'-GAACGACATGGCTACGATCCGACTTCAAATGACTTGCTATTATTGATGGCAA-3'

[0043] NRAS-R

[0044] 5'-TCCTAAGACCGCTTGGCCTCCGACTTGTTATAGATGGTGAAACCTGTTTGTTGG-3'

[0045] NRAS-F

[0046] 5'-GAACGACATGGCTACGATCCGACTTTGGGATCATATTCATCTACAAAGTGGTT-3'

[0047] NRAS-R

[0048] 5'-TCCTAAGACCGCTTGGCCTCCGACTTGATTACTGGTTTCCAACAGGTTCTTG-3'

[0049] Primer sequence pairs for HRAS:

[0050] HRAS-F 5...

Embodiment 2

[0084] Embodiment 2: the pretreatment of sample

[0085] Qualified professionals use a puncture needle to sample tissue. After the biopsy is completed, the tissue should be completely immersed in PBS as soon as possible. Before sample transportation, samples were stored in -20°C or -80°C refrigerators.

Embodiment 3

[0086] Embodiment 3: Construction of library

[0087] The library construction process of this kit is as follows:

[0088] (1) Treatment of punctured tissue samples with nucleic acid releasing agent

[0089] After the puncture tissue sample was taken out, centrifuged at 5000rpm for 3min, the supernatant was removed; 5μl of nucleic acid release agent (400mmol / L NaOH, 1mol / L formamide) was added to the pellet, mixed well, left at room temperature for 10 minutes, and then proceeded to One step experiment.

[0090] (2) Amplification of the target fragment

[0091] The composition of PCR reaction solution is 50mmol / L Tris-HCl, 30mmol / L KCl, 3.5mmol / L MgCl 2 , 600μmol / L dNTPs, 20pmol / L primers and 5U PCR hot start Taq enzyme.

[0092] Prepare the following system:

[0093]

[0094] Set the following conditions on the PCR instrument for the reaction:

[0095]

[0096] (3) The first round of PCR product purification

[0097] 1) After the programmed reaction, the sample wa...

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Abstract

The invention discloses an extraction-free detection kit and detection method for oncogene mutation in human thyroid glands, belonging to the technical field of molecular biology. According to the kit, a specific nucleic acid releasing agent and PCR reaction liquid matched with the specific nucleic acid releasing agent are adopted, a multiplex PCR capture technology and an NGS sequencing technology are cooperatively used, and the kit is used for qualitatively detecting various variations of BRAF, TERT, RET, TP53, NRAS, HRAS, KRAS and PIK3CA genes in a fresh tissue sample with uncertainty according to results of thyroid nodule needle biopsy cytology; and through detection of multiple genes and multiple sites and consideration of clinical pathological analysis results, assistance is providedfor auxiliary diagnosis and treatment of thyroid cancer.

Description

technical field [0001] The invention relates to a detection kit and a detection method for human thyroid extraction-free cancer gene mutation detection in the field of molecular biology technology. Background technique [0002] Thyroid cancer is a common malignant tumor of the head and neck, accounting for 1.0% to 1.5% of all malignant tumors in the whole body. At present, thyroid cancer is mainly evaluated through clinical palpation, ultrasound, radionuclide imaging and fine needle biopsy. To a certain extent, it can improve the accuracy of thyroid cancer diagnosis, but there are still 10% to 30% of which cannot be diagnosed clearly. Both domestic and foreign guidelines for thyroid cancer suggest that if thyroid nodules cannot be diagnosed by thyroid nodule fine-needle aspiration biopsy (FNAB), molecular marker detection can be used to improve the diagnostic accuracy, such as BRAF, RAS, RET / PTC , PAX8 / PPARγ and other mutation detection items have been routine molecular det...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858
CPCC12Q1/6886C12Q1/6858C12Q2600/16C12Q2600/156
Inventor 包文静王宝霞高伙妮何文天张志帅
Owner 上海睿璟生物科技有限公司
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