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Human thyroid cancer gene fusion kit and detection method

A technology for gene fusion and thyroid cancer, applied in biochemical equipment and methods, microbiological measurement/inspection, etc., can solve the problems of less information on fusion gene detection, limited clinical use, and increased experimental operation procedures, so as to improve the accuracy of detection The effect of reducing the risk of opening tubes/pipetting

Inactive Publication Date: 2020-12-01
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

At present, the detection kits based on the next-generation sequencing platform on the market are commonly used to build a library based on the probe capture method at the DNA level, but the cost is high and the operation process is cumbersome; while the RNA-level reverse transcription plus multiple PCR technology combines the sensitivity of qPCR and Specificity and intuitive results of NGS technology, easy operation, short time-consuming, only one sample can detect all fusion types, with outstanding methodological advantages
[0005] However, in many existing technical solutions, most of them reverse RNA to cDNA first, and then use cDNA to construct libraries (patent numbers: CN 111088365A, CN 110241215A, CN109371139A). These methods not only increase the experimental operation process, but also improve the The risk of contamination, and its detection of fusion gene information is relatively small, and its clinical use is limited
The current existing technology (patent number: CN 104894271A) defines the fusion positive threshold based on the value of reads. This method is extremely unstable and is easily affected by various factors such as sequencing data volume and methodology.

Method used

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  • Human thyroid cancer gene fusion kit and detection method
  • Human thyroid cancer gene fusion kit and detection method
  • Human thyroid cancer gene fusion kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 The primer sequence for amplifying the mutation sites of thyroid cancer-related genes was synthesized by Shanghai Sangong.

[0042] Taking CCDC6-RET as an example, the RNA primer design principle of the present invention will be described in detail below. Such as figure 1 As shown, with the position of the breakpoint as the boundary, the exon 1 of the 5' end CCDC6 gene is fused with the exon 12 of the 3' RET gene, so the upstream primer is designed on the exon 1 of the CCDC6 gene, and the downstream primer is designed on the RET gene On exon 12 of the gene; the RET wild-type upstream primer was designed on exon 11, and the downstream was consistent with the downstream primer of the fusion gene.

[0043] (1) RNA-specific primers, the primer sequences of which are shown in Table 2:

[0044] Table 2 fusion gene and its primer sequence

[0045] Gene Primer CCDC6-E1-F 5'-GAACGACATGGCTACGATCCGACTTCAGGAGGAGAACCGCGAC-3' CCDC6-E2-F 5'-GAACG...

Embodiment 2

[0054] Example 2: Sample pretreatment and nucleic acid extraction

[0055] Qualified professionals take samples with a puncture needle, and the samples include puncture tissue samples greater than 1 mg. After the biopsy is completed, infiltrate the tissue completely in RNA preservation solution as soon as possible. Samples were stored at -20°C prior to sample shipment.

[0056] Use the tissue cell RNA extraction kit (spin column method) and refer to the instructions of the kit to extract the RNA in the sample.

[0057] 1. Mechanical tissue sample homogenization:

[0058] Put the sample in a suitable glass tube or centrifuge tube, add 350 μL Buffer RL and DTT mixed lysate (100:1) lysate, insert the probe into the lysate, and homogenize intermittently at high speed, each time for 15-20 seconds until the sample Homogenize completely.

[0059] 2. Extraction of nucleic acid after homogenization

[0060] About 350 μL of the homogenized sample was centrifuged at 14,000 rpm for 3...

Embodiment 3

[0071] Embodiment 3: Construction of library

[0072] The library construction process of this kit is as follows:

[0073] (1) RNA reverse transcription and the first round of PCR amplification (amplification of the target region)

[0074] Using Vazyme's reverse transcriptase and its KAPA multi-reconstruction library mix respectively, the following system was prepared:

[0075]

[0076] Set the following conditions on the PCR instrument for the reaction:

[0077]

[0078] (2) The first round of PCR product purification

[0079] 1) After the programmed reaction, the sample was briefly centrifuged, and the sample was added to 20 μl of nucleic acid-free water, then transferred to 14 μl (0.7×) XP magnetic beads, vortexed and incubated at room temperature for 2 min;

[0080] 2) Place the sample on a magnetic stand. After the solution is clarified, transfer the supernatant to 10 μl (0.5×) XP magnetic beads, vortex and mix, and incubate at room temperature for 5 minutes;

...

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Abstract

The invention discloses a human thyroid cancer gene fusion kit and a detection method in the technical field of molecular biology. According to the kit, a multiple PCR capture technology and an NGS sequencing technology are adopted; the kit is used for qualitatively detecting various variants of CCC6-RET, NCOA4-RET, PAX8 / PPARG and ETV6-NTRK3 fusion genes in a thyroid nodule needle biopsy cytological uncertainty fresh tissue sample, and detection threshold values are determined according to molecular tags. Assistance can be provided for auxiliary diagnosis and treatment of thyroid cancer through detection of fusion sites of the kit in combination with clinical pathological analysis results.

Description

technical field [0001] The invention relates to a human thyroid cancer gene fusion kit and detection method in the field of molecular biology technology. Background technique [0002] Thyroid cancer is the most common tumor in human endocrine organs, accounting for about 4% of all human malignant tumors. In recent years, its incidence has been increasing steadily in China and the world. Thyroid cancer can be divided into papillary carcinoma (PTC), follicular carcinoma (FTC), anaplastic carcinoma (ATC) and medullary carcinoma (MTC) according to pathological type. Among them, PTC and FTC are collectively called differentiated thyroid cancer (DTC), accounting for about 85%-90% of thyroid cancer. Differentiated thyroid cancer (DTC) usually develops from a thyroid nodule. Such nodules are common in the population, especially with age, the incidence rate also increases. However, most thyroid nodules are benign, and it is clinically necessary to identify malignant nodules accura...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686
CPCC12Q1/6886C12Q1/686C12Q2600/158
Inventor 王宝霞胡春旭高伙妮何文天包文静
Owner 上海睿璟生物科技有限公司
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