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Primer for detecting variation of benign and malignant related genes of thyroid nodules, kit and detection method

A technology for thyroid nodules and gene variation, applied in the field of molecular biology, can solve problems such as indefinite diagnosis, achieve good specificity, good repeatability, and improve accuracy

Active Publication Date: 2019-09-17
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 2019 edition of the clinical practice guidelines of the National Comprehensive Cancer Network (NCCN) clearly states: (1) Suspected follicular and Hurthle cell carcinomas that cannot be determined by fine needle aspiration (FNA) (the diagnosis is based on vascular or capsule invasion) (2) For nodules that cannot be clearly diagnosed by cytology, using molecular diagnostic techniques to detect mutations in BRAF, RAS, RET / PTC, PAX8 / PPARγ and other oncogenes is of great help in the diagnosis of thyroid nodules. cancer plays an important role

Method used

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  • Primer for detecting variation of benign and malignant related genes of thyroid nodules, kit and detection method
  • Primer for detecting variation of benign and malignant related genes of thyroid nodules, kit and detection method
  • Primer for detecting variation of benign and malignant related genes of thyroid nodules, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 A primer for detecting gene variation associated with benign and malignant thyroid nodules.

[0049] The benign and malignant thyroid nodules-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 the relevant gene sequences, and the design scope included benign and malignant thyroid nodules-related genes Mutation hotspots.

[0050] As shown in Table 3, in this embodiment, 16 pairs of specific primers were designed for the hotspot mutations of genes related to benign and malignant thyroid nodules. 220-280bp, has the advantages of wide coverage, multiple detection sites, balanced GC content, stable product structure, and less dimer structure. specific,

[0051] Table 3 Primer sequence list

[0052]

[0053]

[0054] Among them, i5 and i7 are 6 nucleotide sequences respectively, and the index numbers are different, and these 6 ...

Embodiment 2

[0057] Example 2 A kit for detecting gene variations associated with benign and malignant thyroid nodules.

[0058] The kit for detecting benign and malignant thyroid nodule related gene variation described in this example mainly includes:

[0059] (1) PCR-specific primers: used to amplify multiple target regions on the target gene of the sample to be tested, the amplification range covers at least the hotspot mutation region of the target gene, and the sequences are shown in SEQ ID NO.1 and SEQ ID NO in Table 3 .29 shown. Preferably, multiple amplification primer pairs are mixed together to form a primer pool;

[0060] (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 to distinguish different samples. The sequence is shown in the table Shown in SEQ ID NO.30 and SEQ ID NO.31 in 3;

[0061] (3) ...

Embodiment 3

[0067] Example 3 A detection method for detecting gene variations associated with benign and malignant thyroid nodules.

[0068] The detection method of the present embodiment comprises the following steps:

[0069] (1) Sample DNA and RNA extraction: Refer to the kit instructions, use the AllPrep DNA / RNA Mini Kit kit to extract DNA and RNA respectively, use NanoDrop to measure the concentration and purity, and use nuclease-free Dilute the nucleic acid sample with water to 10-50ng / μL as the initial nucleic acid concentration for amplification and library construction.

[0070] (2) DNA library construction:

[0071] (2a) DNA target region amplification:

[0072] Carry out the first round of PCR amplification according to the following reaction system and amplification conditions:

[0073]

[0074] DNA multiplex PCR amplification program settings:

[0075]

[0076] (2b) The first round of PCR product purification:

[0077] Purify the first round of PCR products using t...

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Abstract

The invention discloses a primer for detecting variation of benign and malignant related genes of thyroid nodules, a kit and a detection method. According to the primer, the kit and the detection method, variation of 15 loci of the six genes and variation of the three fusion genes can be detected simultaneously, and the BRAF gene, the KRAS gene, the HRAS gene, the NRAS gene, the TERT gene, the EIF1AX gene, RET / PTC1 fusion, RET / PTC3 fusion and PAX8 / PPARgamma fusion are involved. Hotspot mutation and fusion variation of the genes are closely related to the benign and malignant thyroid nodules. Therefore, the primer, the kit and the detection method are used for detecting a sample of a patient and can assist doctors in benign and malignant identification of the thyroid nodules which cannot be clearly diagnosed in the cytology, the accuracy of identifying the benign and malignant thyroid nodules is improved, excessive medical treatment for the patient with the thyroid nodules is reduced, and the primer, the kit and the detection method have important practical significance and high economic benefit for saving medical resources of China.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer, a kit and a detection method for detecting gene variations related to benign and malignant thyroid nodules. 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 and require early surgical treatment in order to obtain a good prognosis. [0003] At present, B-ultrasound is the preferred method for examining thyroid nodules. The nature of nodules can be preliminarily judged according to the imaging characteristics of thyroid nodules, so that the detection rate of thyroid nodules can reach 76%. Among the nodules, benign nodules such as nodular goiter and thyroid adenoma account for about 85%-95%, and only 5%-15% of the nodules...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6883C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q1/6883C12Q1/6886C12Q2600/156C12Q2600/16C12Q2537/143C12Q2531/113C12Q2535/122
Inventor 张宇清赵艳伟裴婷婷
Owner 润安医学科技(苏州)有限公司
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