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PCR (Polymerase Chain Reaction) specific primer, detecting kit and detecting method of benign and malignant thyroid nodule associated gene

A technology of thyroid nodules and detection kits, which is applied in the field of molecular biology, can solve the problems of chip customization, complicated operation, and low accuracy, so as to improve detection efficiency and accuracy, simplify operation steps, and detect sites many effects

Active Publication Date: 2018-07-24
GUANGDONG GENERAL HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thyroid ultrasonography and fine-needle aspiration biopsy have poor specificity and may lead to misjudgment when distinguishing follicular thyroid neoplasia from follicular thyroid carcinoma. The accuracy rate is not high, resulting in a large number of unnecessary malignant diseases treatment case
Chip-based tumor marker detection is mainly provided by the chip provider, which has problems such as high technical cost, complicated operation, low detection sensitivity, poor repeatability, narrow analysis range, and special customization of the chip.

Method used

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  • PCR (Polymerase Chain Reaction) specific primer, detecting kit and detecting method of benign and malignant thyroid nodule associated gene
  • PCR (Polymerase Chain Reaction) specific primer, detecting kit and detecting method of benign and malignant thyroid nodule associated gene
  • PCR (Polymerase Chain Reaction) specific primer, detecting kit and detecting method of benign and malignant thyroid nodule associated gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Multiplex PCR-specific primers, detection kit and detection method for genes related to benign and malignant thyroid nodules based on high-throughput sequencing technology

[0052] 1. Design of primers

[0053] The gene sequences related to benign and malignant thyroid nodules involved in this example were selected from the UCSC (University of California Santa Cruz, University of California, Santa Cruz) database, and hotspot mutation primers were designed based on the relevant gene sequences, and the design range included benign and malignant thyroid nodules. Mutational hotspots in associated genes.

[0054] Such as figure 1 As shown, in this example, a total of 27 pairs of primers were designed for hotspot mutations of genes related to thyroid nodules. Wide coverage, stable structure, and multiple detection sites.

[0055] Specifically, 27 pairs of amplification primers are the amplification primer pairs of 24 genes in Table 1 as follows:

[0056] Table 1...

Embodiment 2

[0081] Example 2 Primer Specificity Verification

[0082] Nucleic acid was extracted from peripheral blood samples (number: 1-3), fine needle puncture samples (number: 4-6), and paraffin tissue samples (number: 7-9) using the method of step S11 in Example 1, and the concentration and purity were determined. After the determination, take qualified samples and use 10mM Tris to dilute each sample to 100ng / μL, and use 1% agarose gel electrophoresis to detect the quality of each sample (the qualification standard is the same as step S11 in Example 1), and enter the group after passing the test and perform mark. Using the method of step S12 in Example 1, the above-mentioned 9 cases of qualified samples were amplified, and the sample volume was 2 μL each. After the amplified product was purified, it was detected by 1% agarose gel electrophoresis (the qualification standard was the same as in Step S13 in Example 1. Step), 9 cases of samples were detected using specific primer amplifi...

Embodiment 3

[0084] Example 3 Primer Detection Sensitivity Verification

[0085] Using the method of step S11 in Example 1, sample nucleic acids were extracted from peripheral blood samples (number: 1), fine needle aspiration samples (number: 4), and paraffin tissue samples (number: 7) that passed the quality inspection to verify the sensitivity of primer detection. The initial concentration of each sample is 100ng / μL, and it is diluted according to the concentration gradient of 5 times, 10 times and 20 times. After dilution, the concentration of each sample is 20ng / μL, 10ng / μL and 5ng / μL respectively, and the sample name and Concentration marks. The method of step S12 in Example 1 was used to amplify the above-mentioned 9 cases of qualified diluted samples, and the sample volume was 2 μL each, and the 9 cases of samples were detected using specific primer amplification and detection methods for benign and malignant thyroid nodules related genes. The control test is the same as in Example...

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) specific primer, a detecting kit and a detecting method of a benign and malignant thyroid nodule associated gene. Sequences of the PCR specific primer of the thyroid nodule benign and malignant associated gene are as shown in SEQ ID NO:1 to SEQ ID NO:54. The benign and malignant thyroid nodule associated gene disclosed by the invention is closely related to pathological indexes of cases of a benign thyroid nodule and a malignant thyroid nodule and can be used as a biomarker for carrying out benign and malignant thyroid nodule classification on a patient suffering from the thyroid nodules, and effective basis can be provided for clinical individual intervention treatment. The PCR specific primer, the detecting kit and the detecting method, disclosed by the invention, are capable of simultaneously detecting multiple benign and malignant thyroid nodule associated genes of multiple samples, and the detecting efficiency and the accuracy can be effectively increased; meanwhile, the cost is reduced, and the operating steps are simplified.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a specific PCR primer, a detection kit and a detection method for genes related to benign and malignant thyroid nodules. Background technique [0002] Thyroid cancer refers to a common malignant tumor that originates in the thyroid gland, which accounts for the first place in the incidence of head and neck tumors, and the incidence has been on the rise in recent years. A study in 2012 found that the incidence of thyroid cancer in women has jumped to the fifth place among women prone to tumors. According to the SSER database of NCI, the incidence of thyroid cancer in 1975 was 4.04 / 100,000 population, and the incidence increased in 2007. to 11.99 / 100,000 population. [0003] At present, overtreatment of thyroid nodules is common. According to the statistics of thyroid surgery in Changchun area, among 9216 cases of thyroid nodules treated by surgery, malignant tumors accounted for...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6883C12Q1/6869C12N15/10C12N15/11
CPCC12N15/1013C12Q1/6869C12Q1/6883C12Q1/6886C12Q2600/156C12Q2535/122
Inventor 邝建朱瑞娟赖水青朱奇陈志江梁雅俊王龙
Owner GUANGDONG GENERAL HOSPITAL
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