阅读说明：本技术 一种检测肥厚型心肌病基因突变的引物组、试剂盒和方法 (Primer group, kit and method for detecting gene mutation of hypertrophic cardiomyopathy ) 是由 张惠丹 戴敬 李阳 赵洪玉 于 2019-12-25 设计创作，主要内容包括：本发明公开了一种检测肥厚型心肌病基因突变的引物组,包括用于检测肥厚型心肌病基因突变的扩增引物组和延伸引物组。本发明还公开了一种检测肥厚型心肌病基因突变的试剂盒,该试剂盒含有用于检测肥厚型心肌病基因突变的扩增引物组和延伸引物组。此外,本发明还公开了一种检测肥厚型心肌病基因突变的方法。采用本发明的引物组进行检测肥厚型心肌病基因突变的方法,检测结果准确,检测速度快,无需荧光标记,且操作便利,检测灵敏度高,节省时间,检测成本大大降低,一天内可以完成对上百个样本的快速检测,适合批量筛查。(The invention discloses a primer group for detecting gene mutation of hypertrophic cardiomyopathy, which comprises an amplification primer group and an extension primer group for detecting gene mutation of hypertrophic cardiomyopathy. The invention also discloses a kit for detecting the gene mutation of the hypertrophic cardiomyopathy, which contains an amplification primer group and an extension primer group for detecting the gene mutation of the hypertrophic cardiomyopathy. In addition, the invention also discloses a method for detecting gene mutation of hypertrophic cardiomyopathy. The method for detecting gene mutation of hypertrophic cardiomyopathy by using the primer group has the advantages of accurate detection result, high detection speed, no need of fluorescent markers, convenience in operation, high detection sensitivity, time saving, great reduction in detection cost, capability of quickly detecting hundreds of samples in one day and suitability for batch screening.)

1. A primer group for detecting gene mutation of hypertrophic cardiomyopathy is characterized by comprising an amplification primer group and an extension primer group, wherein the amplification primer group is used for detecting gene mutation of hypertrophic cardiomyopathy, the amplification primer group comprises one or more pairs of amplification primers with nucleotide sequences shown in Seq ID No. 1-176, and the extension primer group comprises one or more extension primers with nucleotide sequences shown in Seq ID No. 177-264.

2. The primer set for detecting the gene mutation of hypertrophic cardiomyopathy according to claim 1, comprising an amplification primer set and an extension primer set, wherein the amplification primer set comprises all 88 pairs of amplification primers with nucleotide sequences shown in Seq ID No. 1-176, and the extension primer set comprises all 88 extension primers with nucleotide sequences shown in Seq ID No. 177-264.

3. A kit for detecting gene mutation of hypertrophic cardiomyopathy is characterized by comprising an amplification primer group and an extension primer group, wherein the amplification primer group is used for detecting gene mutation of hypertrophic cardiomyopathy, the amplification primer group comprises one or more pairs of amplification primers with nucleotide sequences shown in Seq ID No. 1-176, and the extension primer group comprises one or more extension primers with nucleotide sequences shown in Seq ID No. 177-264.

4. The kit for detecting gene mutation in hypertrophic cardiomyopathy according to claim 3, wherein the kit comprises an amplification primer set and an extension primer set for detecting 88 mutation sites of the pathogenic gene in hypertrophic cardiomyopathy, wherein the amplification primer set comprises nucleotide sequences such as

And all 88 pairs of amplification primers shown in Seq ID No.1 to 176, wherein the extension primer group comprises all 88 extension primers shown in Seq ID No.177 to 264.

5. The kit for detecting gene mutation of hypertrophic cardiomyopathy according to claim 3, further comprising PCR reaction Taq enzyme and PCR buffer solution, wherein the PCR reaction Taq enzyme, the PCR buffer solution and the amplification primer are used for preparing a multiplex PCR amplification reaction system.

6. The kit for detecting gene mutation of hypertrophic cardiomyopathy of claim 3, wherein: the kit also comprises SAP enzyme and SAP buffer solution, wherein the SAP enzyme and the SAP buffer solution are used for preparing an alkaline phosphatase treatment reaction system.

7. The kit for detecting gene mutation in hypertrophic cardiomyopathy of claim 3, further comprising iP L EX enzyme and iP L EX buffer solution, wherein the iP L EX enzyme, the iP L EX buffer solution and the extension primer are used for preparing an extension reaction system.

8. The kit for detecting gene mutation of hypertrophic cardiomyopathy according to claim 3, wherein: the kit also includes a MassARRAY chip.

9. A method for detecting gene mutation of hypertrophic cardiomyopathy is characterized by comprising the following steps:

(1) obtaining an amplification primer and an extension primer for detecting gene mutation of hypertrophic cardiomyopathy;

(2) extracting DNA of a sample to be detected;

(3) taking the DNA extracted in the step (2) as a template, and carrying out PCR amplification by using the amplification primer obtained in the step (1) to obtain a target sequence amplification product;

(4) removing free dNTPs contained in the amplification product obtained in the step (3) by using SAP enzyme;

(5) taking the target sequence amplification product obtained in the step (3) as a template, and carrying out single base extension reaction by using the extension primer obtained in the step (1) to obtain an extension product;

(6) purifying the extension product obtained in the step (5);

(7) and (3) detecting and typing the purified extension product by adopting a time-of-flight mass spectrum genotyping system.

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a primer group, a kit and a method for detecting gene mutation of hypertrophic cardiomyopathy.

Background

Hypertrophic Cardiomyopathy (HCM) is the most common inherited cardiovascular disease and the leading cause of Sudden Cardiac Death (SCD) in adolescents and young athletes. The incidence rate of hypertrophic cardiomyopathy is 1/500, the incidence trend is rising year by year, and at present, 100-200 million hypertrophic cardiomyopathy patients exist in China. With the growing awareness of hypertrophic cardiomyopathy, HCM is considered a disease that can be detected, intervened, and treated, with a better prognosis in most patients.

Many HCM pathogenic genes have been discovered and various gene therapy strategies have been proposed to eliminate genetic defects to achieve the causal treatment of HCM, including genome editing, exon skipping, allele-specific silencing, RNA trans-splicing and gene replacement, among others, and most of these techniques have been tested for efficacy and safety in animal or human-induced models of HCM pluripotent stem cells.

HCM shows clinical heterogeneity due to different pathogenic genes, and nearly 40 genes are related to the occurrence and development of HCM and mainly comprise coding genes of sarcomere structural proteins. B-myosin heavy chain (MYH 7), myosin binding protein C (MYBPC 3) and troponin T (TNNT 2) are the most common three disease-causing genotypes.

The diagnosis of the single-gene disease is mainly based on the second-generation sequencing at present by depending on the development of scientific technology, but the operation steps are complicated, the detection time is long, the requirements on the operation and analysis capability of technical personnel are high, the detection results are easily influenced, meanwhile, the second-generation sequencing detection reagent and the analysis consumable material are expensive, the high cost brings a plurality of challenges to the application of the second-generation sequencing disease molecular diagnosis and the crowd screening, and the popularization of the application is influenced.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a primer group, a kit and a method for detecting gene mutation of hypertrophic cardiomyopathy; the detection method using the primer group has the advantages of accurate detection result, high detection speed, no need of fluorescent markers, greatly reduced detection cost, capability of completing rapid detection of hundreds of samples in one day, and suitability for batch screening.

In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:

a primer group for detecting gene mutation of hypertrophic cardiomyopathy comprises an amplification primer group and an extension primer group, wherein the amplification primer group is used for detecting gene mutation of hypertrophic cardiomyopathy, the amplification primer group comprises one or more pairs of amplification primers with nucleotide sequences shown as Seq ID No. 1-176, and the extension primer group comprises one or more extension primers with nucleotide sequences shown as Seq ID No. 177-264.

Further, the primer group comprises an amplification primer group and an extension primer group, wherein the amplification primer group is used for detecting 88 mutation sites of the hypertrophic cardiomyopathy detection hypertrophic cardiomyopathy pathogenic genes, the amplification primer group comprises all 88 pairs of amplification primers with nucleotide sequences shown as SeqID No. 1-176, and the extension primer group comprises all 88 extension primers with nucleotide sequences shown as Seq ID No. 177-264.

The invention also provides a kit for detecting gene mutation of hypertrophic cardiomyopathy, which comprises an amplification primer group and an extension primer group for detecting gene mutation of hypertrophic cardiomyopathy, wherein the amplification primer group comprises one or more pairs of amplification primers with nucleotide sequences shown as Seq ID No. 1-176, and the extension primer group comprises one or more extension primers with nucleotide sequences shown as Seq ID No. 177-264.

Further, the kit contains an amplification primer group and an extension primer group for detecting 88 mutation sites of pathogenic genes of hypertrophic cardiomyopathy, wherein the amplification primer group comprises all 88 pairs of amplification primers with nucleotide sequences shown in Seq ID No. 1-176, and the extension primer group comprises all 88 extension primers with nucleotide sequences shown in Seq ID No. 177-264.

Further, the kit also comprises PCR reaction Taq enzyme and PCR buffer solution, and the PCR reaction Taq enzyme, the PCR buffer solution and the amplification primer are used for preparing a multiple PCR amplification reaction system.

Further, the kit also comprises SAP enzyme and SAP buffer solution, wherein the SAP enzyme and the SAP buffer solution are used for preparing the alkaline phosphatase treatment reaction system.

Further, the kit also comprises an iP L EX enzyme and an iP L EX buffer solution, wherein the iP L EX enzyme, the i P L EX buffer solution and the extension primer are used for preparing an extension reaction system.

Further, the kit also comprises a MassARRAY chip.

The invention also provides a method for detecting gene mutation of hypertrophic cardiomyopathy, which comprises the following steps:

(1) obtaining an amplification primer and an extension primer for detecting gene mutation of hypertrophic cardiomyopathy;

(2) extracting DNA of a sample to be detected;

(3) taking the DNA extracted in the step (2) as a template, and carrying out PCR amplification by using the amplification primer obtained in the step (1) to obtain a target sequence amplification product;

(4) removing free dNTPs contained in the amplification product obtained in the step (3) by using SAP enzyme;

(5) taking the target sequence amplification product obtained in the step (3) as a template, and carrying out single base extension reaction by using the extension primer obtained in the step (1) to obtain an extension product;

(6) purifying the extension product obtained in the step (5);

(7) and (3) detecting and typing the purified extension product by adopting a time-of-flight mass spectrum genotyping system.

The invention has the beneficial effects that:

the invention adopts different primer design software, combines an agilent early design probe design system, and finally compresses the multiple PCR amplification of 88 mutation sites into 3 wells for detection by continuously optimizing primer sequences, and can complete the detection of 120 patients at most by using a 384-well plate once test, thereby greatly improving the flux of one detection.

The method for detecting gene mutation of hypertrophic cardiomyopathy by using the primer group has the advantages of accurate detection result, high detection speed, no need of fluorescent markers, convenience in operation, high detection sensitivity, time saving, great reduction in detection cost, capability of quickly detecting hundreds of samples in one day and suitability for batch screening.

Detailed Description

The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention, and to clearly and unequivocally define the scope of the present invention.