Kit for detecting heart potassium ion channel coding gene idiopathic ventricular fibrillation risk gene SNP (Single Nucleotide Polymorphism) site

Abstract

The invention relates to a kit for detecting heart potassium ion channel coding gene idiopathic ventricular fibrillation risk gene SNP (Single Nucleotide Polymorphism) loci, which comprises 38 pairs of specific multiplex PCR (Polymerase Chain Reaction) primer pairs with 41 required SNP loci, 41 SNP single-base extension primers, and necessary PCR reaction liquid, SNaPshot reaction liquid, shrimp alkaline phosphatase and exonuclease I. The kit is used for detecting heart potassium ion channel coding gene idiopathic ventricular fibrillation risk gene SNP loci. According to the kit, the multiple PCR technology, the SNaPshot micro-sequencing technology and the capillary electrophoresis technology are combined to screen the risk gene SNP sites related to the idiopathic ventricular fibrillation on the heart potassium ion channel coding genes, the SNP related to the idiopathic ventricular fibrillation on 41 potassium ion channel genes can be detected only through one experiment, a clear conclusion can be obtained, and result interpretation is simple and objective.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and relates to a kit for detecting 41 idiopathic ventricular fibrillation (IVF) risk gene SNP sites on cardiac potassium ion channel encoding genes, which can be used for auxiliary assessment of idiopathic ventricular fibrillation susceptibility risk. Background Art

[0002] Idiopathic ventricular fibrillation (IVF) is a rare malignant ventricular arrhythmia with unknown etiology. It is a non-organic heart disease and ion channel disease. Patients often have polymorphic ventricular tachycardia or ventricular fibrillation, and can cause repeated syncope, cardiac arrest and sudden cardiac death.

[0003] Cardiac ion channels play an important role in different phases of myocardial action potential, and their gene mutations may affect their structure and function. At present, many cardiac ion channel encoding genes are significantly associated with IVF, among which the relationship between sodium, potassium, and calcium ion channel encoding genes and IVF has been studied in depth.

[0004] Studies have shown that changes in the structure and function of the corresponding channel protein caused by mutations in sodium channel coding genes often cause serious arrhythmias. Potassium channels are the most complex channels with the most subtypes discovered so far. More and more studies have shown that mutations in potassium channel coding genes can explain many malignant arrhythmias and sudden cardiac deaths without organic lesions. Common potassium channel coding genes include KCNQ1, KCNH2, KCNJ2, KCNE5, etc.

[0005] SNaPshot technology is a microsequencing technology for detecting SNPs developed by ABI, an American company. This technology uses the end extension method to accurately determine the mutation site and mutation type, can automatically perform typing, and has a high throughput.

[0006] In recent years, SNaPshot technology has been used for a lot of research on SNP detection. In 2011, GGPaneto et al. used SNaPshot technology to perform multiple SNaPshot reactions after single amplification of 42 SNPs to classify mixed populations; in 2013, Sun Yanan et al. used SNaPshot technology to detect the O3-M122 haplogroup of the Y-SNP site. In 2014, Alexandra Coutinho et al. used SNaPshot technology for genotyping of major human mitochondria in the founder lineage of the Americas; in 2015, R. Daniel et al. studied the next generation sequencing SNaPshot for forensic SNP analysis. In the same year, Li Yubi et al. applied the SNaPshot single base extension reaction method to detect the relationship between multiple target gene polymorphisms and susceptibility to non-alcoholic fatty liver disease.

[0007] Since whole genome sequencing technology is expensive and requires specialized analysis, it is currently difficult to popularize. Relatively speaking, the SNaPshot detection system can provide a convenient detection method, and the accuracy of SNaPshot microsequencing is extremely high, and it is widely used in the DNA field. Summary of the invention

[0008] The purpose of the present invention is to provide a kit for detecting the SNP site of the idiopathic ventricular fibrillation risk gene of the cardiac potassium ion channel encoding gene. The kit is used to detect and evaluate the susceptibility risk of idiopathic ventricular fibrillation, with high accuracy and low cost, which is conducive to clinical use and promotion.

[0009] To achieve the above-mentioned purpose of the invention, the kit for detecting the SNP site of the idiopathic ventricular fibrillation risk gene of the cardiac potassium channel encoding gene of the present invention comprises 38 pairs of specific multiplex PCR primer pairs shown in SEQ ID NO.1-76, 41 SNP single base extension primers shown in SEQ ID NO.77-117, and multiplex PCR reaction solution, SNaPshot reaction solution, shrimp alkaline phosphatase and exonuclease I and other reagents required to be configured in the conventional kit, which are used to detect the following 41 idiopathic ventricular fibrillation-related SNP sites: rs786205867, rs2125168897, rs1363307614, rs199472947, rs199472916, rs138776684, rs120074192, rs199472687, rs179489, rs1994 72708, rs120074196, rs199472743, rs397508079, rs397508087, rs199472806, rs120074189, rs121908590, rs778849288, rs104894584 , rs144022753, rs199473648, rs612790, rs2813862, rs605604, rs2798334, rs7539683, rs1805222, rs2120436, rs2034124, rs12090194 , rs4839185, rs6682872, rs4838926, rs4838927, rs1545300, rs11588747, rs2010749, rs12144965, rs3008527, rs3008528, rs2075811.

[0010] Specifically, the 41 idiopathic ventricular fibrillation-related SNP sites are located on genes KCND3, DES, KCNH2, KCNQ1, KCNA5, ABCC9, KCNJ2, and KCNE2, as shown in the following table:

[0011]

[0012]

[0013] The 41 SNP sites described in the present invention are all contained in the target gene fragments of the 38 pairs of specific multiplex PCR primers, and each target gene fragment contains 1 to 2 SNPs.

[0014] Furthermore, the relationship between the SNP sites amplified by the 38 pairs of specific multiplex PCR primers in the kit of the present invention is as follows:

[0015]

[0016]

[0017] Furthermore, the kit of the present invention also contains 41 SNP single base extension primers, and the corresponding extended SNP sites are as follows:

[0018]

[0019]

[0020] The present invention utilizes multiplex PCR technology, SNaPshot microsequencing technology combined with capillary electrophoresis technology to screen SNP sites of risk genes associated with idiopathic ventricular fibrillation on cardiac potassium channel encoding genes. Only one experiment is needed to detect SNPs associated with idiopathic ventricular fibrillation on 41 potassium channel genes and obtain clear conclusions, and the result interpretation is simple and objective.

[0021] The method for detecting the SNP site of the idiopathic ventricular fibrillation risk gene using the kit for detecting the SNP site of the idiopathic ventricular fibrillation risk gene of the cardiac potassium ion channel encoding gene of the present invention specifically comprises:

[0022] 1) Using DNA samples from patients with arrhythmia as templates, a multiplex PCR amplification system was constructed for PCR amplification, and the PCR products were purified;

[0023] 2) Using the purified PCR product as a template, constructing a SNaPshot reaction system for single base extension reaction, and purifying the single base extension reaction product;

[0024] 3) Use a genetic analyzer to analyze the purified single base extension reaction product.

[0025] The test samples involved in the present invention are divided into two categories, with healthy unrelated individuals as the normal control group and samples of patients with malignant arrhythmia as the case group.

[0026] The method for extracting DNA from the sample is specifically as follows: taking 2 mL of fresh venous blood, using OMEGA EZNA TMSE Blood DNA Kit to extract the DNA sample, and performing quantitative analysis on the extracted genomic DNA according to the optical density method to determine the purity and concentration of the genomic DNA.

[0027] The present invention constructs a kit for detecting 41 SNP sites of idiopathic ventricular fibrillation risk genes on cardiac potassium ion channel coding genes based on microsequencing technology, which can be used to detect cardiac potassium ion channel coding gene polymorphisms in patients with idiopathic ventricular fibrillation.

[0028] The present invention does not require special instruments and technical methods, and can simply, quickly and accurately screen 41 SNP sites of idiopathic ventricular fibrillation risk genes. The detection throughput is comparable to direct sequencing, which greatly saves manpower and material resources, and provides genetic data and theoretical basis for precision medicine, eugenics, clinical diagnosis, and prevention of sudden cardiac death. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1It is the result of detecting 41 SNP sites of idiopathic ventricular fibrillation risk genes in random individuals. Implementation

[0030] The following embodiments are further described in detail in conjunction with the accompanying drawings and examples. The following embodiments are only used to more clearly illustrate the technical solution of the present invention so that those skilled in the art can understand and utilize the present invention well, but not to limit the scope of protection of the present invention.

[0031] The experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technicians in the field can understand the conventional process steps based on the names and apply the corresponding equipment to implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0032] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art. Example

[0033] Multiplex PCR technology combined with SNaPshot microsequencing technology was used to detect 41 idiopathic ventricular fibrillation risk gene SNP sites on cardiac potassium channel encoding genes. The 41 idiopathic ventricular fibrillation-related SNP sites specifically included rs786205867, rs2125168897, rs1363307614, rs199472947, rs199472916, and rs138776 684, rs120074192, rs199472687, rs179489, rs199472708, rs120074196, rs199472743, rs397508079, rs397508087, rs199472806, rs120074189, rs121908590, rs778849288, rs 104894584, rs144022753, rs199473648, rs612790, rs2813862, rs605604, rs2798334, r s7539683, rs1805222, rs2120436, rs2034124, rs12090194, rs4839185, rs6682872, rs48 38926, rs4838927, rs1545300, rs11588747, rs2010749, rs12144965, rs3008527, rs3008528, and rs2075811, as described in the previous table, are located on genes KCND3, DES, KCNH2, KCNQ1, KCNA5, ABCC9, KCNJ2, and KCNE2, respectively.

[0034] Based on the above SNP sites, 38 pairs of specific multiplex PCR primer pairs as described in SEQ ID NOs. 1 to 76 and 41 SNP single-base extension primers as described in SEQ ID NOs. 77 to 117 were designed and placed in a kit. At the same time, necessary detection reagents such as multiplex PCR reaction solution, SNaPshot reaction solution, shrimp alkaline phosphatase and exonuclease I were prepared in the kit to construct a kit for detecting 41 idiopathic ventricular fibrillation-related SNP sites according to the specific correspondence in the previous table.

[0035] The specific testing process includes:

[0036] 1) Collect 2 mL of fresh human venous blood, use OMEGA EZNATM SE Blood DNA Kit to extract DNA samples, perform quantitative analysis of the extracted genomic DNA according to the optical density method, determine the purity and concentration of the genomic DNA, and take 10 ng of DNA extracted from the sample as a template for the following reaction;

[0037] 2) Multiplex PCR reaction:

[0038] The PCR reaction included two parallel reactions (system A and system B), both of which were 10 μl systems: 5 μl 2×Multiplex PCR Mix (DNA polymerase 1U, dNTP 0.3mM, Tris-HCl (pH 8.7) 20mM, KCl 100mM, MgCl22.5mM, GC Enhancer), 1 μl PCR primer mixture (final primer concentration 0.2-1μM), and 10 ng DNA template.

[0039] Except for the different primer mixtures, the other components of system A and system B are the same. Among them, system A contains rs120074192, rs397508087, rs138776684, rs6682872, rs144022753, rs4838926, rs120074196, rs2075811, rs612790, rs7539683, rs120074189, rs121908590, rs11588747, rs1363307614, rs2798334, rs3008527, rs199472708, rs4839185, rs179489, rs12090194, rs2010749, rs system B contains the PCR primer pairs of rs1545300, rs2034124, rs1805222, rs397508079, rs199472687, rs3008528, rs104894584, rs605604, rs199473648, rs2813862, rs199472743, rs199472947, rs778849288, rs199472916, rs2125168897, rs786205867, rs12144965, rs4838927, and rs2120436.

[0040] PCR reaction conditions: 95℃ 5min; {94℃ 15s, 62℃ 30s (*-0.5℃ Touchdown), 72℃ 30s; 18 cycles}; {95℃ 20s, 54℃ 30s, 60℃ 60s; 17 cycles}; 60℃ 5min.

[0041] 3) Purification of multiplex PCR reaction products:

[0042] The PCR products of system A and system B were purified using rSAP (shrimp alkaline phosphatase) and ExoⅠ (exonuclease Ⅰ), respectively.

[0043] The purification reaction was a 5 μl system: PCR product 3.3 μl, rSAP 0.5 μl, 10×rSAP buffer 0.5 μl, Exo I 0.2 μl, and 10×Exo I buffer 0.5 μl.

[0044] Purification conditions: 37°C for 1h, 95°C for 15min.

[0045] 4) Single base extension reaction of PCR purified product:

[0046] The PCR purified product was subjected to single base extension reaction using the SNaPshot kit. Except for the single base extension primers added, the other reaction system components and reaction conditions of system A and system B were the same.

[0047] The extension reaction was a 5 μl system: 2 μl of PCR purified product, 2.5 μl of 2×SNaPshot Reaction Mix, and 0.5 μl of single-base extension primer complex (final primer concentration was 0.02-0.8 μM).

[0048] The extension reaction conditions were: 96°C for 5 s, 50°C for 10 s, 60°C for 15 s, for 35 cycles.

[0049] 5) Purification of single base extension reaction products:

[0050] The extension products were purified using rSAP to remove excess ddNTPs in the extension products.

[0051] The purification system is a 5 μl system: 4 μl single base extension reaction product, 0.5 μl rSAP, 0.5 μl 10×rSAP buffer.

[0052] Purification conditions were: 37°C for 1 h, 95°C for 15 min.

[0053] 6) Capillary electrophoresis of the purified products of single base extension reaction and analysis of the results:

[0054] SNPs were detected using capillary electrophoresis.

[0055] The composition system and conditions of the capillary electrophoresis pre-denaturation treatment are as follows: 10 μl formamide (Hi-Di formamide), 1.5 μl purified product, and 0.1 μl internal standard (GeneScan-LIZ 120).

[0056] The pre-denaturation conditions were: 95°C for 5 min and then quickly placed at -20°C for 3 min. The single base extension reaction products were detected using the ABI® 3500 Dx Genetics Detector.

[0057] Gene Mapper software was used to analyze the SNP typing results. SNPs were analyzed and typed based on the fluorescent signals of different colors.

[0058] Figure 1 The results of 41 SNP sites of idiopathic ventricular fibrillation risk genes in random individuals are given, where each SNP corresponds to 1 to 2 fluorescence peaks. Four fluorescently labeled ddNTPs are extended to the 3' end of the single base extension reaction primer through the SNaPshot reaction, so that it has a detectable fluorescent signal: adenine A shows green, guanine G shows blue, cytosine C shows black, and thymine T shows red. CE is used to separate the SBE products to obtain the genotyping of the polymorphic sites.

[0059] The present invention has the following advantages:

[0060] 1) Accurate typing: The detection of target SNPs is more accurate than whole genome sequencing, which reduces the mismatch rate. Since the SNaPshot microsequencing technology uses purified multiplex PCR products as templates, it uses enzymes, four fluorescently labeled ddNTPs, and a single-base extension primer with the 5′ end close to the SNP site as a system for extension reaction. The single-base extension primer terminates after extending one base. After detection by capillary electrophoresis, the SNP site corresponding to the extension product is determined according to the position of the fluorescence peak, and the base type of the SNP can be known according to the color of the fluorescence peak, thereby determining the SNP typing of the sample.

[0061] 2) Compared with traditional SNP detection technology, the detection technology combining multi-site SNP compound typing and SNaPshot microsequencing is more superior. Although the typing ability of a SNaPshot reaction system is limited, this shortcoming can be compensated by establishing multiple extension systems.

[0062] 3) Compared with first-generation sequencing and second-generation sequencing, this method is not limited by laboratory equipment conditions. The instruments and equipment used in this method are commonly used in most forensic DNA laboratories and are easy to popularize.

[0063] 4) This method can detect 41 SNP polymorphisms at one time, and its detection throughput is comparable to direct sequencing. Moreover, this method is not limited to missense mutations or insertions / deletions, and any mutation can be detected without being restricted by SNP site polymorphism.

[0064] 5) It may be used to systematically detect 41 SNP sites of idiopathic ventricular fibrillation risk genes on cardiac potassium channel encoding genes, providing genetic data and theoretical basis for precision medicine, eugenics, clinical diagnosis, and prevention of sudden cardiac death.

[0065] The above embodiments of the present invention do not describe all the details in detail, nor limit the present invention to the above embodiments. Various changes, modifications, substitutions and variations made by ordinary technicians in this field without departing from the principles and purpose of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for detecting SNP sites of idiopathic ventricular fibrillation risk genes in cardiac potassium channel encoding genes, comprising 38 pairs of specific multiplex PCR primer pairs shown in SEQ ID NOs. 1 to 76, and 41 SNP single-base extension primers shown in SEQ ID NOs. 77 to 117, for correspondingly detecting the following 41 idiopathic ventricular fibrillation-related SNP sites: rs786205867, rs2125168897, rs1363307614, rs199472947, rs199472916, rs138776684, rs120074192, rs199472687, rs179489, rs1994 72708, rs120074196, rs199472743, rs397508079, rs397508087, rs199472806, rs120074189, rs121908590, rs778849288, rs104894584 , rs144022753, rs199473648, rs612790, rs2813862, rs605604, rs2798334, rs7539683, rs1805222, rs2120436, rs2034124, rs12090194 , rs4839185, rs6682872, rs4838926, rs4838927, rs1545300, rs11588747, rs2010749, rs12144965, rs3008527, rs3008528, rs2075811.

2. The kit according to claim 1, characterized in that The 41 idiopathic ventricular fibrillation-related SNP sites are located on genes KCND3, DES, KCNH2, KCNQ1, KCNA5, ABCC9, KCNJ2, and KCNE2.

3. The kit according to claim 1, characterized in that The 41 idiopathic ventricular fibrillation-related SNP sites amplified by the 38 pairs of specific multiplex PCR primers correspond to:

4. The kit according to claim 1, characterized in that The 41 SNP single base extension primers correspond to the 41 idiopathic ventricular fibrillation related SNP sites:

5. A method for detecting SNP sites of idiopathic ventricular fibrillation risk genes using the kit for detecting SNP sites of idiopathic ventricular fibrillation risk genes of cardiac potassium ion channel encoding genes according to claim 1, comprising: 1) Using the obtained DNA samples as templates, construct a multiplex PCR amplification system and purify the PCR products; 2) Using the purified PCR product as a template, construct the SNaPshot reaction system and purify the single base extension reaction product; 3) Analyze the purified single base extension reaction product using a genetic analyzer.

6. Use of the kit according to claim 1 in detecting SNP sites of idiopathic ventricular fibrillation risk genes in cardiac potassium channel encoding genes.

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