CN113088571A - SCN5A gene detection kit and detection method - Google Patents
SCN5A gene detection kit and detection method Download PDFInfo
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Abstract
The invention discloses an SCN5A gene detection kit and a detection method, wherein the detection method comprises the following steps: A. extracting DNA; B. performing quality inspection on the genome DNA; C. constructing an amplicon targeted capture library; D. performing on-machine sequencing on the prepared library; E. carrying out standard analysis on the data result; F. controlling the quality of data; G. the mutation detection result is simple to operate and low in cost, the sites such as the full coding region of the SCN5A gene, the front and back 50bp of the coding region, the chr3:38691021 and the like are covered, meanwhile, the amplification of a plurality of pairs of specific primers can be carried out, compared with the traditional one-generation sequencing technology, the complexity of operation is reduced, the same sequencing depth as that of the second-generation sequencing technology is kept, and meanwhile, the cost is reduced.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a SCN5A gene detection kit and a detection method.
Background
Sudden cardiac death refers to the natural death caused by cardiac causes characterized by sudden loss of consciousness occurring within 1 hour after the onset of acute symptoms, and accounts for the majority of sudden death, and whether the dead is born with or without cardiovascular disease. Sudden cardiac death has the characteristics of acute onset, rapid progress, high fatality rate and the like. Every year, 1700 thousands of people die globally due to cardiovascular diseases, and sudden cardiac death accounts for about 25 percent. The incidence of sudden cardiac death in women is 1.40/1000/year and in men 6.68/1000/year. About 50% of cardiac arrest occurs in individuals without known cardiac disease, but most patients have undiagnosed ischemic heart disease. It is estimated that more than 50 million people with sudden cardiac death in our country are getting younger. The vast majority of sudden cardiac deaths suffer from organic heart diseases, mainly including coronary heart disease, hypertrophic and dilated cardiomyopathies, valvular heart disease, myocarditis, non-atherosclerotic coronary artery abnormalities, invasive lesions, conduction abnormalities (QT interval prolongation syndrome, heart block), severe ventricular arrhythmias, etc. In addition, drug intoxication such as digitalis and quinidine can also be caused. Most sudden cardiac death is caused by ventricular tachyarrhythmia. Transient functional factors, such as cardiac instability, platelet aggregation, coronary spasm, myocardial ischemia, and reperfusion, destabilize the original stable cardiac structure. Sudden cardiac death may be triggered by factors such as autonomic nervous system instability, electrolyte imbalance, excessive exertion, emotional depression, and the use of ventricular arrhythmias. Home screening of first degree relatives of sudden cardiac death victims is a very important intervention that not only identifies at-risk families, advises on available treatments, but also adequately prevents sudden death. In relatives of up to 50% of victims of sudden arrhythmic syndrome, a diagnosis of inherited arrhythmogenic diseases can be made.
Long QT syndrome (LQTS) is a familial hereditary electrical activity disorder heart disease. The QT interval is the total time period from the beginning of the QRS complex to the end of the T wave on the electrocardiogram, representing the total time for ventricular depolarization and repolarization, which is a reflection of the conduction of electrical excitation in the ventricle. The clinical manifestations of LQTS are mainly repeated syncope and sudden death caused by torsade de pointes ventricular tachycardia. The symptoms of most patients occur during exercise, emotional stress, agitation, syncope generally lasts for 1-2min, and sudden death of a small percentage of patients occurs during sleep. The electrocardiogram is an important basis for diagnosing LQTS, QT intervals on the electrocardiogram of most patients are prolonged (male is more than or equal to 470ms, female is more than or equal to 480ms), but the QT intervals of a small part of patients can be normal, and the patient is called as 'normal QT interval' or 'hidden type' LQTS. LQTS is the earliest discovered ion channel disease, and has relatively more pathogenic genes and mutation sites, and relates to Na + channels, K + channels, Ca2+ channels, certain subunits and even proteins with a regulation function on the ion channels. Experts recommend the detection of genes associated with LQTS for the following cases: patients who are highly suspected of LQTS by cardiologists based on medical history, family history, and electrocardiogram phenotype; asymptomatic prolongation of the QT interval which is characteristic of this disease; patients with secondary QT interval prolonging factors were excluded.
Brugada syndrome (BrS) is a type of ion channel disease that is prone to sudden cardiac death. The electrocardiogram is characterized in that right bundle branch block, ST segment of thoracic leads from V1 to V3 is elevated, and QT interval is normal. BrS the pathogenic genes involved Na + channels, K + channels, Ca2+ channels and their regulatory subunits, most commonly Na + channels. Given that BrS can cause severe cardiac events such as sudden death, expert consensus recommends that BrS family members and their related relatives should be subjected to specific mutation detection. Patients with clinical suspicion of BrS should be tested for the SCN5A gene.
SCN5A (sodium channel, voltage-gated, type V) is the sodium channel alpha gene, which encodes sodium channel alpha and is a member of the human voltage gated sodium channel gene family. The cardiac sodium channel mediates the sodium ion inflow in the heart to form an express ascending phase of the action potential of the cardiac muscle cell, and plays an important role in excitement conduction. The gene polymorphism has negative influence on the number and the dynamic characteristics of the sodium ion channel protein, so that the internal flow of sodium ions is blocked, the formation of action potential of myocardial cells is influenced, depolarization abnormality and arrhythmia of the myocardial cells are easily caused, the occurrence of heart diseases such as atrial fibrillation, LQT and BrS is easily caused, and the risk of sudden death is increased.
Mutation of the SCN5A gene is one of the causes of sudden cardiac death, and gene diagnosis is also an important means for determining sudden cardiac death. However, the currently accepted sudden cardiac death causing gene variation only aims at the discovered hot spot mutation, and part of the variation is not used as a target for preventing and controlling or treating sudden cardiac death. (however, the mutation at any site of the gene is not related to sudden cardiac death; and the detection result of false negative caused by the fact that sudden cardiac death is inevitable by detecting the existing few gene mutation sites.) the invention firstly clarifies the feasibility of early diagnosis or prognosis prediction of sudden cardiac death by taking the whole coding region and part of the non-coding region of the human SCN5A gene as new screening targets.
Disclosure of Invention
The invention aims to provide an SCN5A gene detection kit and a detection method, which aim to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a detection method of SCN5A gene comprises the following steps:
A. extracting DNA;
B. performing quality inspection on the genome DNA;
C. constructing an amplicon targeted capture library;
D. performing on-machine sequencing on the prepared library;
E. carrying out standard analysis on the data result;
F. controlling the quality of data;
G. and (5) detecting variation.
Preferably, the specific method in step a is as follows: taking 200 microliters of a whole blood sample, extracting whole genome DNA by an extraction method, and detecting the quality and concentration of the DNA type by using Nanodrop 2000; wherein, the ratio of A260/280 is 1.8-2.0, the ratio of A260/230 is 1.7-1.9, the DNA sample is judged to be qualified, and finally the qualified DNA sample is uniformly diluted to 100 ng/microliter.
Preferably, the step B specifically comprises: the Qubit accurately quantifies the DNA concentration, the OD value is generally between 1.8 and 2.0, the total amount of DNA is more than 40ng, and DNA samples meeting the library building standard are used for subsequent library building sequencing.
Preferably, the specific method in step C is as follows: the method comprises the steps of utilizing a multiplex PCR technology and SCN5A gene specificity multiplex PCR primers to simultaneously amplify a plurality of target regions on genome DNA to obtain an amplicon, adding second-generation sequencing joints to two sides of the amplicon in a PCR mode to obtain an amplicon library, and performing second-generation sequencing to obtain sequence information of the target regions.
Preferably, in the step D, illumina nova is used for performing on-machine sequencing, PE150 is used, and the sample size of each sample is not less than 1G.
Preferably, in the step G, based on the bam result of the alignment with the genome reference sequence, samtools and GATK software are used to search for the SNP and the InDel, and then ANNOVAR software is used to annotate the SNP and the InDel sites to determine the gene information, the functional information and the harmfulness corresponding to the mutation sites.
Preferably, the operation is performed according to a library construction process, which comprises the following steps:
1) taking 1 μ l of library, quantifying by using a Qubit dsDNA HS Assay Kit, and recording the concentration of the library, wherein the concentration of the library is about 10-50ng/μ l;
2) a1. mu.l sample was taken for library fragment length determination using the Agilent 2100Bioanalyzer system (Agilent DNA1000Kit), the library length being approximately between 300-450 bp.
Preferably, a small amount of reads containing linker information, low-quality bases or undetected bases exist in the original data obtained by sequencing in step F, Raw reads need to be preliminarily filtered to obtain Clean reads in order to ensure the quality of information analysis, and subsequent analysis is performed based on the Clean reads; the content of data filtering is mainly as follows:
(1) cutting off sequences with the average base quality value less than 20 in an 8bp sliding window mode;
(2) removing the linker sequence at the tail of the sequence;
(3) if the first base or the tail base of the sequence is less than 20, the base is directly sheared off;
(4) and (4) downloading normally, and discarding the pair of sequences if the remaining sequence length is less than 40 after the removal.
Preferably, the kit for detecting the SCN5A gene comprises any one of the detection methods.
Compared with the prior art, the invention has the beneficial effects that: the method is simple to operate and low in cost, covers sites such as the whole coding region of the SCN5A gene, 50bp around the coding region, chr3:38691021 and the like, can amplify multiple pairs of specific primers, reduces the complexity of operation compared with the traditional one-generation sequencing technology, and reduces the cost while keeping the same sequencing depth as the second-generation sequencing technology.
Drawings
FIG. 1 is a flow chart of a standard assay of the present invention;
FIG. 2 is a flow chart of library construction according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front", "rear", "both ends", "one end", "the other end", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it is to be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "disposed," "connected," and the like are to be construed broadly, such as "connected," which may be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
Referring to fig. 1-2, the present invention provides a technical solution: a detection method of SCN5A gene comprises the following steps:
A. extracting DNA;
B. performing quality inspection on the genome DNA;
C. constructing an amplicon targeted capture library;
D. performing on-machine sequencing on the prepared library;
E. carrying out standard analysis on the data result;
F. controlling the quality of data;
G. and (5) detecting variation.
In the invention, the specific method in the step A is as follows: taking 200 microliters of a whole blood sample, extracting whole genome DNA by an extraction method, and detecting the quality and concentration of the DNA type by using Nanodrop 2000; wherein, the ratio of A260/280 is 1.8-2.0, the ratio of A260/230 is 1.7-1.9, the DNA sample is judged to be qualified, and finally the qualified DNA sample is uniformly diluted to 100 ng/microliter.
In the present invention, the step B specifically includes: the Qubit accurately quantifies the DNA concentration, the OD value is generally between 1.8 and 2.0, the total amount of DNA is more than 40ng, and DNA samples meeting the library building standard are used for subsequent library building sequencing.
In the invention, the specific method in the step C is as follows: the method comprises the steps of utilizing a multiplex PCR technology and SCN5A gene specificity multiplex PCR primers to simultaneously amplify a plurality of target regions on genome DNA to obtain an amplicon, adding second-generation sequencing joints to two sides of the amplicon in a PCR mode to obtain an amplicon library, and performing second-generation sequencing to obtain sequence information of the target regions.
In the invention, in the step D, illumina nova is adopted for upper machine sequencing, PE150 is adopted, and the sample data size is not less than 1G.
In the step G, on the basis of a bam result compared with a genome reference sequence, adopting samtools and GATK software to search SNP and InDel, and then adopting ANNOVAR software to annotate the SNP and InDel sites to determine the gene information, the functional information and the harmfulness corresponding to the mutation sites.
In the invention, the operation is carried out according to a library construction process, and the specific process is as follows:
1) taking 1 μ l of library, quantifying by using a Qubit dsDNA HS Assay Kit, and recording the concentration of the library, wherein the concentration of the library is about 10-50ng/μ l;
2) a1. mu.l sample was taken for library fragment length determination using the Agilent 2100Bioanalyzer system (Agilent DNA1000Kit), the library length being approximately between 300-450 bp.
In the invention, a small amount of reads containing joint information, low-quality bases or undetected bases exist in the original data obtained by sequencing in the step F, Raw reads need to be preliminarily filtered to obtain Clean reads in order to ensure the information analysis quality, and subsequent analysis is carried out on the basis of the Clean reads; the content of data filtering is mainly as follows:
(1) cutting off sequences with the average base quality value less than 20 in an 8bp sliding window mode;
(2) removing the linker sequence at the tail of the sequence;
(3) if the first base or the tail base of the sequence is less than 20, the base is directly sheared off;
(4) and (4) downloading normally, and discarding the pair of sequences if the remaining sequence length is less than 40 after the removal.
Wherein the SCN5A gene-specific multiplex PCR primers are from the following tables 1 and 2
TABLE 1
TABLE 2
| Other sites of interest | ForwardPrimer(Fp) | ReversePrimer(Rp) |
| chr3:38691021 | CCTCGGGGAGGAAAGTTgg | GTAGGATGCAGGGATCGCTC |
In conclusion, the method is simple to operate and low in cost, covers sites such as the full coding region of the SCN5A gene, 50bp around the coding region, chr3:38691021 and the like, can amplify multiple pairs of specific primers, reduces the complexity of operation compared with the traditional one-generation sequencing technology, and reduces the cost while keeping the same sequencing depth as the second-generation sequencing technology.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (9)
1. A method for detecting SCN5A gene is characterized in that: the detection method comprises the following steps:
A. extracting DNA;
B. performing quality inspection on the genome DNA;
C. constructing an amplicon targeted capture library;
D. performing on-machine sequencing on the prepared library;
E. carrying out standard analysis on the data result;
F. controlling the quality of data;
G. and (5) detecting variation.
2. The method for detecting SCN5A gene according to claim 1, wherein: the specific method in the step A is as follows: taking 200 microliters of a whole blood sample, extracting whole genome DNA by an extraction method, and detecting the quality and concentration of the DNA type by using Nanodrop 2000; wherein, the ratio of A260/280 is 1.8-2.0, the ratio of A260/230 is 1.7-1.9, the DNA sample is judged to be qualified, and finally the qualified DNA sample is uniformly diluted to 100 ng/microliter.
3. The method for detecting SCN5A gene according to claim 1, wherein: the step B specifically comprises the following steps: the Qubit accurately quantifies the DNA concentration, the OD value is generally between 1.8 and 2.0, the total amount of DNA is more than 40ng, and DNA samples meeting the library building standard are used for subsequent library building sequencing.
4. The method for detecting SCN5A gene according to claim 1, wherein: the specific method in the step C is as follows: the method comprises the steps of utilizing a multiplex PCR technology and SCN5A gene specificity multiplex PCR primers to simultaneously amplify a plurality of target regions on genome DNA to obtain an amplicon, adding second-generation sequencing joints to two sides of the amplicon in a PCR mode to obtain an amplicon library, and performing second-generation sequencing to obtain sequence information of the target regions.
5. The method for detecting SCN5A gene according to claim 1, wherein: and D, performing upper machine sequencing by using illumina nova, wherein the sample data size is not less than 1G by using PE 150.
6. The method for detecting SCN5A gene according to claim 1, wherein: and G, searching SNP and InDel by adopting samtools and GATK software based on the bam result compared with the genome reference sequence, annotating SNP and InDel sites by using ANNOVAR software, and determining the gene information, functional information and harmfulness corresponding to the mutation sites.
7. The method for detecting SCN5A gene according to claim 4, wherein: the operation is carried out according to a library construction process, and the specific process is as follows:
1) taking 1 μ l of library, quantifying by using a Qubit dsDNA HS Assay Kit, and recording the concentration of the library, wherein the concentration of the library is about 10-50ng/μ l;
2) a1. mu.l sample was taken for library fragment length determination using the Agilent 2100Bioanalyzer system (Agilent DNA1000Kit), the library length being approximately between 300-450 bp.
8. The method for detecting SCN5A gene according to claim 1, wherein: in the original data obtained by sequencing in the step F, a small number of reads containing joint information, low-quality bases or undetected bases exist, Raw reads need to be preliminarily filtered to obtain Clean reads in order to ensure the information analysis quality, and subsequent analysis is carried out on the basis of the Clean reads; the content of data filtering is mainly as follows:
(1) cutting off sequences with the average base quality value less than 20 in an 8bp sliding window mode;
(2) removing the linker sequence at the tail of the sequence;
(3) if the first base or the tail base of the sequence is less than 20, the base is directly sheared off;
(4) and (4) downloading normally, and discarding the pair of sequences if the remaining sequence length is less than 40 after the removal.
9. An SCN5A gene detection kit, which is characterized in that: the gene detection kit comprises the detection method according to any one of claims 1 to 8.
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