CN106868126A

CN106868126A - Fluorescent quantificationally PCR detecting kit and detection method

Info

Publication number: CN106868126A
Application number: CN201710089443.6A
Authority: CN
Inventors: 吴少鸿; 肖晶晶; 周文根; 姜萍萍; 李欣
Original assignee: Shenzhen United States Clinical Laboratory Co Ltd
Current assignee: Shenzhen United States Clinical Laboratory Co Ltd
Priority date: 2017-02-20
Filing date: 2017-02-20
Publication date: 2017-06-20
Anticipated expiration: 2037-02-20
Also published as: CN106868126B; WO2018149264A1

Abstract

A kind of fluorescent quantificationally PCR detecting kit, the kit includes fluorescence quantitative PCR detection portion and result treatment evaluation section, and wherein result treatment evaluation section includes the corresponding testing result processing system in site and reference database；The site for having expanded is detected by melting curve peak type figure, obtain the testing result in correspondence site, result treatment is carried out to testing result, the value-at-risk of the testing gene will be drawn after the risk plot point statistics in each site, and the risk class in correspondence database, comprehensively obtain gene risk and associated processing outcomes.The present invention is by the Multiple detection kit using quantitative fluorescent PCR, the testing result of several genes can be estimated, based on the accurate result reference database of science, can obtain very accurate and personalized and strong, result and the suggestion of personalization can be given to detection sample, the purpose of more efficiently genetic test is reached, has been used and promotional value with more extensive.

Description

Fluorescent quantificationally PCR detecting kit and detection method

Technical field

The present invention relates to field of biological detection, more particularly to detecting and evaluating genes system, specifically a kind of fluorescence is determined Amount PCR detection kit and detection method.

Background technology

With continuing to develop for scientific research, after the completion of the Human Genome Project, post genome project is in full swing, with Genetic engineering is that leading biotechnology is widely paid close attention in the application of sports problem domain.Use science of heredity at present Theoretical and method carry out motion selection and had preliminary progress.Conventional gene tester has：Direct Sequencing (direct Sequencing, DS), Ligase detection reaction (ligase detection reaction, LDR), Restriction Fragment Length it is many State property analysis (restriction fragment length polymorphism, RFLP), dhplc analysis (denaturing high performance liquid chromotography, DHPLC), quantitative PCR, wherein using DNA Analyzer direct Sequencing is goldstandard, but the above method is respectively present that high cost, accuracy rate be low, cumbersome and repeatability The problems such as difference.Real time fluorescent quantitative nucleic acid amplification detecting system (Real-time Quantitative PCR Detecting System, qPCR), real-time quantitative gene magnification fluorescence detecting system is also, quantitative fluorescent PCR (realtime PCR) detection exists Fluorophor is added in PCR reaction systems, the whole PCR processes of real-time monitoring are accumulated using fluorescence signal, it is bent finally by standard The method that line carries out quantitative analysis to unknown template, qPCR systems are the PCR detection techniques of three generations, and qPCR has detection sensitivity Height, detects linear wide ranges, accuracy of detection and it is reproducible wait outstanding advantage, therefore be acknowledged as the world today be used for it is clinical Most advanced nucleic acid molecules diagnostic techniques.Therefore the technology can efficiently, accurately be used to move genetic test.

SNP (Single Nucleotide Polymorphisms, SNP) refers to single on genome The variation of nucleotides, including conversion, transversion, missing and insertion, the genetic marker of formation, its quantity are a lot, rich polymorphism. SNP is third generation genetic marker, many phenotypic differences of human body, all may be relevant with SNP to neurological susceptibility of medicine or disease etc., SNP researchs are the important steps that the Human Genome Project moves towards application.This is primarily due to SNP and will provide a strong work Tool, the identification of discovery, disease related gene, the design of medicine and test and the basic research of biology for high risk group Deng.SNP is distributed quite extensively in genome, and research shows that every 300 base-pair is occurred as soon as once in human genome.Largely The SNP site of presence, makes people have an opportunity to find and various diseases, including the related genome mutation of tumour；From experimental implementation From the point of view of, by SNP find disease-correlative gene mutation than by family come it is easy；Some SNP do not directly result in disease The expression of gene, but because it is adjacent with some disease genes, and turn into important mark.SNP is also played in basic research Huge effect, by the analysis to Y chromosome SNP so that in human evolution, the evolution of human population and migrate field and take Obtained a series of important achievements.SNP is widely present in human genome, just has 1 in average every 500~1000 base-pairs It is individual, estimate that its sum is even more more up to 3,000,000.

Existing application angle of the increasing document to fluorescent quantitative PCR technique from every side is illustrated, most of Document discloses the concrete mode method that various single fragments are detected by fluorescent quantitative PCR technique, but enters in inventor During row fluorescence quantitative PCR detection SNP site, it is found that the detection of single fragment is insufficient for detection and requires, for tool There is the comprehensive detection of two or more SNP sites, it is necessary to duplicate detection, the complex steps and operating time is long.

Sudden cardiac death refer to acute symptom breaking-out after in 1 hour occur with realize suddenly lose be characterized by heart The natural death that reason causes.Sudden cardiac death person is most to suffer from organic heart disease, mainly including coronary heart disease, hypertrophic With dilated cardiomyopathy, valvulopathy, myocarditis, non-atherosclerotic coronary artery exception, wellability lesion, conduction abnormalities (QT interval prolongations syndrome, cardiac block) and serious ventricular arrhythmia etc., sudden death correlated inheritance arrhythmia cordis is in breaking-out Before may be asymptomatic, may be fatal if breaking-out.Caused by most of sudden cardiac deaths are then ventricular tachyarrhythmia.One A little temporary transient functional reparations, such as electrocardio are unstable, fill again after platelet aggregation, coronarospasm, myocardial ischemia and ischemic Note etc. makes the cardiac structure of original stabilization that unstable situation to occur extremely.Some factors such as autonomic nerves system is unstable, electrolysis Matter imbalance, overworked, mood are constrained and with medicine for causing VA etc., all can trigger sudden cardiac death.It is existing The research majority of sudden cardiac death gene is the research according to site on ion channel related gene, and these sites are based on Europe The strong association site that crowd's large sample amount GWAS researchs draw, is being applied to asian population, and especially during population of China, site is closed Connection property is weaker, it is difficult to obtain accurate result by detection, and the assessment system for hence setting up Chinese population is that extremely have must Want.

The content of the invention

For this reason, it may be necessary to provide a kind of SNP site to two and above SNP site or two and above gene detect Fluorescent quantificationally PCR detecting kit and detection method.

To achieve the above object, a kind of fluorescent quantificationally PCR detecting kit is inventor provided, the kit includes glimmering Fluorescent Quantitative PCR test section and result treatment evaluation section, wherein result treatment evaluation section include the corresponding testing result treatment in site System and reference database；Reagent, consumption and augmentation detection mode involved by fluorescence quantitative PCR detection portion etc. refer to public In the prior art opened, will not be described here.

Preferably, detection kit includes fluorescence quantitative PCR detection and the result treatment evaluation at least two sites；Also It is to say, the detection kit is multiple fluorescence quantitative PCR detection kit, and multiple sites are not limited to same DNA fragmentation.

Preferably, reference database is made with the East Asia crowd's genomic data in thousand human genomes in result treatment evaluation section It is general population's level, and by the real-time income data storehouse of testing result；Include according to testing gene site in reference database Allelotype value-at-risk and risk rating, the frequency of occurrences of the gene, gene risk and other statistics, and related Proposed projects etc..

Preferably, the genotype and correspondence risk plot point in each site are obtained according to testing result, is calculated and is obtained testing gene Value-at-risk P, and according to average risk value Pa, average variance S, following five kinds of results are obtained accordingly：

P_a+ 0.6S≤P is excessive risk rank；P_a+ 0.2S≤P ＜ P_a+ 0.6S is slightly higher risk class；P_a- 0.2S≤P ＜ P_a + 0.2S is average risk rank；P_a- 0.6S≤P ＜ P_a- 0.2S is lower slightly risk class；P ＜ P_a- 0.6S is low-risk rank.

It is highly preferred that the value-at-risk of testing gene is the product of multiple detection gene locis, that is to say, that P=S1*S2.

As one kind preferred embodiment, the PCR kit for fluorescence quantitative in the present invention is that sudden cardiac death site primer is tried Agent box, the kit detects two SNP sites, and detection site is rs4665058 and rs2824292 sites, rs4665058 sites AA-AC-CC genotype OR values be respectively 3.68-1.98-1；The OR values of the GG-GA-AA genotype in rs2824292 sites point Wei not 3.16-1.78-1；P value >=2.33 are excessive risk rank, and 2.13≤P values ＜ 2.33 is higher risk, 1.93≤P values ＜ 2.13 is average risk, and 1.72≤P values ＜ 1.93 is relatively low risk, and P values ＜ 1.72 is low-risk.

Another object of the present invention is to provide a kind of fluorescent quantitation according to above-mentioned fluorescent quantificationally PCR detecting kit PCR detection method, wherein, detection method is specifically included：

A. the amplification of sample to be tested, using single fluorescence labeling probe, designs the primer and probe sequence of sample to be tested, carries out PCR is expanded；

B. pattern detection, the site for having expanded is detected by melting curve peak type figure, obtains the testing result in correspondence site；

C. result treatment is carried out according to testing result, the wind of the testing gene will be drawn after the risk plot point statistics in each site Danger value, and the risk class in correspondence database；

D. according to the corresponding level of testing result, gene risk and associated processing outcomes are comprehensively obtained.

Preferably, when the testing result of sample carries out result treatment, specific method is as follows：

The genotype and correspondence risk plot point in each site are obtained according to testing result, the value-at-risk for obtaining testing gene is calculated P, and according to average risk value Pa, average variance S, following five kinds of results are obtained accordingly：

It is highly preferred that the calculation of average variance S is as follows：

Wherein i is the detection site quantity of testing gene.

Preferably, the genomic data that average risk value passes through East Asia crowd, wherein crowd's total number of persons are N, the calculating of Pa Formula is：

Prior art is different from, above-mentioned technical proposal, can be to more by the Multiple detection kit using quantitative fluorescent PCR The testing result for planting gene is estimated, and based on the accurate result reference database of science, can obtain very accurate and individual Property and it is strong, result and the suggestion of personalization can be given to detection sample, reached the purpose of more efficiently genetic test, have There are more extensive use and promotional value.

Detection kit of the invention and detection method high specificity, recall rate are high, and efficiency high, low cost are such as applied Whether gene diagnosis field, can be carried testing gene, and assess the wind of the testing gene with analysis with comprehensive detection by inspection crowd Danger, Screening Samples scope, and follow-up guiding opinion is carried out to sample, play more deep directive function.

Brief description of the drawings

Fig. 1 is the detection sample experiments result described in specific embodiment；

Fig. 2 is the detection sample sequencing result described in specific embodiment；

Fig. 3 is the detection sample experiments result described in specific embodiment；

Fig. 4 is the detection sample sequencing result described in specific embodiment；

Fig. 5 is the flow chart being estimated according to detection sample experiments result.

Specific embodiment

To describe technology contents, structural feature, the objects and the effects of technical scheme in detail, below in conjunction with specific reality Apply example and coordinate accompanying drawing to be explained in detail.Experiment reagent is used in following examples unless otherwise specified, can be by commercial means Obtain.

Fig. 1~4 are referred to, sudden cardiac death dependency basis is carried out using fluorescent quantificationally PCR detecting kit in the present embodiment The detection of cause, wherein on two genes related to sudden cardiac death two pleomorphism sites (rs4665058 and Rs2824292) detected, using the primer and probe of specific designs, optimized expansion system and condition are same in same system When complete two genes on two detections of pleomorphism site.

Preferably, using single fluorescence labeling probe, Roche in the present embodiment480 platforms, finally by Melting curve peak type figure detection rs4665058 and rs2824292 sites.

The double fluorescent quantitative PCR detection method in specific rs4665058 and rs2824292 sites is specific as follows：

Primer and the probe design in 1.rs4665058 and rs2824292 sites are as shown in table 1 below：

The primer of table 1 and probe

2. preparation of reagents

3.PCR response procedures

Treating extension increasing sequence carries out pcr amplification reaction, and specific course of reaction (circulation) arrange parameter is as follows：

4. pair amplification detects that as shown in figures 1-4, detection sample rs4665058 experimental results are testing result G, detects sample rs2824292 experimental result AG, is rechecked by sequencing, it was demonstrated that this experimental technique has accuracy and reliability Property.

The article that above-mentioned two site is delivered respectively from Bezzina etc. and doctor Ar-king in the present embodiment, article leads to Cross GWAS researchs and drawn two susceptibility locis directly related with sudden cardiac death, and calculate two risks in site etc. Position genotype value-at-risk, i.e. OR values.GWAS researchs find that the common genetic variation on CXADR and BAZ2B genes may be with ventricle Vibration/SCD occurs relevant.After Bezzina etc. has found that the SNP site (rs2824292) on CXADR genes is acute myocardial infarction AMI The hazards that ventricular fibrillation occurs.Rs2824292 can reduce the Transcript abundance of CXADR, carry the cardiac muscle stalk of the variation Dead animal model shows serious cardiac conduction defects and arrhythmia cordis neurological susceptibility.To inquire between hereditary variation and heart arrest Relation, doctor Ar-king compares discovery, BAZ2B the gene of 4402 sudden cardiac arrest patients and 30,000 normal persons Gene morph (rs4665058) when, the probability of happening of heart arrest significantly increases, and is often lacking any omen In the case of occur, case fatality rate is up to 95%.And there are some researches show, with the disease of CXADR gene-correlations for myocarditis with expand Extensional cardiomyopathy, and with the effect of ventricular heart impulsion, though BAZ2B genes are studied without correlation function, its table in heart Reach.

Assess above-mentioned 2 sites, different genotype and its value-at-risk（OR）For：BAZ2B genes rs4665058, AA-AC-CC base Because the OR values of type are respectively 3.68-1.98-1；The OR values of CXADR genes rs2824292, GG-GA-AA genotype are respectively 3.16-1.78-1.Because the incidence of disease of sudden cardiac death is less than 10%, so OR values are similar to RR values, i.e. value-at-risk.Each position Point value-at-risk then calculated according to genotype, i.e., the value-at-risk of homozygosis risk allele type for OR values square, heterozygosis wind The value-at-risk of dangerous allelotype is OR values, and homozygosis non-risk allele type is 1, for a person under inspection, each SNP site According to the genotype that detection is obtained, there is an easy inductance value, we define this score value for S, then the individual popular feeling source of person under inspection Property the easy inductance value of sudden death be P, P values are two OR value products of genotype：

P=S1*S2.

According to genetic test result, personal sudden cardiac death risk is estimated, and set up the heart source of Chinese population Property the susceptible database of sudden death.

It is general population's level with the genomic data of East Asia crowd in thousand human genomes, total number of persons is defined as N.With thousand people The genotypic mean of East Asia crowd sets up the average mark for being set to crowd separately in genome：

And calculate average variance,

According to Pa and S, the sudden cardiac death value-at-risk of East Asia crowd is classified, by the personal cardiogenic sudden of person under inspection Dead value-at-risk P is corresponded in crowd's classification, the common Pyatyi of crowd's classification, respectively high-slightly higher-general-slightly low-low five risks Rank.

Pa+0.6S≤P is excessive risk rank；Pa+0.2S≤P ＜ Pa+0.6S are slightly higher risk class；Pa-0.2S≤P ＜ Pa+0.2SC is average risk rank；Pa-0.6S≤P ＜ Pa-0.2S are lower slightly risk class；P ＜ Pa-0.6S are low-risk level Not.Based on the sudden cardiac death risk class that East Asia crowd divides, P value >=2.33 are excessive risk rank, 2.13≤P values ＜ 2.33 It is higher risk, 1.93≤P values ＜ 2.13 is average risk, and 1.72≤P values ＜ 1.93 is relatively low risk, and P values ＜ 1.72 is low Risk.

As shown in figure 5, according to genetic test result, two OR values are multiplied and draw the easy inductance value of personal sudden cardiac death, Namely value-at-risk.The sudden cardiac death risk class that individual is obtained by stage division is corresponded to, the P values correspondence according to person under inspection Rank, the risk of the sudden cardiac death of person under inspection is comprehensively obtained, so as to provide individual health Managed Solution.

The present embodiment calculates obtaining for sudden cardiac death risk according to each site to the value-at-risk of sudden cardiac death risk Point, and gone to carry out crowd's classification according to existing database, the sudden cardiac death risk class of individual is obtained by personal score, So as to assess the sudden death risk of person under inspection, guiding excessive risk person makes the life better mode, it is to avoid risk factor, increases physical examination frequency. At the same time two Chinese population databases of gene polymorphism sites are set up, to set up the distinctive sudden cardiac death of Chinese population Assessment lays the foundation.

The present invention can be estimated to the sudden cardiac death relevant risk of person under inspection.The detection kit that the present invention is provided High specificity, recall rate are high, efficiency high, low cost, can comprehensive detection with analysis whether carried " sudden cardiac death by inspection crowd Tumor susceptibility gene ", and its risk for suffering from sudden cardiac death is assessed, Susceptible population is screened from general population, give individual character Change guiding opinion, change bad habits and customs, reach the purpose of prevention.

Although being described to the various embodiments described above, those skilled in the art once know basic wound The property made concept, then can make other change and modification to these embodiments, so embodiments of the invention are the foregoing is only, Not thereby scope of patent protection of the invention, the equivalent structure that every utilization description of the invention and accompanying drawing content are made are limited Or equivalent flow conversion, or other related technical fields are directly or indirectly used in, similarly it is included in patent of the invention Within protection domain.

SEQUENCE LISTING

<110>Shenzhen U.S. is because of Co., Ltd of clinical examination institute

<120>Fluorescent quantificationally PCR detecting kit and detection method

<130> 2017

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 29

<212> DNA

<213> Unknown

<220>

<223>Rs4665058 F ends primer

<400> 1

ctactttaaa gttaggtatt tatacgctc 29

<210> 2

<211> 26

<212> DNA

<213> Unknown

<220>

<223>Rs4665058 R ends primer

<400> 2

ccagactaag atataatgtt gcattg 26

<210> 3

<211> 27

<212> DNA

<213> Unknown

<220>

<223>Rs4665058 probe sequences

<400> 3

caaaatagct tcactgttcc aacttac 27

<210> 4

<211> 35

<212> DNA

<213> Unknown

<220>

<223>Rs2824292 F ends primer

<400> 4

caggccatct agaagtcctt acagg 35

<210> 5

<211> 20

<212> DNA

<213> Unknown

<220>

<223>Rs2824292 R ends primer

<400> 5

gtctgtgcac ctgtcccttg 20

<210> 6

<211> 27

<212> DNA

<213> Unknown

<220>

<223>Rs2824292 probe sequences

<400> 6

ccttacaggc tcttatagca ggcaagg 27

Claims

1. a kind of fluorescent quantificationally PCR detecting kit, it is characterised in that the kit include fluorescence quantitative PCR detection portion and Result treatment evaluation section, wherein result treatment evaluation section include the corresponding testing result processing system in site and reference database.

2. fluorescent quantificationally PCR detecting kit according to claim 1, it is characterised in that detection kit is included at least The fluorescence quantitative PCR detection in two sites and result treatment evaluation.

3. fluorescent quantificationally PCR detecting kit according to claim 1, it is characterised in that reference database is with thousand people's bases Because the East Asia crowd's genomic data in group is used as general population's level, and by the real-time income data storehouse of testing result.

4. fluorescent quantificationally PCR detecting kit according to claim 1, it is characterised in that testing result processing system will In testing result correspondence to the risk class of testing gene, wherein the value-at-risk of testing gene is P, and Pa is average risk value, and S is Average variance, obtains following five kinds of results accordingly：

P_a+ 0.6S≤P is excessive risk rank；P_a+ 0.2S≤P ＜ P_a+ 0.6S is slightly higher risk class；P_a- 0.2S≤P ＜ P_a+ 0.2S is average risk rank；P_a- 0.6S≤P ＜ P_a- 0.2S is lower slightly risk class；P ＜ P_a- 0.6S is low-risk rank.

5. fluorescent quantificationally PCR detecting kit according to claim 4, it is characterised in that the value-at-risk of testing gene is The product of multiple detection gene loci.

6. fluorescent quantificationally PCR detecting kit according to claim 1, it is characterised in that the kit is cardiogenic Sudden death site primer kit, detection site is rs4665058 and rs2824292 sites.

7. the quantitative fluorescent PCR of a kind of fluorescent quantificationally PCR detecting kit using according to any one of claim 1~6 Detection method, it is characterised in that detection method is specifically included：

A. the amplification of sample to be tested, using single fluorescence labeling probe, designs the primer and probe sequence of sample to be tested, enters performing PCR Amplification；

C. result treatment is carried out according to testing result, the risk of the testing gene will be drawn after the risk plot point statistics in each site Value, and the risk class in correspondence database；

8. fluorescent quantitative PCR detection method according to claim 7, it is characterised in that the testing result of sample is tied When fruit is processed, specific method is as follows：

The genotype and correspondence risk plot point in each site are obtained according to testing result, the value-at-risk P for obtaining testing gene is calculated, and According to average risk value Pa, average variance S, following five kinds of results are obtained accordingly：

9. fluorescent quantitative PCR detection method according to claim 8, it is characterised in that the calculation of average variance S is such as Under：

S = \sqrt{\frac{Σ_{i = 1}^{n} {(P_{i} - P_{a})}^{2}}{n}}

Wherein i is the detection site quantity of testing gene.

10. fluorescent quantitative PCR detection method according to claim 8, it is characterised in that average risk value passes through gook The genomic data of group, wherein crowd's total number of persons are N, and the computing formula of Pa is：

P_{a} = (Σ_{1}^{N} P_{N}) / N .