CN103233068B

CN103233068B - Primer system for detecting genetic polymorphic sites related to human cytochrome P450 and application of primer system

Info

Publication number: CN103233068B
Application number: CN201310137910.XA
Authority: CN
Inventors: 马庆伟; 赵洪斌; 张海燕; 赵艳梅
Original assignee: Beijing Yixin Bochuang Biological Technology Co Ltd
Current assignee: Zhejiang Rubidium Easy Pharmaceutical Technology Co ltd; Beijing Clin Bochuang Biotechnology Co Ltd
Priority date: 2013-04-19
Filing date: 2013-04-19
Publication date: 2015-04-29
Anticipated expiration: 2033-04-19
Also published as: CN103233068A

Abstract

The invention discloses a primer system for detecting genetic polymorphic sites related to human cytochrome P450. Based on a product prepared by adopting the primer system, seven genetic polymorphic sites related to the human cytochrome P450 can be simultaneously detected. By using the product, through detecting the genetic polymorphic sites related to the human cytochrome P450, clinical medication scheme can be guided and regulated, the basis is provided for clinical personalized medicine, and adverse medicine effects are prevented. The primer system is capable of simultaneously detecting the seven genetic polymorphic sites on different genes in a reaction system, and has the advantages of being lower in cost and more convenient to operate, and increasing the accuracy and the sensitivity in comparison with technologies of sequencing and real-time fluorescence quantification PCR (Polymerase Chain Reaction).

Description

For detecting the primer system in the gene polymorphic site that human-cytochrome P450 is correlated with and uses thereof

Technical field

The invention belongs to biological technical field, relate to a kind of detection method for determining the gene polymorphism sites (SNP) that human-cytochrome P450 is relevant and product, specifically utilize multiple PCR technique, single-basic extension technology and mass-spectrometric technique, the method that 7 gene polymorphic sites of being correlated with to human-cytochrome P450 are detected and corresponding test kit.

Background technology

Cytochrome P 450 enzymes (cytochrome P450, CYP), also known as mixed-function oxidase (mixed function oxidase) and monooxygenase (monooxygenase), mainly be present in liver microsomes, in human body, its hetero-organization such as: brain, lung, kidney, skin, placenta, gi tract, mammary gland etc. also can produce.Cytochrome P450 plays a very important role in the metabolism of exogenous material (comprising medicine and poisonous substance), its activity determines the metabolic rate of medicine, there is direct relation with the clearance rate of medicine, be the phasel enzyme of drug metabolism, be thus also called drug metabolism enzyme.The gene of P450 enzyme system mainly contains CYP1, CYP2, CYP3 tri-families, and relevant has following several important P450 enzyme: CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5.

At present, puzzlement clinical treatment and the modal problem of pharmaceutical industry are that different patient is different to the reaction of same medicine, show as different curative effect of medication and toxic side effect.World Health Organization adds up: in global Died Patients, 1/3rd is die from non-rational use of drug, and the data at China's health ministry adverse drug reaction supervision center are: in inpatient, about have 200,000 people to die from adverse drug reaction every year.

The medicine of CYP family participation metabolism accounts on market sells more than 80% of medicine, once the function of enzyme changes, can affect metabolism and the curative effect of medicine.Adverse drug reaction research in, the medicine selected by about 48% by P450 metabolism, P450 enzyme gene polynorphisms and a large amount of adverse drug reactions closely related.

CYP450 genetic polymorphism may cause CYP450 enzyme disappearance, expression amount reduces or increases, and substrate specificity sexually revises, thus causes the change of pharmacokinetics.The genetic polymorphism of CYP450 causes Different Individual to produce different pharmaceutical response to same medicine, wherein CYP2C9, CYP2C19, CYP2D6 and CYP1A2 have obvious genetic polymorphism, play a significant role in drug metabolism processes, relate to the multiple medications such as gastrointestinal disorder medicine, cardiovascular agent, anodyne, muscle and bone medication, ophthalmological.

CYP450 gene pleiomorphism causes the change of enzymes metabolism function, and this change exists the difference between significant individual patients.Medication crowd can be divided into four kinds of metabolic phenotypes: slow inactivation (PM), intermediary metabolism type (IM), fast metabolic pattern (EM) and ultra-rapid metabolism type (UM).Normal wild type shows as fast metabolic pattern: the ability of liver metabolism medicine is normal, and the speed of drug metabolism is normal; Most saltant type is slow inactivation: excessively slow to drug metabolic rate, causes medicine Plasma Concentration too high, maximum to the toxicity of liver, causes adverse drug reaction.Intermediary metabolism type: the ability of liver metabolism medicine is weak, the speed of drug metabolism is slow, and the toxicity of medicine to liver is large; Only a few saltant type Yin Jiyin multiple copied repeats the overexpression causing enzyme, is ultra-rapid metabolism type: the ability of liver metabolism medicine is comparatively strong, and the speed of drug metabolism is very fast, causes medication effect poor.CYP450 genetic polymorphism is the one of the main reasons producing poisonous side effect of medicine, reduction or lose curative effect of medication.

Can the guidance of clinical application scheme and adjustment be carried out by CYP450 genotype detection, for clinical individual medication provides foundation, prophylactic agent untoward reaction etc.

CYP2C19 gene extron 5 the 681st bit base G>A suddenlys change (rs4244285), transcription Exon 5 ' is caused to hold 40bp(643-682bp) disappearance, 215-227 amino acids is lost in translation process, and there is frameshit from 215 amino acids, in its downstream, the 20th amino acid place produces abnormal end password, albumen synthesis premature end, synthesis, only containing 234 amino acid whose short albumen, is the nonfunctional albumen of disappearance haem bonding pad.The PM phenotype individuals of 73-83% carries rs4244285 sudden change.

CYP2C19 gene extron 4 the 636th bit base G>A suddenlys change (rs4986893), and the codon mutation of codes for amino acid tryptophan is terminator codon, causes albumen to synthesize premature termination, forms the nonfunctional albumen without protoheme and Binding Capacity district.In Chinese population, the occurrence frequency of rs4986893 sudden change is the 1/9-1/10 of rs4244285 sudden change, and these two kinds sudden changes almost can explain all PM phenotypes.

CYP2C9 gene has genetic polymorphism, wherein modal is CYP2C9*2(rs1799853), CYP2C9*3(rs1057910) etc. gene polymorphic site, after these site mutations, the activity of gene encoding enzyme comparatively wild-type significantly reduces, patient is caused significantly to reduce the metabolism of medicine and Scavenging activity, clinical manifestation goes out the susceptibility to medicine, needs decrement administration to reduce the generation of untoward reaction.

In sum, gene polymorphic site causes Different Individual to produce one of factor of different response to same medicine, and the genetic polymorphism detection of medication advance line correlation, in conjunction with clinical factor, contributes to for patient education's medication, reduces the incidence of serious adverse reaction.In detection technique conventional at present, order-checking need detect one by one to multiple site, complicated operation, costly.Though chip method can detect multiple site, operating process is loaded down with trivial details, and testing cost is high.

Such as, Chinese patent application 201010609217.4, " a kind of method and test kit detecting gene mutation of human cytochrome P 450 CYP 2 C 19 ", it relates to PCR and combines sequencing technologies, only detects for 2 SNP site.And the shortcoming of sequencing technologies be can not Multiple detection, operation is slow.Therefore above technical problem can not be solved.

Chinese patent application 200610072593.8, " a kind of oligonucleotide probe and gene chip detecting cytochrome P 450 Enzyme mutational site ", relate to chip method, because the oligonucleotide probe described in the method adopts fluorescent mark or biotin labeling, therefore have that synthesising probing needle cost is high, process is complicated, therefore can not solve above technical problem.

Described in comprehensive, the technical problem of current existence is: lack method and the product that once can detect the gene polymorphic site that multiple CYP450 is correlated with simultaneously, common detection technique, as order-checking, real-time fluorescence quantitative PCR etc., loci is all needed to detect one by one, the complicated operation when site is more, costly, therefore a kind of detection technique different from the past is needed at present, can detect by the multiple SNP for same individuality in same system, by the Detection Information of this technology, for rational use of drug provides reference frame.

Summary of the invention

The principle of the invention is: provide a kind of associating multiple PCR technique, single-basic extension technology and mass spectrum detection, detect the detection scheme of warfarin dosage genes involved polymorphic site (SNP).Wherein: nearly 7 DNA fragmentations containing SNP of amplification simultaneously in multiplex PCR; In single-basic extension process, multiple single-basic extension is carried out to the purified product of multiplex PCR, extend primer and extend a Nucleotide respectively at 7 SNP places, make extended nucleotide type, relevant to the genotype at SNP place respectively; Single-basic extension produces the mixture to be checked be made up of extension primer and extension products, with mass spectrum, mixture to be checked is detected, each molecular weight of material in mixture to be checked is determined by mass spectra peak, and compare with the theoretical molecular of precalculated each extension primer and extension products, thus determine whether mixture to be checked comprises specific material, and then determine the genotype at each SNP place.

Therefore, the present invention's first object is to provide primer system or the primer sets in the gene polymorphic site relevant to human-cytochrome P450, a kind of detection 7 places, and its sequence is as shown in table 1.

Wherein, described 7 SNPs relevant to human-cytochrome P450 respectively: CYP2C19 gene rs56337013 site (CYP2C9*5), CYP2C19 gene rs4986893 site (CYP2C19*3), CYP2C9 gene rs1799853 site (CYP2C9*2), CYP2C19 gene rs12248560 site (CYP2C19*17), CYP2C9 gene rs1057910 site (CYP2C9*3), CYP2C19 gene rs4244285 site (CYP2C19*2), CYP2C19 gene rs28399504 site (CYP2C19*4).

Wherein, the extension primer that each site is corresponding and extension products molecular weight as shown in table 2.

In one embodiment, above-mentioned PCR primer sequence is core sequence, and it can comprise protection base sequence, a preferred 5-15 base at 5 ' end.In a specific embodiment; protection base sequence is selected from the tag(ACGTTGGATG adding 10bp at 5 ' end); the sequence of protection base makes the molecular weight of PCR primer (i.e. core primers) increase; can avoid reacting remaining PCR primer enters in mass spectrometric detection process, to avoid interference Detection results in the lump.In addition, in a specific embodiment, the 5' end extending primer also can increase base sequence (as above-mentioned protection base sequence) in right amount.Increase the object of base sequence be when extension primer corresponding to two gene polymorphic sites and product molecular weight close to time, all base is increased by extending primers to one of them extension primer or two, change the molecular weight of primer and product thereof, and widen gap between other molecular weight extending primer and product, improve Detection results.And the primer after increase and molecular weight of product, exceed detection window scarcely.

Such as, PCR primer SEQ ID NO:1 is 5 '-CATGAGGAGTAACTTCTCCC-3 '.In another embodiment, extend primer 5 ' holds the base sequence that also can increase as joint.

The present invention's second object there is provided the product for detecting the gene polymorphic site that human-cytochrome P450 is correlated with prepared by above-mentioned primer system.

In one embodiment, this product is detection kit, comprising:

(1) for the reaction reagent of PCR, comprising: Specific PCR primers, resistant to elevated temperatures archaeal dna polymerase, dNTPs, PCR reaction buffer;

(2) for the reagent of PCR primer purifying;

(3) for the reagent of single base extension, comprising: extend primer, resistant to elevated temperatures single-basic extension enzyme, ddNTPs, extension damping fluid.

In a specific embodiment, this test kit also can comprise: negative quality control product, positive quality control product, purifying resin, and point sample and mass spectrometric detection target sheet, excision enzyme, human gene group DNA extracts the reagent such as reagent.

In another embodiment, the reagent for PCR primer purifying: alkaline phosphatase, or alkaline phosphatase and excision enzyme ExoI, or running gel reclaims reagent, or PCR primer purification column.Wherein when comprising the purified reagent of alkaline phosphatase and excision enzyme ExoI, the PCR primer used is without the need to comprising protection base.

The present invention's the 3rd object uses above-mentioned primer, product or test kit to the method in the gene polymorphic site detected human-cytochrome P450 and be correlated with, and comprises the steps:

(1) multiplex PCR: use specific PCR primer, in a reaction system, increase in the gene polymorphic site place region of DNA territory relevant to human-cytochrome P450 to 7 places simultaneously, obtains the PCR primer containing 7 region of DNA territories, place, gene polymorphic site, place;

(2) PCR primer purifying: carry out purifying to the PCR primer that step (1) obtains, to reduce the interference to subsequent reactions;

(3) single-basic extension: use 7 specific extension primers, in a reaction system, after the purifying obtain step (2), PCR primer carries out multiple single-basic extension, extend primer and extend a Nucleotide at the SNP site place of correspondence, the genotype complementary pairing at this Nucleotide and SNP site place;

(4) extension products purifying: carry out purifying to the extension products that step (3) obtains, to obtain high-purity extension products, avoids the impurity such as salt ion on the impact of subsequent detection;

(5) mass spectrograph detects: purified product point step (4) obtained, on the target sheet containing matrix, is put into mass spectrograph and carried out detecting;

In one embodiment, the purge process of step 2 can be selected from alkaline phosphatase enzymic digestion, alkaline phosphatase and excision enzyme ExoI digest, cut glue purification, PCR purification column crosses post etc.In a specific embodiment, after using alkaline phosphatase enzymic digestion or alkaline phosphatase and excision enzyme ExoI digestion to carry out purifying, the process of high temperature enzyme inactivation is carried out.

The present invention's the 4th object is to provide the purposes of the SNP that aforementioned agents box is correlated with at detection 7 people's Cytochrome P450s.

Beneficial effect

Advantage of the present invention and effect as follows:

1. responsive: the present invention combines the technology such as multiplex PCR, single-basic extension, mass spectrometric detection and is integrated, both by round pcr amplification detection template, trace sample is detected again by mass-spectrometric technique, combine the advantage of two kinds of technology, be far superior to be used alone PCR and detect polymorphism SNP, therefore its detection sensitivity is very high.

2. special: single-basic extension is also called " micrometering sequence ", use specific probe DNA molecular to be identified to have the high accuracy of sequencing technologies, the features such as specificity is good, false positive is low; Especially, be different from sequencing technologies and extend hundreds of bases, this technology only extends single base, and error probability is lower;

3. handy and safe: simple to operate, safety, level of automation are high, anti-pollution;

4. quick: speed is fast, high-throughput, can complete the detection of hundreds of samples in 5-6 hour.

5, the present invention can detect multiple known patient, obtains the detected result with different SNP site respectively, and wherein patient can be that single SNP site is undergone mutation, and also can be that multiple SNP site is undergone mutation.This represents that patient can carry one or more SNP and suddenly change, thus for selecting suitable dose to provide reference information.

6, instant invention overcomes the defect that conventional art one-time detection SNP site is very few, with low cost.

Principle and definition

The invention provides the technology such as a kind of associating multiplex PCR, single-basic extension and mass spectrometric detection, detect the detection scheme with human-cytochrome P450 genes involved polymorphic site (SNP).Its principle is:

In multiplexed PCR procedures, by designing and use suitable primer thus nearly 7 the SNP site place DNA fragmentations that can increase simultaneously.

In single-basic extension step, purifying and multiple single-basic extension are carried out successively to the product of previous step multiplex PCR.Wherein, extend primer totally 7, corresponding with 7 SNP site respectively, and extend a Nucleotide at the SNP site place of correspondence, the genotype complementary pairing (if certain SNP site place is A genotype, extending T Nucleotide by the extension primer of correspondence) at this Nucleotide and SNP site place.In single-basic extension step, adopt ddNTP to replace dNTP, therefore, after extension base, extend primer and termination is extended.

In mass spectrometric detection process, single base extension product is after desalting and purifying, and point extremely contains the target sheet of matrix, and by laser excitation in vacuum environment, by tof tube to detector.Different substances is negative correlation by time of tof tube with their molecular weight, and namely molecular weight is larger, and flight velocity is slower, and the time of arrival detector is more late.

Terms " rs1799853 " etc. are all human-cytochrome P450SNP site Unified numbers at ncbi database.Term " CYP2C19*2 " etc. is then the popular call in site, wherein: CYP2C19*2, CYP2C19*3 etc. are the another kind of naming systems to polymorphic site a series of on CYP2C19 gene, and rs4244285 site as corresponding in CYP2C19*2.

Term " protection base ", refers to the extra base increased of 5 ' end in PCR primer.Owing to protecting the sequence of base to make the molecular weight of PCR primer (i.e. core primers) increase, can avoid reacting remaining PCR primer and enter mass spectrometric detection window, to avoid interference Detection results.In addition; 5 ' the end extending primer also can increase base sequence in right amount, but its effect is not as the protection base of PCR primer, makes it exceed detection window; but suitably adjustment extends the molecular weight of primer, makes extension primer and product thereof be in a rational position in detection window.Such as, when extension primer corresponding to two gene polymorphic sites and product molecular weight close to time, base is increased by extending primer to one of them, change the molecular weight of primer and product thereof, and widen gap between other molecular weight extending primer and product, produce interference to avoid regional area mass spectra peak too to concentrate and differentiate unclear, thus improving Detection results.Therefore, increase the molecular weight of the extension primer after base and product, can detection window be exceeded scarcely.The Extra bases of above-mentioned extension primer can be described as primer joint.

Term " alkaline phosphatase enzymic digestion ", its effect is remaining dNTP in the rear system of degraded PCR reaction, its principle makes the 5 '-P end of dNTP convert 5 '-OH end to, thus lose the ability being combined with primer and making primer extension, avoids the impact on next step single-basic extension.

Term " excision enzyme ExoI digests ", its effect be from one end of single stranded DNA according to the order of sequence catalytic hydrolysis composition DNA dNTP between 3,5-phosphodiester bonds, make single stranded DNA finally be hydrolyzed to dNTP.For PCR primer remaining after PCR reaction of degrading in the technical program.Because the PCR primer of strand can be excised by excision enzyme, can't occur in detection window, when therefore using this excision enzyme, the PCR primer used is without the need to comprising protection base.

Term " single-basic extension ", be referred to as again micrometering sequence (mini sequence), refer to add in system and extend primer and ddNTP, ddNTP and extend primer 3 ' holds and is connected to form extension products (i.e. primer extension a base), according to base pair complementarity principle, determine concrete which kind of ddNTP of connection by SNP site genotype, this process is similar to dNTP in PCR process and, according to the based composition of complementary strand, adds to one by one in PCR primer.Due to " ddNTP " and common dNTP unlike, a hydroxyl is lacked in 3 ' position of ribodesose, phosphodiester bond can not be formed with follow-up ddNTP, thus, extend primer and only connect a ddNTP at SNP site place, and as PCR, constantly toward downward-extension, therefore can not be referred to as single-basic extension.Single-basic extension and sequencing procedure closely similar, what add in order-checking system is the mixture of dNTP and ddNTP, and continuations extends after connecting dNTP by sequencing primer, after only having connection ddNTP, side stops extending, the mixture of what therefore order-checking produced is nucleotide fragments different in size; Add in single-basic extension system and only have ddNTP, extend primer and can only connect a ddNTP, and stop extending, what therefore single-basic extension produced is extend the nucleotide fragments that primer only extends a base.

Term " testing product ", refers to, for detecting the genotypic any conventional products of SNP site, comprising: detection reagent, detection chip, detection carrier, and detection kit etc.

Term " ddNTP " is a kind of special Nucleotide, the technical program adopts four kinds altogether, there is molecular weight difference between them, the molecular weight as ddATP, ddCTP, ddGTP, ddTTP be respectively 271.2Da, 247.2Da, 287.2Da, 327.1Da(wherein ddTTP be modify after molecular weight).When extension primer extends different Nucleotide according to the genotype of SNP site, molecular weight difference will be formed.By mass spectrometric detection, distinguishable go out this species diversity.Such as, if certain SNP site C/T is polymorphic, corresponding extension primer length is 17 bases (molecular weight 5019.3Da), when this SNP site place is C genotype, extend primer by extension G Nucleotide and stop extend, form the extension products of 18 bases length, molecular weight 5306.5Da, when this SNP site place is T genotype, extend primer by extension A Nucleotide and stop extend, form the extension products of 18 bases length, molecular weight 5290.5Da, between two kinds of products, there is the molecular weight difference of 16Da.Namely to this experimental program of this SNP site, the mass spectra peak of the corresponding 5306.5Da of C genotype, the mass spectra peak of the corresponding 5290.5Da of T genotype.In actual testing process, user by software to 5019.3Da, 5306.5Da, 5290.5Da tri-place observe: if mass spectra peak appears in 5019.3Da place, be then have partly or entirely to extend primer and be not combined with ddNTP; No matter whether 5019.3Da place there is mass spectra peak, if 5306.5Da and 5290.5Da locates appearance one place mass spectra peak, then the genotype of this SNP site is homozygous, and it is corresponding with the position of mass spectra peak, as previously mentioned, the corresponding C genotype of mass spectra peak of 5306.5Da, the corresponding T genotype of mass spectra peak of 5290.5Da; If 5306.5Da and 5290.5Da two place mass spectra peak all occurs, then the genotype of this SNP site is heterozygous; If 5306.5Da and 5290.5Da two place mass spectra peak does not all occur, then the failure of an experiment.

Term " purifying ", refers to for reducing in system to be checked other materials to the treatment step of the impact of subsequent reactions.PCR primer purifying of the present invention has two kinds of modes: one is separating impurity and abandons, and two is that impurity is lost activity.Wherein, cutting glue purification, crossing purification column etc. is all by the separating impurity such as electrophoresis, purification column, and reclaims relatively pure PCR primer, and can think the first way of purification, which is generally consuming time, complicated operation, when particularly sample size is large; The effect of alkaline phosphatase is degraded (also known as " digestion ") dNTP, makes it the substrate that can not continue as archaeal dna polymerase or single-basic extension enzyme and participates in PCR or single base extension, thus not interfere with subsequent reaction, the second way of purification can be thought.It should be noted that, independent excision enzyme ExoI does not play purification, when it and alkaline phosphatase is used in combination time, its effect is in advance by single stranded DNA (in PCR primer system after completion of the reaction, mainly remaining PCR primer) be degraded into dNTP, then make dNTP continue degraded by alkaline phosphatase.Because PCR primer is degraded, last mass spectroscopy detection step can not be entered, therefore, if increase the process of ExoI excision enzyme in plan purification step, so without the need to using the PCR primer with protection base.In addition, before single-basic extension step, because excision enzyme and alkaline phosphatase all pass through high temperature deactivation, the extension primer, ddNTP etc. of its non-degradable strand added in single-basic extension step, therefore avoid having an impact to subsequent experimental.

Term " detection window ", refers to the scope that can be used for mass spectrometric detection nucleic acid molecule amount, is usually directed to the design reference scope of primer.Wherein, when design extension primer, for different SNP site, according to the sequence characteristic in these region of DNA territories, place, site, and the genotype of SNP site, the different extension primer of molecular weight and extension products can be designed, different extension between primer and product is avoided to there is interference because molecular weight is close, thus at a relatively broad detection window, as 4000-9000Da, the detection to multiple SNP site can be realized.

Term " SNP " genotype, refers to the type representing single nucleotide polymorphism in human genome.Wherein, in actual inspection, both can come from contrast human genome for the genotype detected in contrast, also can control oneself and be cloned into the carrier tool of plasmid, and the latter has reproducible and preserves convenient, steady sources and be subject to the welcome of actual user.

Term " SNP mutation frequency ", refers to the probability that SNP site is undergone mutation.In theory, the present invention can detect in single individuality simultaneously and there are 7 SNP sudden changes simultaneously.But investigator finds in practice, there is certain mutation frequency in different SNP sudden change in individuality.HapMap project is the project that international organization is detected in different crowd SNP site in human genome, there is the detailed frequency information of each site in different crowd in ncbi database, with the data of HCB sample in this project (Chinese Han descendants sample), the distribution frequency of following site in Chinese population is described.Such as:

(1) rs1799853, detects 45 samples, and wild homozygous (C/C) accounts for 100%, and namely the frequency of saltant type (T) in colony is 0%;

(2) rs1057910, detects 45 samples, and wild homozygous (A/A) accounts for 91.1%, and heterozygous (A/C) accounts for 8.9%, and namely the frequency of saltant type (C) in colony is 4.4%(8.9/2=4.45);

(3) rs4244285, detects 45 samples, and wild homozygous (G/G) accounts for 48.9%, and heterozygous (G/A) accounts for 51.1%, and namely the frequency of saltant type (A) in colony is 25.55%(51.1/2=25.55);

(4) rs4986893, lack HapMap data, in the test item of another 99 sample (sample is not limited to Chinese Han descendants), wild homozygous (G/G) accounts for 94.9%, heterozygous (G/A) accounts for 5.1%, and namely the frequency of saltant type (A) in colony is 2.55%(5.1/2=2.55);

(5) rs28399504, detects 43 samples, and wild homozygous (A/A) accounts for 97.7%, and heterozygous (A/G) accounts for 2.3%, and namely the frequency of saltant type (G) in colony is 1.15%(2.3/2=1.15);

(6) rs56337013, lacks HapMap data, there is no the research of mutation frequency;

(7) rs12248560, detects 45 samples, and wild homozygous (C/C) accounts for 95.6%, and heterozygous (C/T) accounts for 4.4%, and namely the frequency of saltant type (T) in colony is 2.2%(4.4/2=2.2).See:

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=1799853

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=1057910

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=4244285

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=4986893

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=28399504

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=12248560

To sum up, the site detected is except the saltant type of rs4244285 is except crowd's medium frequency is higher (25.55%), and all the other are all below 5%, the saltant type in indivedual site is extremely rare (rs1799853), almost lose, therefore, the probability that many places sudden change occurs in 7 sites is simultaneously extremely low.But, too low owing to studying the sample size related to above, well below the quantity of expection, therefore, above data only show the situation that the sudden change of multiple SNP site occurs simultaneously, and its occurrence frequency is extremely low, but not mean that the present invention can not detect the situation that 7 SNP site sudden changes occur simultaneously.In practice, we find, PCR sequencing or fluorescence quantitative PCR method, often increase the reaction that is detected SNP site, will increase corresponding expense and time.By contrast, mass spectroscopy detects advantage and is no matter how many sites to be checked, as long as can at a reaction inner reaction tube, then the reagent spent and time be constant, and this is also the advantage that the present invention detects the sudden change of multiple SNP site simultaneously.

Accompanying drawing explanation

Fig. 1 is in embodiment four, to the detected result in C1 sample 7 sites.

Fig. 2 is in embodiment four, to the detected result in C2 sample 7 sites.

Fig. 3 is in embodiment four, to the detected result in C3 sample 7 sites.

Fig. 4 is in embodiment four, to the detected result in C4 sample 7 sites.

Fig. 5 is in embodiment four, to the detected result in C5 sample 7 sites.

Fig. 6 is in embodiment four, to the detected result in C6 sample 7 sites.

Fig. 7 is in embodiment four, to the detected result in C7 sample 7 sites.

Fig. 8 is in embodiment four, to the detected result in C8 sample 7 sites.

Fig. 9 is in embodiment four, to the detected result in C9 sample 7 sites.

Figure 10 is in embodiment four, to the detected result in C10 sample 7 sites.

Figure 11 is in embodiment four, to the detected result in C8 sample rs1799853 site, three dotted lines are the theoretical peak extending primer, wild-type (C) extension products, saltant type (T) extension products respectively from left to right, and it is that CC isozygotys that detected result shows this sample.

Figure 12 is in embodiment four, to the detected result in C8 sample rs1057910 site, three dotted lines are the theoretical peak extending primer, saltant type (C) extension products, wild-type (A) extension products respectively from left to right, and it is that AA isozygotys that detected result shows this sample.

Figure 13 is in embodiment four, to the detected result in C8 sample rs4244285 site, three dotted lines are the theoretical peak extending primer, saltant type (A) extension products, wild-type (G) extension products respectively from left to right, and it is AG heterozygosis that detected result shows this sample.

Figure 14 is in embodiment four, to the detected result in C8 sample rs4986893 site, three dotted lines are the theoretical peak extending primer, saltant type (A) extension products, wild-type (G) extension products respectively from left to right, and it is that GG isozygotys that detected result shows this sample.

Figure 15 is in embodiment four, and to the detected result in C8 sample rs28399504 site, three dotted lines are the theoretical peak extending primer, saltant type (G) extension products, wild-type (A) extension products respectively from left to right, and detected result shows this sample AA and isozygotys.

Figure 16 is in embodiment four, to the detected result in C8 sample rs56337013 site, three dotted lines are the theoretical peak extending primer, saltant type (T) extension products, wild-type (C) extension products respectively from left to right, and it is that CC isozygotys that detected result shows this sample.

Figure 17 is in embodiment four, to the detected result in C8 sample rs12248560 site, three dotted lines are the theoretical peak extending primer, wild-type (C) extension products, saltant type (T) extension products respectively from left to right, and it is that CC isozygotys that detected result shows this sample.

Figure 18 is the detected result of the plasmid A1-A7 7 sites being to wild-type, wherein:

Peak 1(5306.5) represent rs56337013 site wild-type (C),

Peak 2(6076) represent rs4986893 site wild-type (G),

Peak 3(6280.1) represent the extension primer SEQ ID No:17 that rs1799853 site unreacted is complete,

Peak 4(6393.2) represent the extension primer SEQ ID No:18 that rs12248560 site unreacted is complete,

Peak 5(6527.3) represent rs1799853 site wild-type (C),

Peak 6(6640.3) represent rs12248560 site wild-type (C),

Peak 7(7080.6) represent rs1057910 site wild-type (A),

Peak 8(7475.9) represent rs4244285 site wild-type (G),

Peak 9(7684.9) represent rs28399504 site wild-type (A),

Figure 19 is the detected result of the plasmid B1-B7 7 sites being to saltant type, wherein:

Peak 1(5290.5) represent rs56337013 site mutation type (T),

Peak 2(6060) represent rs4986893 site mutation type (A),

Peak 3(6393.2) represent the extension primer SEQ ID No:18 that rs12248560 site unreacted is complete,

Peak 4(6607.2) represent rs1799853 site mutation type (T),

Peak 5(6720.3) represent rs12248560 site mutation type (T),

Peak 6(7056.6) represent rs1057910 site mutation type (C),

Peak 7(7459.9) represent rs4244285 site mutation type (A),

Peak 8(7605) represent rs28399504 site mutation type (G),

Figure 20 is in comparative examples one, to the sequencing result in C3 sample rs1799853 site, is shown as C and isozygotys.Figure 21 is in comparative examples one, to the sequencing result in C3 sample rs1057910 site, is shown as A and isozygotys.

Figure 22 is in comparative examples one, to the sequencing result in C3 sample rs4244285 site, is shown as G and isozygotys.

Figure 23 is in comparative examples one, to the sequencing result in C3 sample rs4986893 site, is shown as G and isozygotys.

Figure 24 is in comparative examples one, to the sequencing result in C3 sample rs28399504 site, is shown as A and isozygotys.

Figure 25 is in comparative examples one, to the sequencing result in C3 sample rs56337013 site, is shown as C and isozygotys.

Figure 26 is in comparative examples one, to the sequencing result in C3 sample rs12248560 site, is shown as C and isozygotys.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in further detail.

Embodiment one: design of primers and synthesis.

For CYP2C19 gene rs56337013 site (CYP2C9*5), CYP2C19 gene rs4986893 site (CYP2C19*3), CYP2C9 gene rs1799853 site (CYP2C9*2), CYP2C19 gene rs12248560 site (CYP2C19*17), CYP2C9 gene rs1057910 site (CYP2C9*3), CYP2C19 gene rs4244285 site (CYP2C19*2), the gene polymorphic site that 7, CYP2C19 gene rs28399504 site (CYP2C19*4) etc. are relevant to human-cytochrome P450, design corresponding Specific PCR primers core sequence (SEQ ID No:1 to SEQ ID No:14) and specificity extension primer core sequence (SEQ ID No:15 to SEQ ID No:21).

Wherein, in order to avoid PCR primer enters mass spectrograph detection window and Interference Detection effect, 5 ' end of every bar PCR primer can increase the base of certain number on the basis of core sequence (SEQ ID No:1 to SEQ ID No:14), the common tag(ACGTTGGATG as 10bp), to make the molecular weight of PCR primer increase, thus exceed mass spectrograph detection window.

Relevant primer synthesizes in Sangon Biotech (Shanghai) Co., Ltd..

Following testing process operates with reference to " cytochrome P450 gene mutation detection kit (time-of-flight mass spectrometry (TOFMS)) specification sheets " (hereinafter referred to as " specification sheets ") of YiXin Industry (Beijing) Science and Technology Ltd..

Embodiment two: sample DNA extracts.

Collect clinical patients, totally 10 examples.Wherein, sample collection, DNA extraction etc. be requirement to specifications, gathers people's venous blood, and collects with EDTA anticoagulant tube.Requirement to specifications, the blood of collection is preserved at 2-8 DEG C should more than one week, and preserving for-20 DEG C should more than one month, and the on the rocks or bubble chamber sealing on the rocks of curling stone can be adopted to transport, and extracting genome DNA is carried out in suggestion as far as possible employing fresh blood.Because this test kit does not provide human gene group DNA to extract reagent, therefore business-like nucleic acid extraction kit (the DNeasy Blood and Tissue kit as QIAGEN company) is adopted, human gene group DNA is extracted from the 200ul whole blood of every patient, with NanoDrop2000(Thermo company) quantitatively, and markization is respectively C1-C10 to 30ng/ul().Wherein, this test kit is recommended to detect the human gene group DNA that concentration is 30ng/ul, but contrast experiment shows, this test kit also can detect positive findings to the human gene group DNA that concentration is low to moderate 10ng/ul.Requirement to specifications, the human gene group DNA after extraction, preserving at 2-8 DEG C should more than one week, and-20 DEG C of preservations more than 2 years, should can not preserved for-80 DEG C, should avoid multigelation for a long time, and are placed in ice chest and transport.

Embodiment three: Bioexperiment.

Use ABI9700 type PCR instrument, by specification is tested to 7 gene polymorphic sites relevant to cytochrome P450 gene.

In test kit for the component of PCR, PCR primer purifying and single-basic extension be:

Sequence number ingredient names	Main component	Specification
			1PCR mixture	DNTPs, MgCl2, PCR primer	360ul/ pipe x1 manages
2PCR enzyme	Taq enzyme	24ul/ pipe x1 manages
			3SAP enzyme mixture	SAP enzyme	24ul/ pipe x1 manages
4 extend primer mixed solution	Extend primer	24ul/ pipe x1 manages
			5 extend enzyme mixture	IPLEX enzyme, ddNTPs	24ul/ pipe x1 manages
6 positive quality control product	Human gene group DNA (30ng/ul)	40ul/ pipe x1 manages

By specification, concrete operation method is as follows:

1.PCR increases

1.1 in PCR dosing district, prepares 200ul PCR reaction tubes, and mark sample number according to measuring samples number (containing positive quality control product, negative control, blank) on pipe;

1.2 take out PCR mixture, PCR enzyme from test kit, and make it naturally thaw, vortex oscillation makes it fully mix, at the bottom of brief centrifugation to pipe;

1.3 according to number of samples, and the ratio of according to the form below takes out PCR primer mixed solution and PCR reaction solution, is placed in a centrifuge tube and mixes, add 16ul mixture carry out packing by every PCR reaction tubes.Due in point process of assembling, suction pipette head the factor such as to remain and may cause and be not enough to point take on required number, the dose volume of the suitable amplification mixture of suggestion.Such as, when having 10 parts of testing samples, can by 10.5-11 increment product preparating mixture.

Ingredient names	Single reaction volume
		PCR mixture	15ul
PCR enzyme	1ul
		Add up to	16ul

1.4 add 4ul testing sample in pcr amplification district in every pipe mixture, make every part of PCR reaction system cumulative volume be 20ul.Wherein, negative control is purified water, and blank is not for add template.

PCR reaction tubes is placed in PCR amplification instrument by 1.5, and the program of according to the form below carries out pcr amplification reaction.

2.SAP enzymic digestion

After PCR, get 5ul PCR primer successively to new pipe, often pipe adds SAP enzyme mixture 1ul, then PCR reaction tubes is placed in PCR amplification instrument, performs lower list procedure.

Temperature	Time (dividing)	Cycle number
			37	45	1
85	15	1

3. extend

3.1 in PCR dosing district, and according to number of samples, the ratio of according to the form below is taken out and extended primer mixed solution and extend enzyme mixture, is placed in a centrifuge tube and mixes.Due in point process of assembling, suction pipette head the factor such as to remain and may cause and be not enough to point take on required number, the dose volume of the suitable amplification mixture of suggestion.Such as, when having 10 parts of digestion products, can by 10.5-11 increment product preparating mixture.

Ingredient names	Single reaction volume
		Extend primer mixed solution	1ul
Extend enzyme mixture	1ul
		Add up to	2ul

3.2 in pcr amplification district, adds 2ul mixture carry out packing by every pipe digestion products.

PCR reaction tubes is placed in PCR amplification instrument by 3.3, and the program of according to the form below carries out extension.

4. purifying

In every pipe extension products, add 16ul purified water, 6mg resin in pcr amplification district, put upside down mixing 30 minutes.

5. point sample

Use micropipet, draw 1ul purified product, point sample is to target sheet.

Embodiment four: upper machine testing and result interpretation.

The Clin-TOF type time-of-flight mass spectrometer using YiXin Industry (Beijing) Science and Technology Ltd. to produce detects the target sheet after point sample and result judges.

In addition, the wild type control A1-A7 in above site, saltant type contrast B1-B7 are set respectively.Wherein, wild type control A1-A7, saltant type contrast B1-B7 are respectively from artificial plasmid that is commercially available or Laboratories Accession.Wild type control plasmid A1-A7 used in the present invention and saltant type control plasmid B1-B7, for in commercialization plasmid pMD18-T Vector(Takara company) basis on, according to the ordinary method that " molecular cloning " is recorded, after carrying out PCR with primer and normal people DNA, PCR primer is inserted pMD18-T Vector, namely build wild plasmid A1-A7, then distinguish rite-directed mutagenesis, namely build 7 mutant plasmids B1-B7.Described plasmid A1-A7 and B1-B7 can be stored in-20 DEG C of glycerine for a long time, and the used time activates and extracts plasmid DNA.

As shown in Table 2 above, article 7, extend primer and their extension products of producing according to respective genotype on 7 gene polymorphic sites have different molecular weight, the mass spectra peak that these molecular weight are corresponding respective, if there is mass spectra peak at certain molecular weight place, be then judged as there is the material (extend primer or product) corresponding with this molecular weight:

Judging criterion:

(1) if wild-type and mass spectra peak corresponding to saltant type all do not occur, no matter extend the mass spectra peak that primer pair answers and whether exist, be all judged as the failure of an experiment;

(2) if wild-type or mass spectra peak corresponding to saltant type only occur one, then corresponding genotypic homozygous of occurred mass spectra peak is judged as;

(3) if wild-type or mass spectra peak corresponding to saltant type all occur, then heterozygous is judged as.

As shown in figures 1-19, wherein Figure 18 is the mass spectrum that 7 SNP site are the plasmid A1-A7 of wild-type to mass spectral results, and Figure 19 is the mass spectrum that 7 SNP site are the plasmid B1-B7 of saltant type.

Extend the mass spectral results (Fig. 1-10) of the molecular weight inspection sample C1-C10 of primer and extension products with site each shown in aforementioned table 2, determine the genotype of each SNP site, result is as shown in table 3:

C1

C2

C3

C4

C5

C6

C7

C8

C9

C10

rs1799853

C

rs1057910

A

rs4244285

G

A

G

AG

G

AG

G

rs4986893

AG

G

rs28399504

A

rs56337013

C

rs12248560

C

Table 3

Namely, in 10 routine patients, rs4244285 site AG heterozygous 2 example is detected altogether, AA homozygous mutant 1 example, rs4986893 site AG heterozygous 1 example.

Comparative examples one

One, according to embodiment 1, use following primer as shown in table 4, checked order in each site:

Table 4

Two, sample DNA source

For making the data produced between different experiments have comparability, sequence verification adopts the human gene group DNA (C1-C10) extracted the venous blood gathered in 10 routine patient bodies in embodiment two.

Three, order-checking qualification

1, PCR reaction system is 25 μ l

2, reaction conditions: react and carry out on ABI company 9700 thermal cycler, reaction conditions is 94 DEG C of denaturations 5 minutes, 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, and 72 DEG C extend 40 seconds, 35 circulations, 72 DEG C of extensions 7 minutes again after reaction terminates, 4 DEG C of preservations.

3, PCR primer purifying and order-checking

(1) in 96 orifice plates that PCR primer is housed, 50 microlitre lavation buffer solutions are added, mixing.

(2) transferred in Millipore purifying plate, to be put on vacuum pump suction filtration about 3 minutes, to see in purifying plate there is no water.

(3) in purifying plate, again add the lavation buffer solution of 50 microlitres, continue suction filtration, until there is no water in purifying plate.

(4) purifying plate is taken off from vacuum pump, in plate, add the deionized water of 20 microlitres, leave standstill 15 minutes.

(5) shake 15 minutes again, be then drawn onto in new 96 orifice plates.

Needed for sequencing reaction, reagent should be Fresh, needs can to use after sterilizing through autoclaved reagent.Equipment needed for sequencing reaction (as first-class in 384 orifice plates, tip) should be cleaning sterile equally.

(6) in order to ensure the fresh of sample and reaction reagent that check order, should operate on ice during application of sample.

(7) sequencing reaction system is 5 μ l, and all ingredients add-on is as follows: PCR primer 3-10ng, BigDyev3.10.25 μ l, 5*BigDye buffer0.875 μ l, primer 1.6pmol;

(8) sample is put in PCR instrument and does following reaction:

Step: 95 DEG C, 5 points;

95 DEG C, 10 seconds; 60 DEG C, 4 points; Repeat 30 circulations;

4 DEG C keep until prepare purifying.

(9) in each hole, 20 μ l80% ethanol are added, the centrifugal 30min of 4,000rpm;

(10) sample panel is placed on the paper handkerchief rolled well, gets rid of in centrifuges, speed 1000rpm when getting rid of;

(11) in every hole, add 30 μ l70% ethanol, the centrifugal 10min of 4000rpm, gets rid of;

(12) operation 2 times of the 11st step is repeated;

(13) sample panel is put in clean drawer, the dry 30min of lucifuge;

(14) add people 5 μ l methane amide, sealer, be centrifugally placed in-20 DEG C of refrigerators;

(15) the front 95 DEG C of sex change of sequenator 5 minutes, place 2 minutes on ice, centrifugal rear loading.

(16) ABI3730xl type genetic analyzer is used to carry out sequencing

Four, result

To C1-C10 sample, use the primer described in table, respectively to rs1799853, rs1057910, rs4244285, rs4986893, rs28399504, rs56337013, rs12248560(numbered sequence 1-7 respectively) check order, amount to 70 order-checkings.According to the accompanying drawing 20-26 of order-checking, final sequencing result is as shown in table 5:

Patient code

Sequence 1

Sequence 2

Sequence 3

Sequence 4

Sequence 5

Sequence 6

Sequence 7

C1

C

A

G

AG

A

C

C2

C

A

G

A

C

C3

C

A

G

A

C

C4

C

A

G

A

C

C5

C

A

G

A

C

C6

C

A

AG

G

A

C

C7

C

A

G

A

C

C8

C

A

AG

G

A

C

C9

C

A

G

A

C

C10

C

A

G

A

C

Table 5

Through comparing, the result of table 3 and table 5 completely the same, the accuracy of detection method is described.

Claims

1. detect a Primer composition for the gene polymorphic site detection that human-cytochrome P450 is correlated with, its sequence is:

Gene polymorphic site amplimer extends primer

CYP2C19 gene rs56337013 site (CYP2C19*5), SEQ ID No:1-2, SEQ ID No:15;

CYP2C19 gene rs4986893 site (CYP2C19*3), SEQ ID No:3-4, SEQ ID No:16;

CYP2C9 gene rs1799853 site (CYP2C9*2), SEQ ID No:5-6, SEQ ID No:17

CYP2C19 gene rs12248560 site (CYP2C19*17), SEQ ID No:7-8SEQ ID No:18;

CYP2C9 gene rs1057910 site (CYP2C9*3), SEQ ID No:9-10, SEQ ID No:19

CYP2C19 gene rs4244285 site (CYP2C19*2), SEQ ID No:11-12, SEQ ID No:20;

CYP2C19 gene rs28399504 site (CYP2C19*4), SEQ ID No:13-14, SEQ ID No:21.

2. the Primer composition of claim 1; wherein PCR primer sequence is core sequence, and it comprises protection 5-15 base sequence at 5 ' end, and it is by increasing the molecular weight of core primers; avoid reacting remaining PCR primer and enter mass spectrometric detection window, to avoid interference Detection results.

3. the Primer composition of claim 2, wherein this protection base sequence is the tag:ACGTTGGATG of 10bp.

4. the testing product for detecting human-cytochrome P450 genes involved polymorphic site prepared by the Primer composition of claims 1 to 3, wherein said testing product comprises detection reagent, detection chip, detection carrier, and detection kit.

5. the testing product of claim 4, wherein product is detection kit, comprising:

(2) for the reagent of PCR primer purifying;

6. the testing product of claim 5, wherein test kit also comprises: negative quality control product, positive quality control product, purifying resin, and point sample and mass spectrometric detection target sheet, excision enzyme, human gene group DNA extracts the reagent such as reagent.

7. the testing product of claim 5, the reagent wherein for PCR primer purifying is selected from alkaline phosphatase, or alkaline phosphatase and excision enzyme ExoI, or running gel reclaims reagent, or PCR primer purification column.

8. the testing product of claim 7, wherein when comprising the purified reagent of alkaline phosphatase and excision enzyme ExoI, the PCR primer used is without the need to comprising protection base.