CN102690888B - Primer system for detecting gene SNP (single nucleotide polymorphism) related to warfarin dosage and application of primer system - Google Patents

Abstract

The invention discloses a primer system for detecting 7 gene polymorphic sites related to warfarin dosage. A product prepared based on the primer system can be used for realizing the detection of 7 gene polymorphic sites related to warfarin dosage. The product is used so as to detect the genetypes of a warfarin user at the 7 gene polymorphic sites; and the detection result is combined with other clinical indexes so that a reference can be provided for a clinical doctor to reasonably define the warfarin dosage, and the side effect of a medicament is avoided. According to the invention, the primer system can be used for simultaneously detecting the 7 gene polymorphic sites on different genes in one reaction system; and compared with technologies such as sequence measurement and real-time fluorescencent quantitative PCR (polymerase chain reaction), the primer system disclosed by the invention has the advantages of lower cost, simplicity and convenience in operation and improved accuracy and sensitivity.

Description

For detection of primer system of the gene SNP relevant to the warfarin dosage and uses thereof

Technical field

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

Background technology

Warfarin is a present most widely used marcumar derivatives class oral anticoagulation, be mainly used in clinically prevention and treatment thrombotic disease, also need to take warfarin and reduce the phlebothrombosis incidence after the surgical operations such as cardiac valve replacement, stentplacement.But effective therapeutic domain of warfarin is narrower and Different Individual between dosage have larger difference, reach the same function effect, high low dosage person can differ more than 10 times.The excessive hemorrhage complication that easily causes of dosage, severe patient are with threat to life, and underdosage is difficult to reach expection anti-freezing curative effect, easily causes thrombus, and severe patient is threat to life also.Clinically often with prothrombin time (PT) and INR (INR) as monitoring index, use safely and effectively it to reach.So normal first some clinical indices according to the patient of clinician, as height, body weight, age, sex etc., give certain experiences dosage, then according to the situation of each patient INR value, repeatedly adjust dosage and monitoring, until INR reaches zone of reasonableness, thereby definite maintenance dose that needs is kept treatment for a long time.

Cause the reason of warfarin consumption individual difference a lot, can be divided into non-genetic factor and inherited genetic factors.Non-genetic factor mainly contains age, height, body weight, the interaction of medicine, food habits, morbid state etc.Studies show that in recent years, inherited genetic factors plays a role in the performance of warfarin consumption individual difference.The inherited genetic factors that affects warfarin consumption individual difference refers to that the albumen relevant with pharmacodynamics with the pharmacokinetics of warfarin or the gene of enzyme morph, affect the activity of protein expression or enzyme, thereby strengthen or reduce the curative effect of warfarin, make interindividual consumption produce difference, these inherited genetic factorss comprise the gene polymorphic site on the genes such as VKORC1, CYP2C9, CYP4F2, GGCX, CALU.

Warfarin suppresses vitamin K synthetic thrombin in liver cell mainly by acting on the albumen of VKORC1 genes encoding, thus the performance anticoagulation.A plurality of reports show, the G/A gene polymorphic site (rs9923231) that the VKORC1 gene-1 is 639 can cause gene promoter activity difference, compares with the AA genotype, and GG and AG genotype promoter activity are high, thrombin generates more, and patient clinical shows the warfarin opposing.

Vitamin K-dependent clotting factor contains the gamma-glutamic acid residue, after the carboxylation of the vitamin k dependent carboxylase of necessary process GGCX genes encoding, just can become activated thrombin, and then causes the blood coagulation reaction.There are some researches show, on the GGCX encoding gene, rs11676382 gene polymorphic site will cause that enzymic activity changes, and then change the effective concentration that factor Xa is arranged, and affect the variation of the required warfarin dosage of patient.

The CALU of CALU genes encoding has the function of regulating the VKORC1 gene product, has report to show, if rs339097 site G genotype on this gene, the more warfarin dosage of needs of patients.

Warfarin metabolism in vivo is mainly by liver cytochrome oxydase P450(CYP) system.Wherein, CYP2C9 is one of Major Enzymes that affects the warfarin dosage.People CYP2C9 gene has genetic polymorphism, wherein modal is CYP2C9*2(rs1799853), CYP2C9*3(rs1057910), CYP2C9*6(rs9332131) etc. the gene polymorphic site, after these site mutations, the activity of gene encoding enzyme significantly reduces than wild-type, cause the patient that metabolism and the removing ability of warfarin are significantly reduced, clinical manifestation goes out warfarin responsive, needs the decrement administration to reduce the generation of untoward reaction.

CYP4F2 is the monooxygenase of vitamin K, recently finds CYP4F2 gene V433M(rs2108622) site TT type needs of patients raising warfarin dosage, can reach identical anti-freezing effect.

In sum, the gene polymorphic site is to cause the warfarin dosage requirements to have one of factor of individual difference, the advance gene pleiomorphism of line correlation of medication detects, in conjunction with clinical factor, help to make rational warfarin dosage for the patient, shorten the adjustment time, reduce the incidence of serious adverse reaction.Be used at present instructing the gene polymorphic site testing product of warfarin dosage less, and detect target spot and concentrate on VKORC1(rs9923231) and CYP2C9*2(rs1799853), CYP2C9*3(rs1057910) etc. a few gene polymorphic site, relate to less to other sites.And in the detection technique of commonly using at present, order-checking and real-time fluorescence quantitative PCR all need detect one by one a plurality of sites, complicated operation, and expense is higher.

For example, Chinese patent application 200710135388.6, " warfarin Chinese population individual with medicament method for determination of amount " disclose a kind of Restriction Enzyme cutting method of utilizing and have determined that VKORC1 (1639G/A) and the genotypic method of CYP2C9.Because the method operation is slow, accuracy is low, and clinical easy pollution can not Multiple detection, therefore can not solve above technical problem.

Chinese patent application 201010234597.8, " a kind of test kit and pcr amplification method thereof for the SNP locus of gene associated with individualized medication of warfarin detection ", relate to PCR associating electrophoretic detection, can only detect for 2 SNP sites, can not carry out Multiple detection, and its accuracy is low, therefore clinical easy pollution can not solve above technical problem.

Chinese patent application 201110211124.0, " detecting test kit and multiplex PCR amplification method and the detection method in warfarin personalized medicine related SNP site ", the method that a kind of PCR associating electrophoretic method can detect 4 SNP sites is disclosed, the method has had certain progress with respect to conventional art, but still the quantity that exist to detect the SNP site is few, and the intrinsic accuracy that the electrophoresis showed result exists is low, therefore the defective of clinical easy pollution can not solve above technical problem.

Chinese patent application 200910053954.8, " warfarin anticoagulation medicine examination of curative effect " disclose a kind of test kit that detects individual warfarin anticoagulation medicine curative effect, it relates to quantitative fluorescent PCR, the genotype in the SNP site that can determine to detect according to the power of different sequence fluorescence probe signals.Because the method only relates to two SNP sites, and fluorescence quantitative PCR detection exists that the synthesising probing needle cost is high, process copies and overlong time, although therefore be different from above technology, can not solve above technical problem.

Chinese patent application 201110328422.8, " a kind of test kit for detection of VKORC1 and CYP2C9 gene pleiomorphism " disclose a kind of test kit that warfarin medication related gene polymorphism detects that carries out, it relates to the DNA sequencing technology, although in surveyed area, each site can be detected, and may instruct clinical application, but the shortcoming of sequencing technologies is that sensitivity is low, can not Multiple detection, operation is slow.Therefore, can not solve above technical problem.

Comprehensively described, the technical problem that exists at present is: lack method and the product that once can detect simultaneously a plurality of relevant to warfarin dosage gene polymorphics site, common detection technique, as order-checking, real-time fluorescence quantitative PCR etc., all need loci to detect one by one, complicated operation when the site is more, expense is higher, therefore, present existing detection scheme, great majority are only for VKORC1(rs9923231), CYP2C9*2(rs1799853) and a few polymorphic site such as CYP2C9*3(rs1057910).

Therefore need at present a kind of detection technique different from the past, can detect SNP site as much as possible and can be in same system for a plurality of SNP of same individuality or for the technology of the one or more different SNP in a plurality of individualities, by the detection information of this technology, for determining that warfarin dosage provides reference frame.

Summary of the invention

The principle of the invention is: a kind of associating multiple PCR technique, single-basic extension technology and mass spectrum detection are provided, have detected the detection scheme of warfarin dosage genes involved polymorphic site (SNP).Wherein: amplification reaches 7 DNA fragmentations that contain SNP simultaneously in multiplex PCR; In the single-basic extension process, the purified product of multiplex PCR is carried out multiple single-basic extension, extend primer and extend respectively a Nucleotide at 7 SNP places, make the Nucleotide type of extending, respectively with the genotypic correlation at SNP place; Single-basic extension produces the mixture to be checked that forms by extending primer and extension products, with mass spectrum, mixture to be checked is detected, determine each molecular weight of material in mixture to be checked by mass spectra peak, and compare with the precalculated theoretical molecular that respectively extends primer and extension products, thereby determine whether mixture to be checked comprises specific material, and then determine the genotype at each SNP place.

Therefore, first purpose of the present invention is to provide primer system or the primer sets in the gene polymorphic site relevant to the warfarin dosage, a kind of detection 7 places, and its sequence is as shown in table 1.

Numbering	Sequence (5' → 3')	For the site	Purposes
				SEQ ID No：1	TCCAGGGTTCAAGTGGTTCT	rs9923231	The PCR primer
SEQ ID No：2	TTCATGCAGGGACATCTTTG	rs9923231	The PCR primer
				SEQ ID No：3	TCAACTCCTCCACAAGGCAG	rs1799853	The PCR primer
SEQ ID No：4	ATGACGCTGCGGAATTTTGG	rs1799853	The PCR primer
				SEQ ID No：5	CTACACAGATGCTGTGGTGC	rs1057910	The PCR primer
SEQ ID No：6	TGTCACAGGTCACTGCATGG	rs1057910	The PCR primer
				SEQ ID No：7	ACATGAACAACCCTCAGGAC	rs9332131	The PCR primer
SEQ ID No：8	CAAGCAGTCACATAACTAAGC	rs9332131	The PCR primer
				SEQ ID No：9	GGACAAAAACAGAGAGAGGG	rs2108622	The PCR primer
SEQ ID No：10	CATCAGTGTTTTCGGAACCC	rs2108622	The PCR primer
				SEQ ID No：11	TCTAGAGTTACTCTCCCCAG	rs11676382	The PCR primer
SEQ ID No：12	GCCGCAGGTAAGTTCACAAC	rs11676382	The PCR primer
				SEQ ID No：13	CCTTGGATTCTGAATCTGGC	rs339097	The PCR primer
SEQ ID No：14	CCCCTTTAGCCTTATATAGC	rs339097	The PCR primer
				SEQ ID No：15	GCGTGAGCCACCGCACC	rs9923231	Extend primer
SEQ ID No：16	CGGGCTTCCTCTTGAACAC	rs1799853	Extend primer
				SEQ ID No：17	CACGAGGTCCAGAGATAC	rs1057910	Extend primer
SEQ ID No：18	TTCCTGATGAAAATGGAGA	rs9332131	Extend primer
				SEQ ID No：19	CACCTCAGGGTCCGGCCACA	rs2108622	Extend primer
SEQ ID No：20	AGGGGAAAGTTACCAAG	rs11676382	Extend primer
				SEQ ID No：21	TTCTGAATCTGGCCAATACTTA	rs339097	Extend primer

Table 1

Wherein, described 7 relevant to warfarin dosage gene polymorphic sites are respectively: (1639G/A), CYP2C9 gene rs1799853 site (CYP2C9*2), CYP2C9 gene rs1057910 site (CYP2C9*3), CYP2C9 gene rs9332131 site (CYP2C9*6), CYP4F2 gene rs2108622 site (V433M), GGCX gene rs11676382 site, CALU gene rs339097 site, VKORC1 gene rs9923231 site;

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

Table 2

In one embodiment, above-mentioned PCR primer sequence is core sequence, and it can comprise the protection base sequence at the 5' end, preferred 5-15 base.In a specific embodiments, the protection base sequence is selected from the tag(ACGTTGGATG that adds 10bp in the 5' section), for example, PCR primer SEQ ID NO:1 is 5'-ACGTTGGATGTCCAGGGTTCAAGTGGTTCT-3 '.In another embodiment, the 5' that extends primer holds the base sequence that also can increase as joint.

Second purpose of the present invention has been to provide by the prepared product for detection of warfarin dosage genes involved polymorphic site of above-mentioned primer system.

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

(1) be used for the reaction reagent of PCR, comprise: specific PCR primer, resistant to elevated temperatures archaeal dna polymerase, dNTPs, PCR reaction buffer;

(2) be used for the reagent of PCR product purification;

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

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

In another embodiment, be used for the reagent of PCR product purification: alkaline phosphatase, or alkaline phosphatase and excision enzyme ExoI, or running gel recovery reagent, or PCR product purification post.Wherein when comprising the purified reagent of alkaline phosphatase and excision enzyme ExoI, the PCR primer that uses need not to comprise the protection base.

The 3rd purpose of the present invention is the method for warfarin dosage genes involved polymorphic site that detects with above-mentioned primer, product or test kit, comprises the steps:

(1) multiplex PCR: use specific PCR primer, in a reaction system, increased simultaneously in the DNA zone, place, gene polymorphic site that 7 places are relevant to the warfarin dosage, obtain containing the PCR product in 7 DNA zones, place, gene polymorphic site, place;

(2) PCR product purification: the PCR product that step (1) is obtained carries out purifying, to reduce the interference to subsequent reactions;

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

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

(5) mass spectrograph detects: the purified product point that step (4) is obtained is put into mass spectrograph and is detected containing on the target sheet of matrix;

Wherein, described 7 relevant to warfarin dosage gene polymorphic sites are respectively: (1639G/A), CYP2C9 gene rs1799853 site (CYP2C9*2), CYP2C9 gene rs1057910 site (CYP2C9*3), CYP2C9 gene rs9332131 site (CYP2C9*6), CYP4F2 gene rs2108622 site (V433M), GGCX gene rs11676382 site, CALU gene rs339097 site, VKORC1 gene rs9923231 site.

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

The 4th purpose of the present invention is to provide the aforementioned agents box in the purposes that detects the relevant SNP of 7 warfarin dosages.

Beneficial effect

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

2. the characteristics such as special: single-basic extension is called again " micrometering order ", uses specific probe that DNA molecular is identified, and has the high accuracy of sequencing technologies, and specificity is good, false positive is low; Especially, be different from sequencing technologies and extend hundreds of bases, this technology is only extended 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 a plurality of known patients, obtains respectively having the detected result in different SNP site, and wherein the patient can be to undergo mutation in single SNP site, can be also to undergo mutation in a plurality of SNP site.This expression patient can carry one or more SNP sudden changes, thereby for selecting appropriate dose that reference information is provided.

6, the present invention has overcome the very few defective in conventional art one-time detection SNP site, and is 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 warfarin dosage genes involved polymorphic site (SNP).Its principle is:

In the multiplex PCR step, by designing and use suitable primer, reach place, 7 SNP sites DNA fragmentation thereby can increase simultaneously.

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

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

Terms " rs9923231, rs2108622 " etc. are all that people SNP site is at the Unified number of ncbi database.Term " 1639G/A, V443M, CYP2C9*2 " is some popular calls in each site, and wherein: (1)-1639G/A represents that the rs9923231 site is positioned at place, VKORC1 genetic transcription starting point upstream-1639bp, for G/A polymorphic; (2) V443M represents that the sudden change of rs2108622 site generation causes the 443rd amino acid generation α-amino-isovaleric acid (Val, V) of CYP4F2 gene coded protein to the variation of methionine(Met) (Met, M), is presented as that in the DNA aspect C/T is polymorphic; (3) CYP2C9*2, CYP2C9*3, CYP2C9*6 etc. are the another kind of naming systems to a series of polymorphic sites on the CYP2C9 gene, as the corresponding rs1799853 of CYP2C9*2 site.

Should be pointed out that in different documents, there are two kinds of form of presentations in the gene polymorphic site (rs9923231) about 639 of VKORC1 gene-1s, i.e. the SNP site of G/A and C/T.As, in ncbi database, report is the SNP site of C/T polymorphism, and in a lot of documents, is expressed as G/A polymorphic.This is because adopted the cause of different directions for this site order-checking.When using a certain DNA chain to check order as template, this site is detected and is wild-type T or mutant C, be expressed as C/T, and when using complementary with it DNA chain to check order as template, wild-type T or mutant C to this site, detected result is their complementary pairing base A or G, is expressed as G/A.Based on identical reason, also there is the A/G(document in the SNP site for rs339097 in the CALU gene) and the T/C(NCBI database) difference.In the present invention, unified use the gene polymorphic site (rs9923231) of 639 of VKORC1 gene-1s polymorphic as G/A, in the CALU gene, the SNP site of rs339097 is that A/G is polymorphic.

Term " protection base " refers in the extra base that increases of the 5' of PCR primer end.Make the molecular weight increase of PCR primer (being core primers) due to the sequence of protection base, can avoid reacting remaining PCR primer and enter the mass spectrometric detection window, to avoid interference the detection effect.In addition; the 5' end that extends primer also can increase base sequence in right amount, but its effect is not the protection base as the PCR primer, makes it exceed detection window; but suitably adjust the molecular weight that extends primer, make extension primer and product thereof be in a rational position in detection window.For example, when the molecular weight of extension primer corresponding to two gene polymorphic sites and product near the time, increase base by extending primer to one of them, change the molecular weight of primer and product thereof, and widen gap between the molecular weight of other extension primers and product, generation is disturbed and it is unclear to differentiate to avoid the regional area mass spectra peak too to concentrate, and detects effect thereby improve.Therefore, the extension primer after the increase base and the molecular weight of product can exceed detection window scarcely.The extra base of above-mentioned extension primer can be described as the primer joint.

Term " alkaline phosphatase enzymic digestion ", its effect is remaining dNTP in the rear system of degraded PCR reaction, its principle is to make the 5'-P end of dNTP convert the 5'-OH end to, thereby loses the ability that makes primer extension of being combined with primer, has avoided the impact on next step single-basic extension.

Term " excision enzyme ExoI digestion ", its effect are that the end from single stranded DNA begins catalytic hydrolysis according to the order of sequence and forms between the dNTP of DNA 3, and the 5-phosphodiester bond makes single stranded DNA finally be hydrolyzed to dNTP.Be used for the rear remaining PCR primer of degraded PCR reaction in the technical program.Because excision enzyme can can't the PCR primer excision of strand occur in detection window, when therefore using this excision enzyme, the PCR primer that uses need not to comprise the protection base.

Term " single-basic extension ", be referred to as again micrometering order (mini sequence), finger adds extension primer and ddNTP in system, ddNTP and the 3' end that extends primer are connected to form extension products (be primer extension a base), according to the base complementrity pair principle, determine concrete which kind of ddNTP of connection by SNP site genotype, this process is similar to that in the PCR process, dNTP according to the based composition of complementary strand, adds on the PCR primer one by one.Due to " ddNTP " different from common dNTP be, 3' position at ribodesose lacks a hydroxyl, can not form phosphodiester bond with follow-up ddNTP, thereby, extend primer and only connect a ddNTP in the SNP site, and can not as PCR, constantly toward downward-extension, therefore be referred to as single-basic extension.Single-basic extension and order-checking process are closely similar, what add in the order-checking system is the mixture of dNTP and ddNTP, and sequencing primer will continue to extend after connecting dNTP, after only having the ddNTP of connection, stop to extend the side, and therefore order-checking produces is the mixture of nucleotide fragments different in size; Add in the single-basic extension system and only have ddNTP, extend primer and can only connect a ddNTP, and stop extending, so the single-basic extension generation is to extend primer only to extend the nucleotide fragments of a base.

Term " testing product " refers to any conventional products for detection of the SNP loci gene type, 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, have molecular weight difference between them, as the molecular weight of ddATP, ddCTP, ddGTP, ddTTP be respectively 271.2Da, 247.2Da, 287.2Da, 327.1Da(wherein ddTTP be molecular weight after modifying).Extend different Nucleotide when extending primer according to the genotype in SNP site, will form molecular weight difference.By mass spectrometric detection, distinguishablely go out this species diversity.For example, if certain SNP site A/G is polymorphic, corresponding extension primer length is 22 bases (molecular weight 7200Da), when this SNP site is the A genotype, extending primer will extend a T Nucleotide and stop extending, form the extension products of 23 bases length, molecular weight 7487.2Da, when this SNP site is the G genotype, extending primer will extend a C Nucleotide and stop extending, form that 23 bases are long, the extension products of molecular weight 7447.2Da, have the molecular weight difference of 40Da between two kinds of products.Namely for this SNP site, if use the extension primer of this 7200Da, the G genotype is with the mass spectra peak of corresponding 7447.2Da, and the A genotype is with the mass spectra peak of corresponding 7487.2Da.In actual testing process, the user can observe 7200Da, 7447.2Da, 7487.2Da three places by software: if mass spectra peak appears in the 7200Da place, be to have part or all of extension primer to be combined with ddNTP; No matter whether the 7200Da place mass spectra peak occurs, if place's mass spectra peak only appears in 7447.2Da and 7487.2Da place, the genotype in this SNP site is homozygous, and corresponding with the position of mass spectra peak, as previously mentioned, 7447.2Da the corresponding G genotype of mass spectra peak, the mass spectra peak corresponding A genotype of 7487.2Da; If 7447.2Da and 7487.2Da two place's mass spectra peaks all occur, the genotype in this SNP site is heterozygous; If 7447.2Da and 7487.2Da two place's mass spectra peaks all do not occur, the failure of an experiment.

Term " purifying " refers to be used to reducing the treatment step of other materials on the impact of subsequent reactions in system to be checked.PCR product purification of the present invention has dual mode: the one, and separating impurity also abandons, and the 2nd, impurity is lost activity.Wherein, cutting glue purification, mistake purification column etc. is all to separate impurity by electrophoresis, purification column etc., and reclaims relatively pure PCR product, can think the first purifying mode, and this mode is generally consuming time, and complicated operation is when particularly sample size is large; The effect of alkaline phosphatase is degraded (also claiming " digestion ") dNTP, and the substrate that makes it can not continue as archaeal dna polymerase or single-basic extension enzyme participates in PCR or single base extension, thereby not interfere with subsequent reaction can be thought the second purifying mode.Should be noted that, independent excision enzyme ExoI does not play purification, mix with alkaline phosphatase when it when using, its effect is in advance with single stranded DNA (in the PCR product system after reaction is completed, mainly remaining PCR primer) be degraded into dNTP, then make dNTP continue degraded by alkaline phosphatase.Because the PCR primer is degraded, can not enter last mass spectrometric detection step, therefore, process if increase the ExoI excision enzyme in the plan purification step, need not so to use the PCR primer with protection base.In addition, before the single-basic extension step, because excision enzyme and alkaline phosphatase all pass through high temperature deactivation, therefore the extension primer of its non-degradable strand that adds in the single-basic extension step, ddNTP etc. avoid subsequent experimental is exerted an influence.

Term " detection window " refers to can be used for the scope of mass spectrometric detection nucleic acid molecule amount, is usually directed to the design reference scope of primer.Wherein, when the design extension primer, for different SNP sites, according to the sequence characteristic in these DNA zones, place, site, and the genotype in SNP site, can design molecular weight different extension primer and extension products, avoid different the extension between primer and product to have interference because molecular weight approaches, thereby can at a relatively broad detection window, as 4000-9000Da, realize the detection to a plurality of SNP site.

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

Term " SNP mutation frequency " refers to the probability of undergoing mutation in the SNP site.In theory, the present invention can detect simultaneously and have simultaneously 7 SNP sudden changes in single individuality.But in practice, the investigator finds, there is certain mutation frequency in different SNP sudden changes in individuality.The HapMap project is the project that international organization is detected in different crowd in the site SNP in human genome, the detailed frequency information of each site in different crowd arranged 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.For example:

(1) rs9923231 detects 43 samples, and wild homozygous (T/T) accounts for 88.4%, and heterozygous (C/T) accounts for 11.6%, and namely the frequency of mutant (C) in colony is 5.8%(11.6/2=5.8);

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

(3) 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 mutant (C) in colony is 4.4%(8.9/2=4.45);

(4) rs9332131, lack the HapMap data, in the test item of another 86 sample (sample is not limited to the Chinese Han descendants), wild homozygous (A/A) accounts for 98.8%, heterozygous (A/-) accounts for 1.2%, be mutant (disappearance, del) frequency in colony is 0.6%(1.2/2=0.6);

(5) rs2108622 detects 42 samples, and wild homozygous (C/C) accounts for 54.8%, and heterozygous (C/T) accounts for 40.5%, and suddenly change homozygous (T/T) accounts for 4.8%, and namely the frequency of mutant (T) in colony is 25%(40.5/2+4.8=25.05);

(6) rs11676382 detects 45 samples, and wild homozygous (C/C) accounts for 100%, and namely the frequency of mutant (G) in colony is 0%;

(6) rs339097 detects 43 samples, and wild homozygous (T/T) accounts for 95.3%, and heterozygous (C/T) accounts for 4.7%, and namely the frequency of mutant (C) in colony is 2.3%(4.7/2=2.3).Referring to:

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi rs=9923231；

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=9332131；

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi rs=2108622；

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi rs=11676382；

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi rs=339097。

To sum up, the site of detecting is except the mutant (25%) crowd's medium frequency is higher of rs2108622, and all the other are all below 6%, the mutant extremely rare (rs1799853, rs11676382) in indivedual sites, almost lose, therefore, the probability that the many places sudden change occurs simultaneously in 7 sites is extremely low.Yet because the sample size that above research relates to is too low, the quantity well below expection for example for the lower site of occurrence frequency (as 0.5%), needs to detect 200 karyomit(e)s of 100 people in theory, expected result could occur.Therefore, above data only show the situation that a plurality of SNP site mutations occur simultaneously, and its occurrence frequency is extremely low, but are not to mean that the present invention can not detect the situation that 7 SNP site mutations occur simultaneously.In practice, we find, PCR sequencing or fluorescence quantitative PCR method, and reaction that detects the SNP site of every increase will increase corresponding expense and time.By contrast, what sites to be checked are mass spectroscopy detection advantage no matter be, as long as can be at a reaction tubes internal reaction, reagent and the time of cost be constant, and this is also the advantage that the present invention detects a plurality of SNP site mutations simultaneously.

Should be pointed out that the detection method that the present invention relates to the relevant SNP of how to confirm warfarin dosage, do not relate to how to confirm warfarin dosage.Mainly by acting on the albumen of said gene coding, suppress vitamin K due to warfarin, thus the performance anticoagulation.The target site of this detection scheme, as (1639G/A), CYP4F2 gene rs2108622 site (V433M), GGCX gene rs11676382 site, CALU gene rs339097 site etc., VKORC1 gene rs9923231 site, all relevant with vitamin K, therefore, if the patient does not note at ordinary times, eat some more and contain vitamin K abundant food or vegetables, as tomato, all might cause discomfort, at this moment, need to take more warfarin and could eliminate the impact that too much vitamin K brings, this has been clinical common phenomenon.This proves absolutely, uses this programme only can provide reference as clinical application, some non-genetic factors (as medication person's food habits), and even random occurrence (as eating once in a while tomato) all might affect drug effect and dosage.In addition, because the present invention takes or taken at known needs to detect its related SNP in the patient of warfarin, and according to detected result and other clinical indices, judge dosage or dosage is carried out retrospective study, therefore this detection method does not belong to the diagnostic method of disease.And after learning patient's SNP somatotype, can only roughly predict needs of patients adjustment warfarin dosage, and concrete dosage need to design a model or further research according to this intermediate information, just can obtain dosage accurately.As previously mentioned, in Chinese patent application 200710135388.6, " the individual with medicament method for determination of amount of warfarin Chinese population ", for resulting SNP genotype, need to and introduce the dosage that just can estimate warfarin in designed regression equation in conjunction with patient age and body weight.In addition, after the people such as Xie Shuan (Chinese Journal of New Drugs the 20th the 13rd phase of volume in 2011) have also reported and have known the related SNP genotype, need the relevant medication model of design and revise at any time, could obtain appropriate dose before patient's administration according to the intermediate information that detects.Comprehensively described, the method of detection SNP involved in the present invention, it is neither the diagnostic method of disease, resulting intermediate information can not be directly used in definite dosage, can not be namely directly that therapeutic process is used, therefore it does not belong to methods for the treatment of yet, should be considered as identical with common SNP detection method.

Description of drawings

Fig. 1 is in embodiment four, to the detected result in 7 sites of A1 sample.

Fig. 2 is in embodiment four, to the detected result in 7 sites of A2 sample.

Fig. 3 is in embodiment four, to the detected result in 7 sites of A3 sample.

Fig. 4 is in embodiment four, to the detected result in 7 sites of A4 sample.

Fig. 5 is in embodiment four, to the detected result in 7 sites of A5 sample.

Fig. 6 is in embodiment four, to the detected result in 7 sites of A6 sample.

Fig. 7 is in embodiment four, to the detected result in 7 sites of A7 sample.

Fig. 8 is in embodiment four, to the detected result in 7 sites of A8 sample.

Fig. 9 is in embodiment four, to the detected result in 7 sites of A9 sample.

Figure 10 is in embodiment four, to the detected result in 7 sites of A10 sample.

Figure 11 is in embodiment four, detected result to A9 sample rs9923231 site, three dotted lines are respectively the theoretical peak of extending primer, mutant (G) extension products, wild-type (A) extension products from left to right, and detected result shows that this sample is the AG heterozygosis.

Figure 12 is in embodiment four, detected result to A9 sample rs1799853 site, three dotted lines are respectively the theoretical peak of extending primer, mutant (T) extension products, wild-type (C) extension products from left to right, and detected result shows that this sample is that CC isozygotys.

Figure 13 is in embodiment four, detected result to A9 sample rs1057910 site, three dotted lines are respectively the theoretical peak of extending primer, mutant (C) extension products, wild-type (A) extension products from left to right, and detected result shows that this sample is that AA isozygotys.

Figure 14 is in embodiment four, detected result to A9 sample rs9332131 site, three dotted lines are respectively the theoretical peak of extending primer, wild-type (A) extension products, mutant (Del, disappearance) extension products from left to right, and detected result shows that this sample is that AA isozygotys.

Figure 15 is in embodiment four, and to the detected result in A9 sample rs2108622 site, three dotted lines are respectively the theoretical peak of extending primer, wild-type (C) extension products, mutant (T) extension products from left to right, and detected result shows this sample CT heterozygosis.

Figure 16 is in embodiment four, detected result to A9 sample rs11676382 site, three dotted lines are respectively the theoretical peak of extending primer, wild-type (C) extension products, mutant (G) extension products from left to right, and detected result shows that this sample is that CC isozygotys.

Figure 17 is in embodiment four, and to the detected result in A9 sample rs339097 site, three dotted lines are respectively the theoretical peak of extending primer, mutant (G) extension products, wild-type (A) extension products from left to right, and detected result shows that this sample is that AA isozygotys.

Figure 18 is for being the detected result of the plasmid B1-B7 of wild-type, wherein to 7 sites:

Peak 1(5141.3) the complete extension primer SEQ ID No:15 of expression rs9923231 site unreacted,

Peak 2(5468.4) expression rs9923231 site wild-type (A),

Peak 3(5539.7) expression rs11676382 site wild-type (C),

Peak 4(5788.8) expression rs1057910 site wild-type (A),

Peak 5(5875.9) the complete extension primer SEQ ID No:18 of expression rs9332131 site unreacted,

Peak 6(6029.6) expression rs1799853 site wild-type (C),

Peak 7(6147.1) expression rs9332131 site wild-type (A),

Peak 8(6295.1) expression rs2108622 site wild-type (C),

Peak 9(6684.4) the complete extension primer SEQ ID No:21 of expression rs339097 site unreacted,

Peak 10(7011.5) expression rs339097 site wild-type (A).

Figure 19 is for being the detected result of the plasmid C1-C7 of mutant, wherein to 7 sites:

Peak 1(5141.3) the complete extension primer SEQ ID No:15 of expression rs9923231 site unreacted,

Peak 2(5292.5) the complete extension primer SEQ ID No:20 of expression rs11676382 site unreacted,

Peak 3(5388.5) expression rs9923231 site mutation type (G),

Peak 4(5579.7) expression rs11676382 site mutation type (G),

Peak 5(5764.8) expression rs1057910 site mutation type (C),

Peak 6(5875.9) the complete extension primer SEQ ID No:18 of expression rs9332131 site unreacted,

Peak 7(6010.9) expression rs1799853 site mutation type (T),

Peak 8(6163.1) expression rs9332131 site mutation type (Del),

Peak 9(6375) expression rs2108622 site mutation type (T),

Peak 10(6931.6) expression rs339097 site mutation type (G).

Figure 20 is in comparative examples one, to the sequencing result in A7 sample rs9923231 site, is shown as A and isozygotys.

Figure 21 is in comparative examples one, to the sequencing result in A7 sample rs1799853 site, is shown as C and isozygotys.

Figure 22 is in comparative examples one, to the sequencing result in A7 sample rs1057910 site, is shown as A and isozygotys.

Figure 23 is in comparative examples one, to the sequencing result in A7 sample rs9332131 site, is shown as A and isozygotys.

Figure 24 is in comparative examples one, to the sequencing result in A7 sample rs2108622 site, is shown as C and isozygotys.

Figure 25 is in comparative examples one, to the sequencing result in A7 sample rs11676382 site, is shown as C and isozygotys.

Figure 26 is in comparative examples one, to the sequencing result in A7 sample rs339097 site, is shown as A and isozygotys.

Embodiment

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

Embodiment one: design of primers and synthetic.

for VKORC1 gene rs9923231 site (1639G/A), CYP2C9 gene rs1799853 site (CYP2C9*2), CYP2C9 gene rs1057910 site (CYP2C9*3), CYP2C9 gene rs9332131 site (CYP2C9*6), CYP4F2 gene rs2108622 site (V433M), GGCX gene rs11676382 site, 7 gene polymorphic sites relevant to the warfarin dosage such as CALU gene rs339097 site, design corresponding specific PCR primer core sequence (SEQ ID No:1 to SEQ ID No:14) and specificity and extend primer core sequence (SEQ ID No:15 to SEQ ID No:21).

Wherein, enter the mass spectrograph detection window and the Interference Detection effect for fear of the PCR primer, the 5' end of every PCR primer can increase the base of certain number on the basis of core sequence (SEQ ID No:1 to SEQ ID No:14), common tag(ACGTTGGATG as 10bp), so that the increase of the molecular weight of PCR primer, thereby exceed the mass spectrograph detection window.

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

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

Embodiment two: sample DNA extracts.

Collecting clinical manifestation is the patient of warfarin resistance, totally 10 examples.Wherein, the requirements to specifications such as sample collection, DNA extraction gather people's venous blood, and collect with the EDTA anticoagulant tube.Requirement to specifications, the blood of collection should not surpass a week 2-8 ℃ of preservation, and-20 ℃ of preservations should not surpass one month, and can adopt the on the rocks or bubble chamber sealing on the rocks of curling stone to transport, and suggestion adopts fresh blood to carry out extracting genome DNA as far as possible.Because not providing the human gene group DNA, this test kit do not extract reagent, therefore adopt business-like nucleic acid extraction kit (as the DNeasy Blood and Tissue kit of QIAGEN company), extract the human gene group DNA from every patient's 200ul whole blood, with NanoDrop 2000(Thermo company) quantitatively, and markization is respectively A1-A10 to 30ng/ul().Wherein, the human gene group DNA that it is 30ng/ul to concentration that this test kit is recommended detects, but the 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 should not surpass a week 2-8 ℃ of preservation, and-20 ℃ of preservations should not surpass 2 years, but-80 ℃ of prolonged preservation should be avoided multigelation, and be placed in ice chest and transport.

Embodiment three: biological experiment.

Use ABI 9700 type PCR instrument, by specification is tested to 7 relevant to warfarin dosage gene polymorphic sites.

The component that is used for PCR, PCR product purification and single-basic extension in test kit is:

Sequence number	The component title	Main component	Specification
					1	PCR primer mixed solution	The PCR primer	24ul/ pipe x1 pipe
2	The PCR reaction solution	Taq enzyme, dNTP	72ul/ pipe x1 pipe
				3	Endonuclease reaction liquid	The SAP enzyme	48ul/ pipe x1 pipe
4	Extend the primer mixed solution	Extend primer	24ul/ pipe x1 pipe
				5	Extension liquid	Single-basic extension enzyme, ddNTP	24ul/ pipe x1 pipe
6	Positive quality control product	Human gene group DNA (30ng/ul)	10ul/ pipe x1 pipe

By specification, concrete operation method is as follows:

1.PCR amplification

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

1.2 take out PCR primer mixed solution, PCR reaction solution from test kit, it is thawed naturally, the vortex vibration makes its abundant mixing, and is instantaneous centrifugal to managing the end;

1.3 according to number of samples, the ratio of according to the form below is taken out PCR primer mixed solution and PCR reaction solution, is placed in a centrifuge tube mixing, adds the 4ul mixture to carry out packing by every PCR reaction tubes.Due in minute process of assembling, the factor such as suction pipette head is residual may cause and be not enough to minute take on required umber, and suggestion is the dose volume of amplification mixture suitably.When 10 parts of testing samples are for example arranged, can be by 10.5-11 duplicate samples preparating mixture.

The component title	The single reaction volume
		PCR primer mixed solution	1ul
The PCR reaction solution	3ul
		Add up to	4ul

1.4 add the 1ul testing sample in the pcr amplification district in every pipe mixture, making every part of PCR reaction system cumulative volume is 5ul.Wherein, negative control is purified water, and blank is not for adding template.

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

2.SAP enzymic digestion

Add 2ul endonuclease reaction liquid in the PCR reaction tubes, then the PCR reaction tubes is placed in the pcr amplification instrument, carry out lower list procedure.

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

3. extend

3.1 in PCR dosing district, according to number of samples, the ratio of according to the form below is taken out and is extended primer mixed solution and extension liquid, is placed in a centrifuge tube mixing.Due in minute process of assembling, the factor such as suction pipette head is residual may cause and be not enough to minute take on required umber, and suggestion is the dose volume of amplification mixture suitably.When for example having 10 parts of enzymes to cut product, can be by 10.5-11 duplicate samples preparating mixture.

The component title	The single reaction volume
		Extend the primer mixed solution	1ul
Extension liquid	1ul
		Add up to	2ul

3.2 in the pcr amplification district, cut product by every pipe enzyme and add the 2ul mixture to carry out packing.

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

4. purifying

The district adds the 16ul purified water in every pipe extension products at pcr amplification, and the 6mg resin was put upside down mixing 30 minutes.

5. point sample

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

Embodiment four: upper machine testing and interpretation as a result.

Target sheet after using Clin-TOF type time-of-flight mass spectrometer that YiXin Industry (Beijing) Science and Technology Ltd. produces to point sample detects with result and judges.

Wild-type contrast B1-B7, the mutant contrast C1-C7 in above site are set respectively in addition.Wherein, wild-type contrast B1-B7, mutant contrast C1-C7 are respectively from the artificial plasmid of commercially available or laboratory preservation.Wild-type control plasmid B1-B7 and mutant control plasmid C1-C7 used in the present invention, for in commercialization plasmid pMD18-T Vector(Takara company) the basis on, ordinary method according to " molecular cloning " record, after carrying out PCR with primer and normal people DNA, the PCR product is inserted pMD18-T Vector, namely build wild plasmid B1-B7, then distinguish rite-directed mutagenesis, namely build 7 mutant plasmid C1-C7.But described plasmid B1-B7 and C1-C7 prolonged preservation are in-20 ℃ of glycerine, and the used time activates and extract plasmid DNA.

As shown in aforementioned table 2, article 7, extend primer and they and have different molecular weight according to the extension products that genotype separately produces on 7 gene polymorphic sites, these molecular weight correspondences mass spectra peak separately, if mass spectra peak appears in the place at certain molecular weight, be judged as and have the material corresponding with this molecular weight (extension primer or product):

Judging criterion:

(1) if mass spectra peak corresponding to wild-type and mutant all occurs, no matter extend the mass spectra peak that primer pair answers and whether exist, all be judged as the failure of an experiment;

(2) if mass spectra peak corresponding to wild-type or mutant only occurs one, be judged as corresponding genotypic homozygous of mass spectra peak appear;

(3) if mass spectra peak corresponding to wild-type or mutant all occurs, be judged as heterozygous.

The mass spectrum result is as shown in Fig. 1-19, and wherein Figure 18 is the mass spectrum that 7 SNP sites are the plasmid B1-B7 of wild-type, and Figure 19 is the mass spectrum that 7 SNP sites are the plasmid C1-C7 of mutant.

The molecular weight that extends primer and extension products with each site shown in aforementioned table 2 checks the mass spectrum result (Fig. 1-10) of sample A1-A10, determines the genotype in each SNP site, and result is as shown in table 3:

A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

rs9923231

A

A

GA

A

GA

A

A

A

GA

A

rs1799853

C

C

C

C

C

C

C

C

C

C

rs1057910

A

A

A

A

A

A

A

A

A

A

rs9332131

A

A

A

A

A

A

A

A

A

A

rs2108622

CT

CT

CT

CT

CT

CT

C

CT

CT

CT

rs11676382

C

C

C

C

C

C

C

C

C

C

rs339097

A

A

A

A

A

A

A

A

A

A

Table 3

Be namely in the patient of warfarin resistance in 10 routine clinical manifestations, detect altogether rs9923231 site GA heterozygous 3 examples, rs2108622 site CT heterozygote 9 examples.

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 that produce between different experiments have comparability, sequence verification adopts the human gene group DNA (A1-A10) who extracts the venous blood that gathers in embodiment two in 10 routine patient bodies.

Three, order-checking is identified

1, the PCR reaction system is 25 μ l

2, reaction conditions: reaction is carried out on ABI company 9700 thermal cyclers, and reaction conditions is 94 ℃ of denaturations 5 minutes, 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 40 seconds, 35 circulations, after reaction finishes again 72 ℃ extended 7 minutes, 4 ℃ of preservations.

3, PCR product purification and order-checking

(1) add 50 microlitre lavation buffer solutions, mixing in 96 orifice plates that the PCR product is housed.

(2) it is transferred in Millipore purifying plate, be put on vacuum pump suction filtration approximately 3 minutes, see in the purifying plate not having water to get final product.

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

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

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

The required reagent of sequencing reaction should be fresh preparation, need to can use after autoclaved reagent must be sterilized.The required equipment of sequencing reaction (as first-class in 384 orifice plates, tip) should be cleaning sterile equally.

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

(7) the sequencing reaction system is 5 μ l, and all ingredients add-on is as follows: PCR product 3-10ng, BigDyev3.1 0.25 μ l, 5*BigDye buffer 0.875 μ l, primer 1.6pmol.

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

Step: 95 ℃, 5 minutes;

95 ℃, 10 seconds; 60 ℃, 4 minutes; Repeat 30 circulations;

4 ℃ keep until prepare purifying.

(9) add 20 μ l 80% ethanol, the centrifugal 30min of 4,000rpm in each hole.

(10) sample panel is placed on the paper handkerchief of rolling well, gets rid of in whizzer, speed 1000rpm when getting rid of.

(11) add 30 μ l 70% ethanol in every hole, the centrifugal 10min of 4000rpm gets rid of.

(12) repeat the 11st operation that goes on foot 2 times.

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

(14) add 5 μ l methane amides, sealer is in ℃ refrigerator of centrifugal being placed on-20.

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

(16) use ABI3730xl type genetic analyzer to carry out sequencing.

Four, result

To the A1-A10 sample, the described primer of table in use is respectively to carrying out rs9923231, rs1799853, rs1057910, rs9332131, rs2108622, rs11676382, rs339097(numbered sequence 1-7 respectively) order-checking, 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's numbering

Sequence

1

Sequence 2

Sequence 3

Sequence 4

Sequence 5

Sequence 6

Sequence 7

A1

A

C

A

A

CT

C

A

A2

A

C

A

A

CT

C

A

A3

GA

C

A

A

CT

C

A

A4

A

C

A

A

CT

C

A

A5

GA

C

A

A

CT

C

A

A6

A

C

A

A

CT

C

A

A7

A

C

A

A

C

C

A

A8

A

C

A

A

CT

C

A

A9

GA

C

A

A

CT

C

A

A10

A

C

A

A

CT

C

A

Table 5

Through comparing, result and the table 5 of table 3 are in full accord, and the accuracy of detection method of the present invention is described.

Embodiment five, the present invention are for the advisory opinion of determining the warfarin dosage

Result according to embodiment four and comparative examples one shows:

First group: patient A7 does not carry the mutator gene type, advises that this patient only considers its non-genetic factor when first decision dosage, as height, body weight, age, sex etc., and closely monitors the INR value, adjusts at any time;

Second group: patient A1, A2, A4, A6, A8, A10 all carry the mutator gene type on a site, decision on the warfarin dosage has certain impact, suggestion is when first decision dosage, except the factors such as the height of considering the patient, body weight, age, sex, also need consider the impact that transgenation brings, and closely monitor the INR value, adjust at any time;

The 3rd group: patient A3, A5, A9 all carry the mutator gene type on two sites, decision on the warfarin dosage has larger impact, suggestion is when first decision dosage, except the factors such as the height of considering the patient, body weight, age, sex, should consider emphatically the impact that transgenation brings, and closely monitor the INR value, adjust at any time;

Noticeable, the warfarin medicining condition is not simply to be determined by the gene test result, the patient of same gene test result, possible dosage is approximate, also may be also inconsistent, also need to consider clinical other indexs, as factors such as height, body weight, age, sexes.Therefore actual this how medication need the clinician comprehensively in many ways factor decide.

SEQUENCE LISTING

＜110〉to China

＜120〉for detection of primer system of the gene SNP relevant to the warfarin dosage and uses thereof

<130> 2012

<160> 35

<170> PatentIn version 3.5

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＜213〉artificial sequence

<400> 35

ttccatgagc gattcttccg 20

Claims (9)

1. primer sets compound that detects the gene polymorphic site relevant to the warfarin dosage, 7 places, wherein said primer sequence is:

2. the primer sets compound of claim 1; wherein above-mentioned PCR primer sequence is core sequence; and comprise that at the 5' end 5-15 of extra increase is protected base sequence; it is by increasing the molecular weight of core primers; avoid reacting remaining PCR primer and enter the mass spectrometric detection window, to avoid interference the detection effect.

3. the primer sets compound of claim 2, wherein this protection base sequence tag:ACGTTGGATG that is 10bp.

4. the primer sets compound of claim 3, wherein extend the increase of primer 5' end as 1-15 base sequence of joint.

5. by the prepared testing product for detection of warfarin dosage genes involved polymorphic site of the primer sets compound of claim 1 to 4 any one, wherein said testing product comprises detection reagent, detection chip, detection carrier, and detection kit.

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

(1) be used for the reaction reagent of PCR, comprise: described specific PCR primer, resistant to elevated temperatures archaeal dna polymerase, dNTPs, PCR reaction buffer;

(2) be used for the reagent of PCR product purification;

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

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

8. the testing product of claim 6, the reagent that wherein is used for the PCR product purification is selected from alkaline phosphatase, or alkaline phosphatase and excision enzyme ExoI, or running gel reclaims reagent, or PCR product purification post.

9. the product of claim 8, wherein when comprising the purified reagent of alkaline phosphatase and excision enzyme ExoI, the PCR primer that uses need not to comprise the protection base.

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