CN109402249A - A kind of method and kit detecting CYP2C19 gene pleiomorphism - Google Patents

Abstract

The present invention provides a kind of methods and kit for detecting CYP2C19 gene pleiomorphism, specifically, the present invention is combined using ARMS-PCR method with addition PCR amplification blocking agent, and the kit of a kind of high specific, high sensitivity, high throughput, a kind of detection CYP2C19 gene pleiomorphism easy to operate is provided.It is combined by R & D design ARMS primer with PCR amplification blocking agent, substantially increases specificity and accuracy.Therefore, the present invention provides a kind of high sensitivities, primer, the probe of the detection CYP2C19 gene pleiomorphism of high specificity, and provide it is a kind of it is easy to operate, result is accurate, convenient for popularization detection CYP2C19 gene pleiomorphism kit and method.

Description

A kind of method and kit detecting CYP2C19 gene pleiomorphism

Technical field

The invention belongs to biotechnologys and detection field, specifically, the present invention relates to a kind of detection CYP2C19 gene is more The method and kit of state property

Background technique

Cytochrome P450 (cytochrome P450, referred to as CYP450) is a kind of heme-thiolate proteins Superfamily, it participate in endogenous material and the exogenous material including pharmaceutical environment compound metabolism.And CYP2C19 It is one of most important drug metabolic enzyme in CYP450 family, it is appropriate that genetic polymorphism affects clopidogrel, Omeprazole, benzene The metabolism of many drugs such as English sodium and diazepam.The CYP2C19 assignment of genes gene mapping in No. 10 chromosome q24.1-24.3 zone of people, Wild type is CYP2C19*1/*1 type.Often there is gene mutation in Chinese population, common mutations type is CYP2C19*2 type (outside the 5th Show sub- G681A) and CYP2C19*3 type (the 4th exon G636A).Sim in 2006 etc. has found a kind of new CYP2C19 equipotential Gene C YP2C19*17, the mutational site having is: C806T, CYP2C19*17 allele also have higher frequency in crowd Rate occurs.

CYP2C19 gene pleiomorphism is mainly caused by the mutation of two classes: one kind is to reduce the mutation of enzymatic activity, such as CYP2C19* 2, (specific mutation can refer to document Tantry US, Bliden KP, Wei C, et al.First analysis to CYP2C19*3 of the relation between CYP2C19 genotype and pharmacodynamics in patients treated with ticagrelor versus clopidogrel:The ONSET/OFFSET and RESPOND genotype studies.Circulation:Cardiovascular Genetics,2010,3(6):556-566.)；It is another Class is zymosthenic mutation, and only discovery has CYP2C19*17 (specific mutation can refer to document Frere C, Cuisset at present T,Gaborit B,et al.The CYP2C19*17 allele is associ-ated with better platelet response to clopidogrel in patients admitted for non-ST acute coronary syndrome.J Thromb Haemost,2009,7(8):1409-1411.).CYP2C19 hereditary variation can lead to enzymatic activity Individual difference can be divided into fast metabolic pattern (extensive metabolizer, EM), intermediate supersession (intermediary Metabolizer, IM) and slow inactivation (poormetabo lizer, PM).Slow inactivation individual carries two reductions The mutational site of CYP2C19 enzymatic activity, such as CYP2C19*2/*2, CYP2C19*3/*3；Fast metabolic pattern person then carries two enhancings The mutational site of enzymatic activity, such as CYP2C19*17/*17；Intermediate supersession type person only carries reduction or zymosthenic prominent Conjugate point, CYP2C19*1/*2, CYP2C19*1/*3, CYP2C19*1/*17.CYP2C19*2 type and CYP2C19*3 type can cause The enzymatic activity of CYP2C19 gene coding is lost, and the reduced capability of metabolism substrate increases blood concentration, in Normal therapeutic dose Shi Yi causes accumulation of poisoning.Poor metabolizer 99% in Chinese population is CYP2C19*2 type and CYP2C19*3 type allele, The enzymatic activity enhancing that CYP2C19*17 type then causes CYP2C19 gene to encode, the ability enhancing of metabolism substrate, it is therefore desirable to increase Drug dose.

Clopidogrel is current clinical common platelet receptor P2Y12 inhibitor, belongs to Thienopyridines inactive precursor Drug is converted into activated product after liver cytochrome enzyme P450 systemic metabolism, by blocking adenosine diphosphate (ADP) (ADP) Receptor inhibits platelet aggregation, and then pathologic thrombus is prevented to be formed.Clopidogrel is widely used in acute coronary in clinic Superior mesenteric artery syndrome (acute coronarysyndrome, ACS) and percutaneous coronary artery stent implantation (percutaneous Coronary stent implantation, PCI) patient afterwards, can significantly reduce the non-revascularization patient of ACS case fatality rate and Other cardiovascular event incidences.Clopidogrel metabolism related gene polymorphism is the important of antiplatelet drug individuation difference The polymorphism of internal factor, especially CYP2C19 afunction gene (Loss of function LOF).After being implanted into bracket Using in the cardiovascular patient of clopidogrel, CYP2C19*2, * 3 gene carrier's ratio CYP2C19*1 gene carriers are more Easily thrombus after generation bracket, but cardiovascular event and bleeding rate are suitable.And take Effect of Clopidogrel in Treating CYP2C19*17 gene mutation cardiovascular patient, cardiovascular event occurrence risk is relatively low, but bleeding episodes are sent out Raw risk is relatively high.In the patient for receiving the Effect of Clopidogrel in Treating of routine dose, it is low that drug effect occurs in about 4%-30%, Such event is considered as that the reactivity attenuating of clopidogrel or anergy, this phenomenon are referred to as clopidogrel Resistant (clopidogrel resistance,CR).Correlative study shows, CYP2C19*2, * 3 gene carriers are more in patients with coronary heart disease It is easy to happen clopidogrel Resistant.According to the requirement of U.S. FDA, before patient is using clinical applications drugs such as clopidogrels, it is proposed that CYP2C19 gene pleiomorphism is detected, to ensure drug safety.

The method of currently used detection CYP2C19 gene pleiomorphism has:

(1) PCR- direct sequencing is the goldstandard for detecting point mutation, and this method belongs to qualitative detection, and advantage is Directly acquire sequence, it is possible to find new variant sites, but sensitivity is not high, and complicated for operation, the cost is relatively high, and speed is slow, logical It measures low.

(2) PCR- high-resolution melting curve (HRM) method, this method is easy to operate, quick, flux is big, use cost is low, But it has higher requirements to the sensitivity and resolution ratio of instrument.

(3) PCR- gene chips, this method flux is high, can detect simultaneously to multiple SNP sites to be measured, but cost Height needs specific apparatus and equipment.

(4) real-time fluorescence PCR method, flux is higher, easy to operate, and instrument and equipment is easily universal, but probe is costly.

(5) PCR- restriction fragment length polymorphism method, without special instrument and equipment, cost is relatively low, experimentation Simply, strong operability, but flux is low, is only applicable to part SNP parting.

(6) ARMS-PCR method, this method can be used for detecting various types of SNP, and advantage is high sensitivity, especially suitable It is detected together in the somatic mutation in tumor tissues；The disadvantage is that being likely to occur false positive.

Therefore, there is an urgent need in the art to develop high specific, high sensitivity, high throughput, detection CYP2C19 easy to operate The technology of gene pleiomorphism.

Summary of the invention

The purpose of the present invention is to provide a kind of methods and kit for detecting CYP2C19 gene pleiomorphism.

In the first aspect of the present invention, a kind of kit for detecting CYP2C19 gene pleiomorphism, the kit are provided It include: the first component, first component includes targeting the primer pair and targeting CYP2C19*2 gene of CYP2C19*2 gene Probe；Wherein, the primer pair of the targeting CYP2C19*2 gene includes forward primer shown in SEQ ID NO.:1, the target Probe to CYP2C19*2 gene includes that probe is shared shown in SEQ ID NO.:2.

In another preferred example, the primer pair of the targeting CYP2C19*2 gene further include: shown in SEQ ID NO.:3 Wild type ARMS reverse primer.

In another preferred example, the primer pair of the targeting CYP2C19*2 gene further include: shown in SEQ ID NO.:4 Saltant type ARMS reverse primer.

In another preferred example, first component further includes targeting shown in the SEQ ID NO.:5 of CYP2C19*2 gene Wild type PCR amplification blocking agent.

In another preferred example, first component further includes targeting shown in the SEQ ID NO.:6 of CYP2C19*2 gene Saltant type PCR amplification blocking agent.

In another preferred example, the kit further include: the second component, second component include targeting CYP2C19* The primer pair of 3 genes and the probe of targeting CYP2C19*3 gene；Wherein, the primer pair of the targeting CYP2C19*3 gene includes The probe of forward primer shown in SEQ ID NO.:7, the targeting CYP2C19*3 gene includes shown in SEQ ID NO.:8 Share probe.

In another preferred example, the primer pair of the targeting CYP2C19*3 gene further include: shown in SEQ ID NO.:9 Wild type ARMS reverse primer.

In another preferred example, the primer pair of the targeting CYP2C19*3 gene further include: shown in SEQ ID NO.:10 Saltant type ARMS reverse primer.

In another preferred example, first component further includes targeting shown in the SEQ ID NO.:11 of CYP2C19*3 gene Wild type PCR amplification blocking agent.

In another preferred example, first component further includes targeting shown in the SEQ ID NO.:12 of CYP2C19*3 gene Saltant type PCR amplification blocking agent.

In another preferred example, the kit further include: third component, the third component include targeting CYP2C19* The primer pair of 17 genes and the probe of targeting CYP2C19*17 gene；Wherein, the primer pair of the targeting CYP2C19*17 gene Including forward primer shown in SEQ ID NO.:13, the probe of the targeting CYP2C19*17 gene includes SEQ ID NO.:14 Shown in share probe.

In another preferred example, the primer pair of the targeting CYP2C19*17 gene further include: shown in SEQ ID NO.:15 Wild type ARMS reverse primer.

In another preferred example, the primer pair of the targeting CYP2C19*17 gene further include: shown in SEQ ID NO.:16 Saltant type ARMS reverse primer.

In another preferred example, first component further includes targeting the SEQ ID NO.:17 institute of CYP2C19*17 gene The wild type PCR amplification blocking agent shown.

In another preferred example, first component further includes targeting the SEQ ID NO.:18 institute of CYP2C19*17 gene The saltant type PCR amplification blocking agent shown.

In another preferred example, the kit further include interior label primer to and internal standard probe.

In another preferred example, the interior label primer is to including internal standard forward primer and SEQ shown in SEQ ID NO.:19 The internal standard reverse primer of ID NO.:20.

In another preferred example, the internal standard probe is as shown in SEQ ID NO.:21.

In another preferred example, the kit further includes hot start Taq polymerase, UNG enzyme and dUTPs.

In another preferred example, the kit includes the first container, includes in the first container:

The SEQ for targeting forward primer shown in the SEQ ID NO.:1 of CYP2C19*2 gene, targeting CYP2C19*2 gene Wild type ARMS shown in the SEQ ID NO.:3 of probe, targeting CYP2C19*2 gene is shared shown in ID NO.:2 reversely to draw Object targets saltant type PCR amplification blocking agent shown in the SEQ ID NO.:6 of CYP2C19*2 gene；And/or

The SEQ for targeting forward primer shown in the SEQ ID NO.:7 of CYP2C19*3 gene, targeting CYP2C19*3 gene Wild type ARMS shown in the SEQ ID NO.:9 of probe, targeting CYP2C19*3 gene is shared shown in ID NO.:8 reversely to draw Object targets saltant type PCR amplification blocking agent shown in the SEQ ID NO.:12 of CYP2C19*3 gene；And/or

It targets forward primer shown in the SEQ ID NO.:13 of CYP2C19*17 gene, target CYP2C19*17 gene Probe is shared shown in SEQ ID NO.:14, targets wild type ARMS shown in the SEQ ID NO.:15 of CYP2C19*17 gene Reverse primer targets saltant type PCR amplification blocking agent shown in the SEQ ID NO.:18 of CYP2C19*17 gene.

It in another preferred example, also include interior label primer and internal standard probe in the first container；Preferably, the internal standard Primer includes reverse primer shown in forward primer shown in SEQ ID NO.:19 and SEQ ID NO.:20；Preferably, described Internal standard probe is as shown in SEQ ID NO.:21.

In another preferred example, the kit includes second container, includes in the second container:

The SEQ for targeting forward primer shown in the SEQ ID NO.:1 of CYP2C19*2 gene, targeting CYP2C19*2 gene Saltant type ARMS shown in the SEQ ID NO.:4 of probe, targeting CYP2C19*2 gene is shared shown in ID NO.:2 reversely to draw Object targets wild type PCR amplification blocking agent shown in the SEQ ID NO.:5 of CYP2C19*2 gene；And/or

The SEQ for targeting forward primer shown in the SEQ ID NO.:7 of CYP2C19*3 gene, targeting CYP2C19*3 gene Saltant type ARMS shown in the SEQ ID NO.:10 of probe, targeting CYP2C19*3 gene is shared shown in ID NO.:8 reversely to draw Object targets wild type PCR amplification blocking agent shown in the SEQ ID NO.:11 of CYP2C19*3 gene；And/or

It targets forward primer shown in the SEQ ID NO.:13 of CYP2C19*17 gene, target CYP2C19*17 gene Probe is shared shown in SEQ ID NO.:14, targets saltant type ARMS shown in the SEQ ID NO.:16 of CYP2C19*17 gene Reverse primer targets wild type PCR amplification blocking agent shown in the SEQ ID NO.:17 of CYP2C19*17 gene.

It in another preferred example, also include interior label primer and internal standard probe in the second container；Preferably, the internal standard Primer includes reverse primer shown in forward primer shown in SEQ ID NO.:19 and SEQ ID NO.:20；Preferably, described Internal standard probe is as shown in SEQ ID NO.:21.

In another preferred example, the kit further includes third container, includes in the third container: thermal starting Taq Enzyme, UNG enzyme, and/or dUTPs.

In another preferred example, the kit further includes the 4th container, includes positive control in the 4th container；It is excellent Selection of land, the positive control are the heterozygous containing this 3 kinds of allele of CYP2C19*2, CYP2C19*3, CYP2C19*17 DNA。

In another preferred example, the kit further includes the 5th container, includes negative control in the 5th container；It is excellent Selection of land, the negative control are pure water.

In another preferred example, each probe is respectively provided with fluorescent reporter group and fluorescent quenching group；Preferably, each probe It is respectively provided with different fluorescent reporter groups and different fluorescent quenching groups.Preferably, sharing for CYP2C19*2 gene is targeted Probe has FAM fluorescent reporter group and BHQ1 fluorescent quenching group.Preferably, the shared probe of CYP2C19*3 gene is targeted With VIC fluorescent reporter group and BHQ2 fluorescent quenching group.Preferably, the shared probe for targeting CYP2C19*17 gene has Texas Red fluorescent reporter group and TAMRA fluorescent quenching group.

The second aspect of the present invention, provide it is a kind of detect CYP2C19 gene pleiomorphism method, the method includes with Lower step:

(1) DNA sample of object to be detected is provided；

(2) it prepares quantitative fluorescent PCR reaction system and carries out fluorescence quantitative PCR detection:

Wherein, the quantitative fluorescent PCR reaction system includes that the first quantitative fluorescent PCR reaction system and the second fluorescence are fixed Measure PCR reaction system；

The first quantitative fluorescent PCR reaction system includes:

The SEQ for targeting forward primer shown in the SEQ ID NO.:1 of CYP2C19*2 gene, targeting CYP2C19*2 gene Wild type ARMS shown in the SEQ ID NO.:3 of probe, targeting CYP2C19*2 gene is shared shown in ID NO.:2 reversely to draw Object targets saltant type PCR amplification blocking agent shown in the SEQ ID NO.:6 of CYP2C19*2 gene；And/or

The SEQ for targeting forward primer shown in the SEQ ID NO.:7 of CYP2C19*3 gene, targeting CYP2C19*3 gene Wild type ARMS shown in the SEQ ID NO.:9 of probe, targeting CYP2C19*3 gene is shared shown in ID NO.:8 reversely to draw Object targets saltant type PCR amplification blocking agent shown in the SEQ ID NO.:12 of CYP2C19*3 gene；And/or

It targets forward primer shown in the SEQ ID NO.:13 of CYP2C19*17 gene, target CYP2C19*17 gene Probe is shared shown in SEQ ID NO.:14, targets wild type ARMS shown in the SEQ ID NO.:15 of CYP2C19*17 gene Reverse primer targets saltant type PCR amplification blocking agent shown in the SEQ ID NO.:18 of CYP2C19*17 gene；

The second quantitative fluorescent PCR reaction system includes:

The SEQ for targeting forward primer shown in the SEQ ID NO.:1 of CYP2C19*2 gene, targeting CYP2C19*2 gene Saltant type ARMS shown in the SEQ ID NO.:4 of probe, targeting CYP2C19*2 gene is shared shown in ID NO.:2 reversely to draw Object targets wild type PCR amplification blocking agent shown in the SEQ ID NO.:5 of CYP2C19*2 gene；And/or

The SEQ for targeting forward primer shown in the SEQ ID NO.:7 of CYP2C19*3 gene, targeting CYP2C19*3 gene Saltant type ARMS shown in the SEQ ID NO.:10 of probe, targeting CYP2C19*3 gene is shared shown in ID NO.:8 reversely to draw Object targets wild type PCR amplification blocking agent shown in the SEQ ID NO.:11 of CYP2C19*3 gene；And/or

It targets forward primer shown in the SEQ ID NO.:13 of CYP2C19*17 gene, target CYP2C19*17 gene Probe is shared shown in SEQ ID NO.:14, targets saltant type ARMS shown in the SEQ ID NO.:16 of CYP2C19*17 gene Reverse primer targets wild type PCR amplification blocking agent shown in the SEQ ID NO.:17 of CYP2C19*17 gene.

In another preferred example, the first quantitative fluorescent PCR reaction system further include: hot start Taq polymerase, UNG enzyme, And/or dUTPs.

In another preferred example, the second quantitative fluorescent PCR reaction system further include: hot start Taq polymerase, UNG enzyme, And/or dUTPs.

In another preferred example, during the fluorescence quantitative PCR detection, fluorescent quantitative PCR condition are as follows:

50 DEG C 2 minutes, 94 DEG C 5 minutes；94 DEG C 15 seconds, 58 DEG C 32 seconds, 40 circulation.

In another preferred example, the method is non-diagnostic purpose.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 shows 1 in CYP2C19*2-GG wild type 7 testing result.

Fig. 2 shows 1 in CYP2C19*2-AG heterozygous 2 testing result.

Fig. 3 shows CYP2C19*2-AA saltant type 1 testing result.

Fig. 4 shows 1 in CYP2C19*3-GG wild type 6 testing result.

Fig. 5 shows 1 in CYP2C19*3-AG heterozygous 2 testing result.

Fig. 6 shows CYP2C19*3-AA saltant type 1 testing result.

Fig. 7 shows 1 in CYP2C19*17-CC wild type 9 testing result.

Fig. 8 shows CYP2C19*17-TC heterozygous 1 testing result.

Fig. 9 shows CYP2C19*17-TT saltant type 1 testing result.

The testing result of Figure 10 visualizingre agent box positive quality control product.

The testing result of Figure 11 visualizingre agent box feminine gender quality-control product.

Figure 12 visualizingre agent box is using 15 whole bloods as the result of wherein 1 detection of template.

Figure 13 visualizingre agent box detects wherein 1 testing result for being limited to 0.5ng/ μ L.

Specific embodiment

The present inventor obtains a kind of method and examination for detecting CYP2C19 gene pleiomorphism by extensive and in-depth research Agent box, two amplification systems realize the polymorphic detection of three gene locis, have detection quickly, it is high-throughput, highly sensitive and The characteristics of reducing cost.

Specifically, the present invention is combined using ARMS-PCR method with addition PCR amplification blocking agent, is provided a kind of high special Property, high sensitivity, high throughput, a kind of detection CYP2C19 gene pleiomorphism easy to operate kit.Pass through R & D design ARMS primer is combined with PCR amplification blocking agent, substantially increases specificity and accuracy.Therefore, the present invention provides one kind High sensitivity, high specificity detection CYP2C19 gene pleiomorphism primer, probe, and provide a kind of easy to operate, result Accurately, convenient for the kit and method of the detection CYP2C19 gene pleiomorphism of popularization.

The present invention is based on ARMS-PCR technical fields, while PCR amplification blocking agent is added, in particular to a kind of detection Method, primer, probe and the kit including primer and probe mixing of CYP2C19 gene pleiomorphism.

The method of CYP2C19 gene pleiomorphism, primer, probe and including the primer and spy are detected the invention discloses a kind of The kit of needle mixing.Provide detection tri- gene locis of CYP2C19*2, CYP2C19*3 and CYP2C19*17 primer and Probe.The present invention is based on ARMS-PCR technical field, two amplification systems are realized the polymorphic detection of three gene locis, are had The characteristics of detecting quick, high-throughput, high sensitivity and reducing cost.In addition, the present invention is protected by the way that PCR amplification blocking agent is added Specificity is substantially increased while having stayed ARMS-PCR technological merit, and can be directly template amplification with whole blood, has exempted from DNA Extract cumbersome step, stopped pipe operation avoids the advantages such as cross contamination between sample.

Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because this Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and And it is not intended to be restrictive, the scope of the present invention will be limited only by the claims which follow.

Unless otherwise defined, otherwise whole technologies used herein and scientific term all have such as fields of the present invention The normally understood identical meanings of those of ordinary skill.As used herein, in use, term in mentioning the numerical value specifically enumerated " about " mean that the value can change not more than 1% from the value enumerated.For example, as used herein, statement " about 100 " includes 99 Hes 101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).

Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method And material, place enumerates preferred method and material herein.

The present invention is based on the technical fields that ARMS-PCR is combined with addition PCR amplification blocking agent, provide a kind of detection Method, primer, probe and the kit including primer and probe mixing of CYP2C19 gene pleiomorphism.Detection of the invention The method of CYP2C19 gene pleiomorphism, required number of probes is few, and optimization process is simple, it is easy quickly, precisely effectively, and can be with It is used for quickly detecting by whole blood for template.

It is preferably carried out in mode at of the invention one, the present invention provides a kind of detection CYP2C19 gene pleiomorphisms Primer and probe, for tri- gene polymorphism sites of CYP2C19*2, CYP2C19*3 and CYP2C19*17, design is shared just To primer, shared probe, wild type ARMS reverse primer, saltant type ARMS reverse primer, wild type PCR amplification blocking agent and Saltant type PCR amplification blocking agent, specific nucleotide sequence such as following table.

The primer and probe sequence situation table of 1 CYP2C19*2 genetic test of table

The primer and probe sequence situation table of 2 CYP2C19*3 genetic test of table

The primer and probe sequence situation table of 3 CYP2C19*17 genetic test of table

For the primer and probe of above-mentioned detection CYP2C19 gene pleiomorphism:

In a preferred embodiment of the present invention, for different gene polymorphism sites, saltant type and wild type are designed ARMS reverse primer.

In present aspect preferred embodiment, saltant type PCR amplification blocking agent carries out C6 spacer modification at 3 ' ends, Tm value is improved simultaneously at 60~70 DEG C, and first in conjunction with saltant type template, specific inhibition reversely draws saltant type PCR amplification blocking agent The combination of object and saltant type segment, to block the amplification of saltant type template, probe does not discharge fluorescence signal, when detection wild type When sample, reverse primer normally in conjunction with wild-type template, expands wild type target fragment, so that hydrolysis probes keep its release glimmering Optical signal improves the specificity of wild type system；In the same manner, wild type PCR amplification blocking agent carries out C6 spacer at 3 ' ends Modification, while Tm value is improved at 60~70 DEG C, wild type PCR amplification blocking agent is first in conjunction with wild-type template, specific inhibition The combination of reverse primer and wild-type fragment, to block the amplification of wild-type template, probe does not discharge fluorescence signal, works as detection When saltant type sample, reverse primer normally in conjunction with saltant type template, expands saltant type target fragment, so that hydrolysis probes make it Fluorescence signal is discharged, the specificity of saltant type system is improved.

Primer Tm designed by present aspect, in conjunction with the primer forward or backwards of collocation, avoids and draws near 55 DEG C The generation of object dimer, while MGB probe being selected to improve Tm value, make the Tm value of probe near 65 DEG C, avoids probe Sequence is too long and the combination of other non-specific sequences, at the same also avoid the mutual row of probe at primer dimer and Hairpin structure effectively improves the specificity and efficiency of PCR amplification.

In present aspect preferred embodiment, in the combination of the primer and probe, every primer concentration is 0.1~ 0.5 μm of ol/L, every concentration and probe concentration are 0.1~0.25 μm of ol/L.

In further preferred embodiment of the present invention, to reach testing goal, the equal band of the probe of all gene locis There are fluorescent reporter group and fluorescent quenching group, the wild type in the detection same gene site and the fluorophor of saltant type Identical, the fluorophor of institute's band is different between the detection different genes site, it is preferable that the CYP2C19*2 gene position Point fluorescent reporter group is selected from FAM, and fluorescent quenching group is selected from BHQ1.Preferably, the CYP2C19*3 gene loci fluorescence Reporter group is selected from VIC, and fluorescent quenching group is selected from BHQ2.Preferably, the CYP2C19*17 gene loci fluorescence report Group is selected from Texas Red, and fluorescent quenching group is selected from TAMRA.

It is preferably carried out in mode in of the invention another, the present invention provides a kind of detection CYP2C19 gene polymorphics Property detection kit, it includes above-mentioned primer and probes.Preferably, the kit by CYP2C19 wild type PCR reaction solution A, CYP2C19 saltant type PCR reaction solution A, CYP2C19PCR reaction solution B, CYP2C19 positive quality control product, negative quality-control product composition.

In present aspect preferred embodiment, a kind of detection CYP2C19 genetic polymorphism detection kit, instead Liquid A is answered to further comprise the detection primer and probe of one group of internal standard gene, specific nucleotide sequence is as follows:

The primer and probe sequence situation table of 4 internal standard gene of table detection

In a preferred embodiment of the present invention, a kind of detection CYP2C19 genetic polymorphism detection kit, it is interior It is different from the fluorophor of the target-probe to mark gene probe, it is preferable that the fluorescent reporter group is selected from CY5, glimmering Optical quenching group is selected from BHQ1.

In present aspect preferred embodiment, CYP2C19 wild type PCR reaction solution A be sensed by CYP2C19*2, The shared forward primer of CYP2C19*3, CYP2C19*17 gene pleiomorphism shares probe, wild type ARMS reverse primer, mutation Type PCR amplification blocking agent, interior label primer and probe, PCR reaction solution composition.

In present aspect preferred embodiment, CYP2C19 saltant type PCR reaction solution A be sensed by CYP2C19*2, The shared forward primer of CYP2C19*3, CYP2C19*17 gene pleiomorphism shares probe, saltant type ARMS reverse primer, wild Type PCR amplification blocking agent, interior label primer and probe, PCR reaction solution composition.

In further preferred embodiment of the present invention, wherein the PCR reaction solution is 8.0~8.8 by pH value Tris-HCl, MgCl2, KCl composition, the concentration for the ris-HCl that preferable ph is 8.0~8.8 is 50mmol/L, preferably MgCl2 Concentration be 3~8mmol/L, preferably the concentration of KCl be 150~350mmol/L.

In a preferred embodiment of the present invention, the CYP2C19PCR reaction solution B by hot start Taq polymerase, dUTPs and Antipollution UNG enzyme composition, the dosage that preferably every person-portion PCR reacts hot start Taq polymerase in enzyme system is 5~10U；It is preferred that UNG enzyme Dosage is 1.5~5U；It is preferred that dUTPs is 6~12mmol.

In present aspect preferred embodiment, a kind of detection CYP2C19 genetic polymorphism detection kit is mentioned Supply negative quality-control product for sterilizing pure water, positive quality control product is to contain this 3 kinds of CYP2C19*2, CYP2C19*3, CYP2C19*17 The heterozygous DNA of allele.

It is further carried out in mode in of the invention, the present invention also provides preferably using whole blood as one kind of template amplification The method for detecting CYP2C19 gene pleiomorphism, comprising the following steps:

(1) DNA directly is extracted using whole blood sample to be measured or using nucleic acid extraction kit.

(2) using the primer and probe for detecting CYP2C19 gene pleiomorphism described in first aspect, or using second party A kind of detection CYP2C19 genetic polymorphism detection kit described in face, using the whole blood or DNA to be measured in step (1) as template Carry out fluorescent PCR amplification.

(3) a kind of detection CYP2C19 genetic polymorphism detection kit, PCR amplification condition according to second aspect Are as follows: 50 DEG C 2 minutes, 94 DEG C 5 minutes；94 DEG C 15 seconds, 58 DEG C 32 seconds, 40 circulation.

(4) a kind of detection CYP2C19 genetic polymorphism detection kit according to second aspect, the judgement of PCR result It is determined by each sense channel Ct value, the testing result of the sample of the Ct value less than 35 in the channel internal standard CY5 is considered as effectively, other inspections It surveys channel C t value and is considered as the Air conduct measurement genic mutation type or wild-type positive less than 35.

Concrete outcome interpretation mode is as follows:

In present aspect preferred embodiment, the judgement of PCR result determines that internal standard CY5 is logical by each sense channel Ct value The testing result of the sample of the Ct value less than 35 in road is considered as effectively, other FAM, VIC, Texas Red sense channel Ct value are less than 35 are considered as the Air conduct measurement genic mutation type or wild-type positive.

In present aspect preferred embodiment, for above-mentioned detection method, preferably, being wherein directly using whole blood Template carries out PCR amplification, and whole blood dosage is 1ul~5ul, has exempted from DNA and has extracted cumbersome step, operates easier, stopped pipe operation, Avoid cross contamination between sample.

In present aspect preferred embodiment, it is further preferred that the PCR amplification condition are as follows: 50 DEG C 2 minutes, 94 DEG C 5 minutes；94 DEG C 15 seconds, 58 DEG C 32 seconds, 40 circulations, entirely the upper machine time greatly shortens, it is only necessary to can obtain within 70 minutes As a result.

In present aspect preferred embodiment, ARMS-PCR is high-precision to mutated target sequence progress using specific primer Quasi- PCR amplification amplification, PCR amplification blocking agent is added at the same time, is detected using probe to amplified production, in real-time fluorescence The detection to gene pleiomorphism in sample DNA is realized on quantitative PCR platform, to reach high specific and high sensitivity.

Main advantages of the present invention are:

(1) the present invention is based on the detections of ARMS-PCR technology, while PCR amplification blocking agent is added, and remain ARMS-PCR skill The characteristics of the advantages of art, substantially increases specificity simultaneously, is also provided with highly sensitive and high accuracy.

(2) present invention by two amplification systems may be implemented 3 gene loci CYP2C19*2, CYP2C19*3, The polymorphic detection of CYP2C19*17, relative to can only once detect a gene loci, more high-throughput detection, reduce detection at Sheet and manipulation strength are suitable for clinic.

(3) present invention can directly be the advantage of template amplification using whole blood, exempted from the step of DNA is extracted, operated simpler Just more rapidly, stopped pipe operates, and avoids cross contamination between sample.

(4) internal standard gene is added in two amplification systems of the invention simultaneously, and reducing extraction problem leads to false positive, and is added Yin and yang attribute quality-control product, can quality to reagent and environment with the presence or absence of pollution carry out control, improve the accuracy of kit, As a result more true and reliable.

(5) the present invention provides a kind of quickly upper machine condition, detection time is short, it is whole only need 70 minutes to go out as a result, Reduce at least 20 minutes significantly relative to the existing similar kit in market, easy to operate, detection quickly, and only needs real-time fluorescence Quantitative PCR apparatus can be realized, convenient for promoting.

Method of the invention is equally applicable to non-diagnostic purpose, for example, during new drug development, using of the invention Detection method obtains the gene mutation information for being used as intermediate result, these gene mutation information can be used as public health and manage it It needs, can be used for research and the targeting new drug development of drug metabolism mechanism.

Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip Part such as U.S. Sambrook.J etc. writes " Molecular Cloning: A Laboratory room guide " (Huang Peitang etc. is translated, Beijing: Science Press, 2002) Described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight It calculates.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.

1 kit of embodiment

Present embodiments provide a kind of detection kit for detecting CYP2C19 gene pleiomorphism, detection of the present invention The composition of kit is as follows:

Embodiment 2 is used to detect the detection method of CYP2C19 gene pleiomorphism

Step 1: the preparation of PCR reaction system

From kit take out CYP2C19 wild type PCR reaction solution A, CYP2C19 saltant type PCR reaction solution A and CYP2C19PCR reaction solution B, room temperature vibrates after melting to be mixed, and is used after 8,000rpm brief centrifugations.

The single part wild type PCR reaction tube system of CYP2C19 reagent is formulated as follows table:

The single part saltant type PCR reaction tube system of CYP2C19 reagent is formulated as follows table:

Each component is sufficiently mixed and is configured to corresponding CYP2C19 wild type PCR reaction tube and CYP2C19 saltant type PCR 20 μ l amplification systems are dispensed into PCR pipe by reaction tube, brief centrifugation.

Step 2: processing sample to be tested

Directly it is template using 30 whole blood samples to be measured or is extracted in whole blood sample using nucleic acid extraction kit DNA。

Step 3: sample-adding

Into above-mentioned PCR reaction tube with being separately added into test sample whole blood sample or DNA, CYP2C19 positive quality control product and Negative each 1~5 μ l of quality-control product.Pipe lid is covered tightly, augmentation detection area is transferred to after 8,000rpm brief centrifugations.

Step 4: PCR amplification (7500 fluorescence quantitative PCR instrument of ABI Prism)

Setup window is opened, by corresponding sequence setting positive and negative quality-control product and sample to be measured, and is arranged in the column Name Sample ID.All setting sample wells are chosen, are double-clicked, Add Detector is selected, selects Reporter for FAM and Quencher For none, reselection Reporter be HEX and Quencher is none；Reselection Reporter be Texas Red and Quencher is none；Then select Reporter for Cy5 and Quencher be none after close window.In Passive (none) is selected in Reference.Open instrument window be arranged cycling condition: 50 DEG C 2 minutes, 94 DEG C 5 minutes；94℃ 15 seconds, 58 DEG C 32 seconds, 40 circulation.It is all be provided with after save file, run.

Step 5 interpretation of result

According to the amplification curve that CYP2C19 wild type and saltant type amplification system detect, the results show that CYP2C19*2-GG Type is 7, and CYP2C19*2-AG type is 2, and CYP2C19*2-AA type is 1, and CYP2C19*3-GG is 7, CYP2C19*3- AG be 2, CYP2C19*3-AA type be 1, CYP2C19*17-CC type be 9,1, CYP2C19*17-TC type, CYP2C19* 17 types are 1, and representative genotypic results are as shown in Figure of description 1- Figure 11, with gold mark PCR sequencing PCR Comparative result hair It is existing, using the kit of detection CYP2C19 gene pleiomorphism provided by the invention, detect CYP2C19 gene pleiomorphism result Accuracy is 100%.

Embodiment 3 is using people's clinic whole blood sample as template detection CYP2C19 gene pleiomorphism

The present embodiment uses people's clinic whole blood sample and using it as template, and kit according to the invention is detected CYP2C19*2, CYP2C19*3, CYP2C19*17 gene pleiomorphism.

The PCR reaction system and PCR reaction condition that the present embodiment is had been described above using this specification, concrete operation method with Embodiment 2 is identical.The present embodiment chooses 15 representative Human clinical's whole blood samples, further, respectively 15 Example whole blood sample is directly added in PCR reacting hole, upper machine testing.It is compared by genotypic results and goldstandard sequencing It was found that 14, CYP2C19*1/*1 type are detected simultaneously by, and 2, CYP2C19*1/*2 type, 2, CYP2C19*1/*3 type, 2, CYP2C19*1/*17 type, representative genotypic results demonstrate as shown in Figure of description 12 and are with whole blood sample The accuracy of template detection.The present embodiment demonstrates the kit dependence extracting genome DNA that the present invention does not need current mainstream It with the cumbersome step such as purifying, operates easier, has saved a large amount of time and cost, directly for template be using whole blood sample The big characteristic of of the invention one.

Embodiment 4 is using human gene group DNA's sample as the sensitivity of template detection kit of the present invention

The present embodiment is using human gene group DNA's sample as the sensitivity of template detection kit of the present invention, specific detection method It is as follows:

The PCR reaction system and PCR reaction condition being had been described above using this specification, concrete operation method and embodiment 2 It is identical.Choosing 9 human gene group DNAs is template, and the type of 9 human gene group DNAs is respectively CYP2C19*2-GG, CYP2C19* 2-AG、CYP2C19*2-AA、CYP2C19*3-GG、CYP2C19*3-AG、CYP2C19*3-AA、CYP2C19*17-CC、 CYP2C19*17-TC, CYP2C19*17-TT, at the same be arranged 9 DNA profiling detection limits be respectively 0.1ng/ μ L, 0.5ng/ μ L, 1ng/μL,10ng/μL,100ng/μL.The results show that still can be apparent under the conditions of DNA template concentration is 0.5ng/ μ L The accurate parting in ground, representative genotypic results are as shown in Figure of description 13.Verifying proves through this embodiment, utilizes this The kit of invention, which carries out the detection of CYP2C19*2, CYP2C19*3, CYP2C19*17 Genotyping, has very high sensitivity, spirit Sensitivity is up to 0.5ng/ μ L.

Comparative example 1

The present inventor in the course of the research, has screened tens of groups of quantitative fluorescent PCRs for each mutational site target sequence and has used Primer and probe, wherein the problems such as overwhelming majority display specificity is low, poor sensitivity, multiplex amplification inefficient, is unable to satisfy and answers Use demand.Although simultaneously we have found that some probes only a poor nucleotide can significantly affect detection specificity.It is typical general Logical primer sequence and probe in detecting effect are as follows:

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

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Claims (10)

1. a kind of kit for detecting CYP2C19 gene pleiomorphism, which is characterized in that the kit includes: the first component, institute Stating the first component includes the primer pair for targeting CYP2C19*2 gene and the probe of targeting CYP2C19*2 gene；Wherein, the target Primer pair to CYP2C19*2 gene includes forward primer shown in SEQ ID NO.:1, the targeting CYP2C19*2 gene Probe includes that probe is shared shown in SEQ ID NO.:2.

2. kit as described in claim 1, which is characterized in that the primer pair of the targeting CYP2C19*2 gene further include: Wild type ARMS reverse primer shown in SEQ ID NO.:3；And/or

The primer pair of the targeting CYP2C19*2 gene further include: saltant type ARMS reverse primer shown in SEQ ID NO.:4； And/or

First component further includes targeting wild type PCR amplification shown in the SEQ ID NO.:5 of CYP2C19*2 gene to block Agent；And/or

First component further includes targeting saltant type PCR amplification shown in the SEQ ID NO.:6 of CYP2C19*2 gene to block Agent.

3. kit as described in claim 1, which is characterized in that the kit further include: the second component, described second group Divide includes the primer pair of targeting CYP2C19*3 gene and the probe of targeting CYP2C19*3 gene；Wherein, the targeting The primer pair of CYP2C19*3 gene includes forward primer shown in SEQ ID NO.:7, the spy of the targeting CYP2C19*3 gene Needle includes that probe is shared shown in SEQ ID NO.:8；

Preferably, the primer pair of the targeting CYP2C19*3 gene further include: wild type ARMS shown in SEQ ID NO.:9 is anti- To primer.

4. kit as described in claim 1, which is characterized in that the kit further include: third component, the third group Divide includes the primer pair of targeting CYP2C19*17 gene and the probe of targeting CYP2C19*17 gene；Wherein, the targeting The primer pair of CYP2C19*17 gene includes forward primer shown in SEQ ID NO.:13, the targeting CYP2C19*17 gene Probe include shown in SEQ ID NO.:14 share probe.

5. kit as described in claim 1, which is characterized in that the kit further include interior label primer to and internal standard visit Needle.

6. a kind of kit for detecting CYP2C19 gene pleiomorphism, which is characterized in that the kit includes the first container, institute It states in the first container and includes:

The SEQ ID for targeting forward primer shown in the SEQ ID NO.:1 of CYP2C19*2 gene, targeting CYP2C19*2 gene Wild type ARMS reverse primer shown in the SEQ ID NO.:3 of shared probe, targeting CYP2C19*2 gene shown in NO.:2, Target saltant type PCR amplification blocking agent shown in the SEQ ID NO.:6 of CYP2C19*2 gene；And/or

The SEQ ID for targeting forward primer shown in the SEQ ID NO.:7 of CYP2C19*3 gene, targeting CYP2C19*3 gene Wild type ARMS reverse primer shown in the SEQ ID NO.:9 of shared probe, targeting CYP2C19*3 gene shown in NO.:8, Target saltant type PCR amplification blocking agent shown in the SEQ ID NO.:12 of CYP2C19*3 gene；And/or

The SEQ for targeting forward primer shown in the SEQ ID NO.:13 of CYP2C19*17 gene, targeting CYP2C19*17 gene It is reversed that wild type ARMS shown in the SEQ ID NO.:15 of probe, targeting CYP2C19*17 gene is shared shown in ID NO.:14 Primer targets saltant type PCR amplification blocking agent shown in the SEQ ID NO.:18 of CYP2C19*17 gene.

7. kit as claimed in claim 6, which is characterized in that the kit further includes second container, and described second holds Include in device:

The SEQ ID for targeting forward primer shown in the SEQ ID NO.:1 of CYP2C19*2 gene, targeting CYP2C19*2 gene Saltant type ARMS reverse primer shown in the SEQ ID NO.:4 of shared probe, targeting CYP2C19*2 gene shown in NO.:2, Target wild type PCR amplification blocking agent shown in the SEQ ID NO.:5 of CYP2C19*2 gene；And/or

The SEQ ID for targeting forward primer shown in the SEQ ID NO.:7 of CYP2C19*3 gene, targeting CYP2C19*3 gene Saltant type ARMS reverse primer shown in the SEQ ID NO.:10 of shared probe, targeting CYP2C19*3 gene shown in NO.:8, Target wild type PCR amplification blocking agent shown in the SEQ ID NO.:11 of CYP2C19*3 gene；And/or

The SEQ for targeting forward primer shown in the SEQ ID NO.:13 of CYP2C19*17 gene, targeting CYP2C19*17 gene It is reversed that saltant type ARMS shown in the SEQ ID NO.:16 of probe, targeting CYP2C19*17 gene is shared shown in ID NO.:14 Primer targets wild type PCR amplification blocking agent shown in the SEQ ID NO.:17 of CYP2C19*17 gene.

8. kit as claimed in claim 7, which is characterized in that the kit further includes third container, and the third is held Include in device: hot start Taq polymerase, UNG enzyme, and/or dUTPs；And/or

The kit further includes the 4th container, includes positive control in the 4th container；And/or

The kit further includes the 5th container, includes negative control in the 5th container.

9. a kind of method for detecting CYP2C19 gene pleiomorphism, which is characterized in that the described method comprises the following steps:

(1) DNA sample of object to be detected is provided；

(2) it prepares quantitative fluorescent PCR reaction system and carries out fluorescence quantitative PCR detection:

Wherein, the quantitative fluorescent PCR reaction system includes the first quantitative fluorescent PCR reaction system and the second quantitative fluorescent PCR Reaction system；

The first quantitative fluorescent PCR reaction system includes:

The SEQ ID for targeting forward primer shown in the SEQ ID NO.:1 of CYP2C19*2 gene, targeting CYP2C19*2 gene Wild type ARMS reverse primer shown in the SEQ ID NO.:3 of shared probe, targeting CYP2C19*2 gene shown in NO.:2, Target saltant type PCR amplification blocking agent shown in the SEQ ID NO.:6 of CYP2C19*2 gene；And/or

The SEQ ID for targeting forward primer shown in the SEQ ID NO.:7 of CYP2C19*3 gene, targeting CYP2C19*3 gene Wild type ARMS reverse primer shown in the SEQ ID NO.:9 of shared probe, targeting CYP2C19*3 gene shown in NO.:8, Target saltant type PCR amplification blocking agent shown in the SEQ ID NO.:12 of CYP2C19*3 gene；And/or

The SEQ for targeting forward primer shown in the SEQ ID NO.:13 of CYP2C19*17 gene, targeting CYP2C19*17 gene It is reversed that wild type ARMS shown in the SEQ ID NO.:15 of probe, targeting CYP2C19*17 gene is shared shown in ID NO.:14 Primer targets saltant type PCR amplification blocking agent shown in the SEQ ID NO.:18 of CYP2C19*17 gene；

The second quantitative fluorescent PCR reaction system includes:

The SEQ ID for targeting forward primer shown in the SEQ ID NO.:1 of CYP2C19*2 gene, targeting CYP2C19*2 gene Saltant type ARMS reverse primer shown in the SEQ ID NO.:4 of shared probe, targeting CYP2C19*2 gene shown in NO.:2, Target wild type PCR amplification blocking agent shown in the SEQ ID NO.:5 of CYP2C19*2 gene；And/or

The SEQ ID for targeting forward primer shown in the SEQ ID NO.:7 of CYP2C19*3 gene, targeting CYP2C19*3 gene Saltant type ARMS reverse primer shown in the SEQ ID NO.:10 of shared probe, targeting CYP2C19*3 gene shown in NO.:8, Target wild type PCR amplification blocking agent shown in the SEQ ID NO.:11 of CYP2C19*3 gene；And/or

The SEQ for targeting forward primer shown in the SEQ ID NO.:13 of CYP2C19*17 gene, targeting CYP2C19*17 gene It is reversed that saltant type ARMS shown in the SEQ ID NO.:16 of probe, targeting CYP2C19*17 gene is shared shown in ID NO.:14 Primer targets wild type PCR amplification blocking agent shown in the SEQ ID NO.:17 of CYP2C19*17 gene.

10. method as claimed in claim 9, which is characterized in that the first quantitative fluorescent PCR reaction system further include: heat Start Taq enzyme, UNG enzyme, and/or dUTPs；

The second quantitative fluorescent PCR reaction system further include: hot start Taq polymerase, UNG enzyme, and/or dUTPs.