CN106319040A - Kit for detecting human CYP2C19 genetic typing and detection method - Google Patents
Kit for detecting human CYP2C19 genetic typing and detection method Download PDFInfo
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Abstract
The invention provides a kit for detecting human CYP2C19 genetic typing and a detection method. The kit comprises a reaction solution 1 and a reaction solution 2, wherein the reaction solution 1 and the reaction solution 2 respectively include primers and probes of sites 681 and 636 of a CYP2C19 gene, and the sequences of the primers of the site 681 are as follows: upstream primer 192FP:5'-ACAACCAGAGCTTGGCATATTGTATCT-3', and downstream primer 192RP:5'-ACTTTCTCCAAAATATCACTTTCCA-3'; the sequences of the primers of the site 636 are as follows: upstream primer 193FP:5'-CGTTTCGATTATAAAGATCAGCAA-3', and downstream primer 193RP:5'-TTCTCAGGAAGCAAAAAACTTG-3'.
Description
Technical field
The invention belongs to biomedical sector, relate to PCR fluorescent probe technique, particularly relate to a kind of detection people
The test kit of CYP2C19 gene type and detection method.
Background technology
Cytochrome P450 (Cytochrome P450 proteins, CYP) is a class heme-mercaptan
The 26S Proteasome Structure and Function of salt albumen composition is similar, by the isozyme of superfamily gene code.They are that a class is positioned at
Monooxygenase on people's cytomicrosome, mitochondrion and endoplasmic reticulum, participates in catalyzing endogenous material, as steroidal,
The metabolism of bile acid, fatty acid, biogenic Ammonia etc., also metabolism allogenic material includes most drug.
Cytochrome P450 is distributed widely in each organ of human body, is mainly distributed in liver and the intestinal of people.Carefully
In born of the same parents' cytochrome p 450 superfamily, mainly it is made up of CYP1,2,3 family.In numerous CYP families,
CYP2C family is an important family participating in drug metabolism, and CYP2C19 is in CYP2C family
Individual important enzyme, this enzyme is by CYP2C19 gene code.CYP2C19 gene is made up of 9 exons,
The long 1473bp of encoding gene, encodes the albumen that size is 56kDa of a 490 aminoacid composition.At present,
Finding that CYP2C19 gene has at least 14 kinds of mutant genes and 18 kinds of allele, modal sudden change has
Two kinds, 636 site mutations of CYP2C19 gene extron 5 and the 681 of CYP2C19 gene extron 4
The sudden change in site.And it is aobvious outside the two kinds of allelic mutation CYP2C19*2-the 5th on CYP2C19 gene
(CYP2C19*1 is in G636A sudden change on the G681A sudden change of son, CYP2C19*3-the 4th exon
Wild-type allele) cause enzyme to inactivate, make Different Individual be divided into six class drug metabolism gene types, respectively
For CYP2C19*1/*1 (fast metabolic pattern), CYP2C19*1/*2 (medium metabolic type), CYP2C19*1/*3
(medium metabolic type), CYP2C19*2/*2 (slow inactivation), CYP2C19*3/*3 (slow inactivation),
CYP2C19*2/*3 (slow inactivation).Different genotype is individual different to the metabolic rate of certain drug,
Thus directly affect the power of curative effect of medication and toxic and side effects.
Therefore instruct accurately according to the genotype of CYP2C19, safe medication has important clinical meaning.
At present the method for detection CYP2C19 gene pleiomorphism has multiple, including Manganic pyrophosphate complex initiation method,
PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism), gene chips, quantitative fluorescent PCR
Method.Manganic pyrophosphate complex initiation method, result is accurate, but cost is high time-consuming long；PCR-RFLP is by restriction enzyme digestion and electrophoresis
Method detection gene pleiomorphism, has low cost, and the advantage of visual result, but operating process vulnerable to pollution are led
Cause result mistake.Gene chips detects genotype by Hybridization principle, has the advantage that flux is high, shortcoming
It is relatively costly, complex operation, result difficulty interpretation.Fluorescence quantitative PCR method has simple to operate, the shortest,
Result advantage accurate, reliable, current quantitative fluorescent PCR is applied to clinical examination just by more and more extensive
Use.
Summary of the invention
The present invention uses 2 weight TaqMan-MGB fluorescent probe PCR technology, utilizes specific primer group and spy
Specific probes group, can detect 636 single nucleotide polymorphisms of CYP2C19 exon 5 in single tube reacts
And/or 681 single nucleotide polymorphisms of CYP2C19 exon 4, and testing result have resolution high,
Rapidly, advantage accurately.
Another object of the present invention is to provide the inspection of a kind of test kit detecting people's CYP2C19 gene type
Survey method.
The present invention is achieved in that a kind of test kit detecting people's CYP2C19 gene type, described examination
Agent box includes that reactant liquor 1 and reactant liquor 2, described reactant liquor 1, reactant liquor 2 include CYP2C19 base respectively
Cause 681 sites and the primer in 636 sites, probe, wherein, described CYP2C19 gene 681 site,
The primer sequence in 636 sites is as follows:
CYP2C19 gene 681 site
Forward primer 192FP:5 '-ACAACCAGAGCTTGGCATATTGTATCT-3 '；
Downstream primer 192RP:5 '-ACTTTCTCCAAAATATCACTTTCCA-3 '；
CYP2C19 gene 636 site
Forward primer 193FP:5 '-CGTTTCGATTATAAAGATCAGCAA-3 '；
Downstream primer 193RP:5 '-TTCTCAGGAAGCAAAAAACTTG-3 '.
And, the detection method of a kind of test kit detecting people's CYP2C19 gene type, including following step
Rapid:
Extract people's Whole Blood Genomic DNA and mentioned reagent box；
In proportion 5 μ l people's Whole Blood Genomic DNA extracting solution are added the reaction system of test kit described in 40 μ l
In；
PCR expands；
Result judges.
The test kit of detection people's CYP2C19 gene type that the present invention provides, containing specific primer group and
Specific probe group, 636 locus genes that can detect CYP2C19 exon 5 in single tube reacts are polymorphic
Property and/or 681 single nucleotide polymorphisms of CYP2C19 exon 4, and testing result resolution is high, fast
Fast, accurately.
The detection method of the test kit of detection people's CYP2C19 gene type that the present invention provides, has operation
Simply, rapidly, result advantage accurately and reliably.
Accompanying drawing explanation
Fig. 1 is that the 681 site GG of CYP2C19 in the embodiment of the present invention 8 isozygoty wild pattern detection result
Figure；
Fig. 2 is the 681 site AA homozygous mutation pattern detection results of CYP2C19 in the embodiment of the present invention 8
Figure；
Fig. 3 is the 681 site GA heterozygosis pattern detection result figures of CYP2C19 in the embodiment of the present invention 8；
Fig. 4 is that the 636 site GG of CYP2C19 in the embodiment of the present invention 8 isozygoty wild pattern detection result
Figure；
Fig. 5 is the 636 site AA homozygous mutation pattern detection results of CYP2C19 in the embodiment of the present invention 8
Figure；
Fig. 6 is the 636 site GA heterozygosis pattern detection result figures of CYP2C19 in the embodiment of the present invention 8.
Detailed description of the invention
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearer, with
Under in conjunction with the embodiments, the present invention is further elaborated.Should be appreciated that described herein specifically
Embodiment only in order to explain the present invention, is not intended to limit the present invention.
In order to solve the problem that prior art exists, embodiments provide a kind of detection people CYP2C19
The test kit of gene type, the know-why of employing is 2 weight TaqMan-MGB fluorescent probe PCR methods, root
According to the TaqMan-MGB probe that site to be detected design special primer, wild type and saltant type are special.
TaqMan-MGB probe is introducing MGB modification group, TaqMan on the basis of TaqMan probe
Probe two ends are respectively provided with fluorophor and fluorescent quenching group, in normal state, the fluorescence quilt on probe
Cancellation, does not send fluorescence.After proceeding by PCR amplification, probe by PCR enzyme hydrolysis, quenching group with
Fluorophor separates, and has fluorescence signal to discharge.And the quenching group of TaqMan-MGB probe uses non-
Fluorescent quenching group (Non-Fluorescent Quencher), itself does not produce fluorescence, can be substantially reduced probe
The intensity of background signal.It is also associated with MGB (Minor Groove Binder) modification group on probe simultaneously,
The Tm value of probe can be improved about 10 DEG C.Therefore to obtain same Tm value, TaqMan-MGB
Probe can be designed to shorter than general T aqMan probe, has both reduced synthesis cost, also makes probe set
The success rate of meter greatly improves, because in the case of the DNA base composition of template is undesirable, and short spy
Pin is than long easier design.
Concrete, embodiments provide a kind of test kit detecting people's CYP2C19 gene type,
Described test kit includes that reactant liquor 1 and reactant liquor 2, described reactant liquor 1, reactant liquor 2 include CYP2C19 respectively
Gene 681 site and the primer in 636 sites, probe, wherein, described CYP2C19 gene 681 site,
The primer sequence in 636 sites is as follows:
CYP2C19 gene 681 site
Forward primer 192FP:5 '-ACAACCAGAGCTTGGCATATTGTATCT-3 '；
Downstream primer 192RP:5 '-ACTTTCTCCAAAATATCACTTTCCA-3 '；
CYP2C19 gene 636 site
Forward primer 193FP:5 '-CGTTTCGATTATAAAGATCAGCAA-3 '；
Downstream primer 193RP:5 '-TTCTCAGGAAGCAAAAAACTTG-3 '.
Further, carefully studying through inventor, the most following have a specific probe sets, specifically
, described CYP2C19 gene 681 site, 636 sites probe sequence as follows:
CYP2C19 gene 681 site
Probe 192G:5 '-CATTGATTATTTCCCGGGAA-3 '；
Probe 192A:5 '-CCACTATCATTGATTATTTCCCAGG-3 '；
CYP2C19 gene 636 site
Probe 193G:5 '-CCCCTGGATCCAGGT-3 '；
Probe 193A:5 '-CCCCCTGAATCCA-3 '；
Described probe 5 ' end use FAM, HEX, TET, VIC, ROX, CY5, CY3, JOE,
One in ALEX, CAL, Fluor fluorophor is marked, described probe 3 ' end without fluorophore,
Connect MGB group.
TaqMan-MGB probe used by the embodiment of the present invention, has appropriate Tm value, and commonly
TaqMan probe compares, and its sequential design of the probe in 636 sites obtains shorter, has both reduced synthesis cost, also
The success rate that probe is designed greatly improves, because the DNA base in template forms undesirable situation
Under, short probe is than long easier design.And due to people CYP2C19 gene described in the embodiment of the present invention
681 site target sequences are rich in A/T, and therefore, the embodiment of the present invention designs for this site
TaqMan-MGB probe is for distinguishing even more ideal rich in the template of A/T.Additionally, compare common
TaqMan probe, the TaqMan-MGB probe that the embodiment of the present invention is used than general T aqMan have
There are higher operability, detection specificity and accuracy.
Embodiment of the present invention SNP site specific bond probe uses 2 weight TaqMan-MGB of Bicolor-code to visit
Pin, one of them genotype the most corresponding, such as FAM labelling wild type specific probe and VIC labelling are dashed forward
Modification specific probe.When the corresponding detection site of target nucleic acid is wild type, can be at FAM Air conduct measurement to glimmering
Optical signal；When the corresponding detection site of target nucleic acid is saltant type, can be at VIC Air conduct measurement to fluorescence signal；
When the corresponding detection site of target nucleic acid is heterozygous, FAM and the VIC dual pathways all can detect fluorescence signal.
So, TaqMan-MGB probe list base difference is utilized, according to PCR instrument device amplified signal in certain threshold
Period (Ct value) during value, calculates FAM and VIC interchannel Ct value difference value (Ct) and judges
The nucleotide type of detection site, and then accurately judge the SNP site genotype of CYP 2C19.The method
A kind of type of one specific probe correspondence detection site is set, arranges one to one and make testing result more accurate
Really, more reliable, result judge more convenient quickly.
As a particular preferred embodiment, described test kit also includes UNG enzyme (uracil glycosylase enzyme)
The enzymatic mixture formed with archaeal dna polymerase, test kit reaction system described in the embodiment of the present invention uses
UNG-dUTP decontamination system, wherein, affiliated UNG enzyme can contain the double of dU with selective hydrolysis fracture
Uracil glycosidic bond in chain or single stranded DNA, the DNA having disappearance base of formation, at alkaline medium
And the further hydrolytic cleavage of meeting under high temperature, in reaction system, therefore add appropriate dUTP, expand at PCR
Before increasing, UNG enzyme effect is utilized can effectively to prevent the pollution that amplified production in laboratory causes, thus to whole
Individual detection system includes that the concentration of sample, interference factor carry out quality control.And UNG enzyme can when 95 DEG C
It is inactivated, does not affect subsequent PCR amplification effect.Wherein, described archaeal dna polymerase can select multiple DNA
Polymerase, the embodiment of the present invention specifically preferred Taq Hotstart archaeal dna polymerase.This preferred mixed enzyme body
System, carries out PCR amplification not only by archaeal dna polymerase, at the same time it can also be by described UNG
Enzyme eliminates and pollutes, thus better assures that the success rate of experiment and the accuracy of result.
Additionally, in test kit described in the embodiment of the present invention, described reactant liquor 1 and described reactant liquor 2 all include
Buffer system, MgCl2。
In order to carry out comparative illustration, test kit described in the embodiment of the present invention is provided with positive reference substance and feminine gender is right
According to product, described positive reference substance comprises 4 kinds of plasmids, and described negative controls is TE buffer；Wherein,
Described 4 kinds of plasmids are respectively CYP2C19 gene 681 site G and A detection site recombiant plasmid,
CYP2C19 gene 636 site G and A detection site recombiant plasmid.More specifically, described positive control
Product include CYP2C19 positive reference substance 1 and CYP2C19 positive reference substance 2, wherein, described CYP2C19
Positive reference substance 1 is pressed the ratio of 1:1 by CYP2C19 gene 681 site G and A detection site recombiant plasmid
Example forms, and described CYP2C19 positive reference substance 2 is detected by CYP2C19 gene 636 site G and A
Site recombiant plasmid is formed in the ratio of 1:1.
The test kit of detection people's CYP2C19 gene type that the embodiment of the present invention provides, draws containing specificity
Thing group and specific probe group, can detect 636 site bases of CYP2C19 exon 5 in single tube reacts
Because of polymorphism and/or 681 single nucleotide polymorphisms of CYP2C19 exon 4, and testing result resolution
High, rapidly, accurately.
And, the embodiment of the present invention additionally provides a kind of test kit detecting people's CYP2C19 gene type
Detection method, comprises the following steps:
S01. people's Whole Blood Genomic DNA and mentioned reagent box are extracted；
The most in proportion 5 μ l people's Whole Blood Genomic DNA extracting solution are added the reaction of test kit described in 40 μ l
In system；
S03.PCR expands；
S04. result judges.
Concrete, in above-mentioned steps S01, described test kit is mentioned reagent box, described people's whole blood genome
The extracting method of DNA can refer to following specific embodiment to be carried out, and obtains people's whole blood gene it is of course possible to extract
The method of group DNA, all in range of embodiment of the invention.
In above-mentioned steps S02, test kit described in 40 μ l includes 34.5 μ lPCR reactant liquors and the mixing of 0.5 μ l enzyme
Thing, certainly, concrete volume can adjust accordingly according to this ratio.The pipe number carrying out PCR reaction does not has spy
Do not limit, usually sample number+2 (negative control, positive control).Therefore, this step at least include to
The reaction system of different described test kits is separately added into sample, positive control and negative control.
In above-mentioned steps S03, the reaction condition of described PCR amplification is:
50℃3min；
95 DEG C of denaturations 3min；
95℃15s；62℃45s；40 circulations,
Fluorescence is collected when 62 DEG C of 45s.
In above-mentioned steps S04, by result being analyzed judgement, described CYP2C19 gene can be learned
681 sites, the concrete gene type in 636 sites, described analysis decision method, in following specific embodiment
It is described.
The detection method of the test kit of detection people's CYP2C19 gene type that the embodiment of the present invention provides, tool
There is simple to operate, rapid, result advantage accurately and reliably.
Illustrate below in conjunction with specific embodiment.
Embodiment 1 design of primers synthesizes
636 sites of embodiment of the present invention CYP2C19 gene extron 5 and 681 sites of exon 4
Gene pleiomorphism design of primers, according to NCBI people's CYP2C19 gene 4,5 exon genes sequential design
As follows:
CYP2C19 gene 636 site primer:
Forward primer 193FP:5 '-CGTTTCGATTATAAAGATCAGCAA-3 '；
Downstream primer 193RP:5 '-TTCTCAGGAAGCAAAAAACTTG-3 '；
CYP2C19 gene 681 site primer:
Forward primer 192FP:5 '-ACAACCAGAGCTTGGCATATTGTATCT-3 '；
Downstream primer 192RP:5 '-ACTTTCTCCAAAATATCACTTTCCA-3 '.
The design synthesis of embodiment 2 probe
681, the 636 single nucleotide polymorphisms designs according to NCBI gene bank people's CYP2C19 gene 4,5
Corresponding probe is as follows:
CYP2C19 gene 636 site:
Probe 193G:5 '-CCCCTGGATCCAGGT-3 '；
Probe 193A:5 '-CCCCCTGAATCCA-3 '；
Wherein, the available multiple fluorophor labelling of probe 5 ' end, the embodiment of the present invention selects wild type 636G
Site FAM labelling, saltant type 636A HEX labelling；Without fluorophore held by probe 3 ', connects
MGB group.
CYP2C19 gene 681 site:
Probe 192G:5 '-CATTGATTATTTCCCGGGAA-3 '；
Probe 192A:5 '-CCACTATCATTGATTATTTCCCAGG-3 '；
Wherein, the available multiple fluorophor labelling of probe 5 ' end, the embodiment of the present invention selects wild type 681G
Site FAM labelling, saltant type 681A HEX labelling；Without fluorophore held by probe 3 ', connects
MGB group.
The preparation of embodiment 3 CYP2C19PCR reactant liquor
CYP2C19PCR reactant liquor includes that CYP2C19PCR reactant liquor 1 and CYP2C19PCR reacts
Liquid 2, the composition of two kinds of reactant liquors is buffer, MgCl2, primer, probe, the following institute of concentration of component
Show:
CYP2C19PCR reactant liquor 1:
CYP2C19PCR reactant liquor 2:
Example 4 enzymatic mixture
Enzymatic mixture described in the embodiment of the present invention is by uracil glycosylase enzyme (UNG enzyme) and archaeal dna polymerase
Mixing, wherein, described archaeal dna polymerase can select multiple archaeal dna polymerase, the embodiment of the present invention
Preferential selection Taq Hotstart archaeal dna polymerase.
Embodiment 5 positive reference substance and the preparation of blank
681,636 single nucleotide polymorphisms according to ncbi database CYP2C19 gene extron 4,5
Design positive reference substance sequence, composition sequence, constructed dna recombiant plasmid.Positive reference substance includes four kinds
Plasmid is respectively 681G wild type, 681A saltant type, 636G wild type, 636A saltant type.CYP2C19
Positive reference substance 1 is mixed by 1:1 by 681G wild type, 681A saltant type recombiant plasmid；CYP2C19
Positive reference substance 2 is mixed by 1:1 by 636G wild type, 636A saltant type recombiant plasmid.
The outfit of embodiment 6 reaction system
Using the reaction system of 40 μ l in the present invention, its concrete reactive component is as shown in table 1 below.
Table 1
The setting of embodiment 7 response procedures
In the embodiment of the present invention, preferred response procedures is 50 DEG C, 3min；95 DEG C, 3min；95 DEG C, 15s,
62 DEG C, 45s, 40 circulations.
The detection method of 8 test kit detection clinical samples of embodiment
The detection method of this test kit detection clinical sample, comprises the following steps:
1. the extraction of human blood sample genomic dna, can use Shenzhen HYK Gene Technology Co., Ltd. precious
The poba gene group extraction purification test kit that dutiful company produces, concrete operations are as follows:
(1) from extract test kit takes out 1.5ml centrifuge tube several, each pipe adds anticoagulation 200 μ l.
(2) adding 500 μ l buffer CLG solution in anticoagulation, cover tightly centrifugal lid, whirlpool shakes
More than 20s, the most reverse mixing, it is necessary to fully mixing or whirlpool concussion are to guarantee to discharge genomic DNA.
(3) adding 100 μ l buffer PP solution, whirlpool shakes or overturns 10s back and forth.
(4) 12,000g is centrifuged 5min.
(5) from extracting taking-up DNA purification column test kit, the supernatant in step 4 is transferred to purification
Post, 8000g is centrifuged 1min.For improving DNA extraction amount, after being centrifuged, filtrate can rewind be centrifuged once again.
(6) abandoned filtrate after post, and DNA purification column was put in same collecting pipe, add in purification column
Entering 750 μ l buffer W I, after room temperature places 2min, 8000g is centrifuged 1min and (confirms buffer W I
Add dehydrated alcohol).
(7) abandoned filtrate after post, in purification column, added 750 μ l Buffer buffer W II, 8000g from
Heart 1min (confirms that buffer W II has added dehydrated alcohol).
(8) abandoning filtrate after post, by purification column 12,000g is centrifuged 2min.
(9) being transferred to by purification column in new 1.5ml centrifuge tube, room temperature is placed 5min and is fully volatilized ethanol.
Add 70～100 μ l collect liquid EB (collect liquid EB and be preheating to 65 DEG C of elution efficiencies being conducive to improving DNA),
Room temperature places 1min, and 12,000 are centrifuged 2min eluting genomic DNA.Can by the eluent sucking-off after post excessively again
Being added on purification column film, room temperature places 1min, and 12,000g are centrifuged 1min can improve the elution amount of DNA.
(10) concentration and the quality of genomic DNA should measure OD260 by trace ultraviolet spectrophotometer
With OD280 light absorption value (the NanoPhotometerTMP-Clas trace spectrophotometric of conventional Germany Implen
Meter, the K2800 foranalysis of nucleic acids instrument of Beijing triumphant AudioCodes skill Development Co., Ltd), DNA concentration should be 2
Ng/ μ l≤DNA concentration≤100ng/ μ l scope OD260/OD280 value is between 1.5～2.0.Purification DNA
Sample preserves less than 7 days at 4 DEG C；The DNA sample failing to detect in time is in-20 DEG C of preservations, during preservation
Between less than 3 months；Purification DNA sample multigelation is less than 5 times.
2. the fluorescence quantitative PCR detection of sample
(1) reagent prepares
From test kit, take out CYP2C19PCR reactant liquor, thaw on ice, calculate each reaction system
Required PCR reaction tube number, pipe number is n (n=sample number+2 (negative control, positive control)).
According to the form below 2 takes the PCR reactant liquor of amount of calculation and enzymatic mixture respectively in suitable centrifuge tube, reverse mixing
10 times, preparing reaction system, 2000g is centrifuged 10 seconds.
Table 2
Take 35 μ l reaction systems respectively to PCR reaction tube, the reaction system that labelling is different.
(2) sample-adding
Positive control and negative control take out when receiving, and are put into sample treatment room/sample addition zone.Positive control and
Negative control thaws on ice, mixes at turbula shaker, and 2000g is centrifuged 10 seconds.To set n
Individual PCR reaction tube is separately added into sample, positive control, each 5 μ l of negative control, covers tightly lid, and do
Good record.Mix the PCR reaction system in each PCR reaction tube and added sample.By PCR reaction tube
2000g is centrifuged 10 seconds.Being transferred to detection zone, be placed in PCR instrument, order put by record sample.
(3) PCR amplification
ABI7500 quantitative real time PCR Instrument, selects FAM (Reporter:FAM；Quencher:none)
Sense channel；VIC(Reporter:VIC；Quencher:none) sense channel.Reference fluorescent (Passive
Reference) being set to None, threshold line is set to 50000.
PCR amplification program:
50℃3min；
95 DEG C of denaturations 3min；
95℃15s；62℃45s；40 circulations,
Collect fluorescence, i.e. phosphor collection when 62 DEG C of 45s and be arranged on " step 3:62 DEG C, 45s ".
3. the Analysis of test results of sample
(1) such as sample CYP2C19PCR reactant liquor 1FAM sense channel and the equal nothing of VIC sense channel
Ct or Ct is all higher than 37.5, or CYP2C19PCR reactant liquor 2FAM sense channel and VIC detection are led to
Road is all higher than 37 without Ct or Ct, then the genomic DNA concentration of judgment sample is less than detection limit.
(2) there is sense channel Ct less than or equal to 37.5, CYP2C19PCR at CYP2C19PCR reactant liquor 1
In the case of reactant liquor 2 has sense channel Ct less than or equal to 37, by following judgement.
A.CYP2C19PCR reactant liquor 1 result judges (681 site result)
Record sample FAM channel C t (being designated as CtF), VIC channel C t (is designated as CtV), FAM passage or
When a certain sense channel of VIC passage is without Ct, the Ct without Ct passage is designated as 40.The Ct calculating two passages is poor
Absolute value Ct, Ct=| CtF-CtV | of value.According to Ct size of data, according to the form below 3 result of determination:
Table 3
B.CYP2C19PCR reactant liquor 2 result judges (636 site result)
Record sample FAM channel C t (being designated as CtF), VIC channel C t (is designated as CtV), FAM passage or
When a certain sense channel of VIC passage is without Ct, the Ct without Ct passage is designated as 40.The Ct calculating two passages is poor
Absolute value Ct, Ct=| CtF-CtV | of value.According to Ct size of data, according to the form below 4 result of determination:
Table 4
CYP2C19 gene type is as shown in table 5 below:
Table 5
In embodiment of the present invention test kit, the testing result in 681 sites of CYP2C19 gene extron 4
Having 3 kinds, Fig. 1 show the testing result that 681 sites are GG wild type, and Fig. 2 show 681 sites
For the testing result of AA saltant type, Fig. 3 show the testing result of 681 site GA heterozygous；CYP2C19
The testing result in 636 sites of gene extron 5 also has 3 kinds, and it is wild that Fig. 4 show 636 site GG
The testing result of type, Fig. 5 show the testing result of 636 site AA saltant types, and Fig. 6 show 636
The testing result of site GA heterozygous.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all at this
Any amendment, equivalent and the improvement etc. made within bright spirit and principle, should be included in the present invention
Protection domain within.
Claims (8)
1. the test kit detecting people's CYP2C19 gene type, it is characterised in that described test kit bag
Including reactant liquor 1 and reactant liquor 2, described reactant liquor 1, reactant liquor 2 include CYP2C19 gene 681 respectively
Site and the primer in 636 sites, probe, wherein, described CYP2C19 gene 681 site, 636 sites
Primer sequence as follows:
CYP2C19 gene 681 site
Forward primer 192FP:5 '-ACAACCAGAGCTTGGCATATTGTATCT-3 '；
Downstream primer 192RP:5 '-ACTTTCTCCAAAATATCACTTTCCA-3 '；
CYP2C19 gene 636 site
Forward primer 193FP:5 '-CGTTTCGATTATAAAGATCAGCAA-3 '；
Downstream primer 193RP:5 '-TTCTCAGGAAGCAAAAAACTTG-3 '.
2. the test kit detecting people's CYP2C19 gene type as claimed in claim 1, it is characterised in that
Described CYP2C19 gene 681 site, 636 sites probe sequence as follows:
CYP2C19 gene 681 site
Probe 192G:5 '-CATTGATTATTTCCCGGGAA-3 '；
Probe 192A:5 '-CCACTATCATTGATTATTTCCCAGG-3 '；
CYP2C19 gene 636 site
Probe 193G:5 '-CCCCTGGATCCAGGT-3 '；
Probe 193A:5 '-CCCCCTGAATCCA-3 '；
Described probe 5 ' end use FAM, HEX, TET, VIC, ROX, CY5, CY3, JOE,
One in ALEX, CAL, Fluor fluorophor is marked, described probe 3 ' end without fluorophore,
Connect MGB group.
3. the test kit detecting people's CYP2C19 gene type as claimed in claim 1, it is characterised in that
Described test kit also includes the enzymatic mixture that UNG enzyme and archaeal dna polymerase are formed.
4. the test kit of the detection people's CYP2C19 gene type as described in claim 1-3 is arbitrary, it is special
Levying and be, described test kit is provided with positive reference substance and negative controls, and described positive reference substance comprises 4
Planting plasmid, described negative controls is TE buffer；Wherein, described 4 kinds of plasmids are respectively CYP2C19
Gene 681 site G and A detection site recombiant plasmid, CYP2C19 gene 636 site G and A detects
Site recombiant plasmid.
5. the test kit detecting people's CYP2C19 gene type as claimed in claim 4, it is characterised in that
Described positive reference substance includes CYP2C19 positive reference substance 1 and CYP2C19 positive reference substance 2, wherein,
Described CYP2C19 positive reference substance 1 is recombinated by CYP2C19 gene 681 site G and A detection site
Plasmid is formed in the ratio of 1:1, and described CYP2C19 positive reference substance 2 is by CYP2C19 gene 636
Site G and A detection site recombiant plasmid are formed in the ratio of 1:1.
6. the test kit of the detection people's CYP2C19 gene type as described in claim 1-3 is arbitrary, it is special
Levying and be, described reactant liquor 1 and described reactant liquor 2 all include buffer system, MgCl2。
7. detect a detection method for the test kit of people's CYP2C19 gene type, comprise the following steps:
Extract people's Whole Blood Genomic DNA and the arbitrary described test kit of claim 1-6；
In proportion 5 μ l people's Whole Blood Genomic DNA extracting solution are added the reaction system of test kit described in 40 μ l
In；
PCR expands；
Result judges.
8. the detection method of the test kit of detection people's CYP2C19 gene type as claimed in claim 7,
It is characterized in that, the reaction condition of described PCR amplification is:
50℃3min；
95 DEG C of denaturations 3min；
95℃15s；62℃45s；40 circulations.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107083445A (en) * | 2017-06-20 | 2017-08-22 | 嘉兴雅康博贝南生物科技有限公司 | A kind of multiple fluorescence PCR method detects the kit of CYP2C19 gene pleiomorphisms |
| CN107083447A (en) * | 2017-06-27 | 2017-08-22 | 踏石生物科技（苏州）有限公司 | Using the site GG types of people CYP2C19*2 genes 681 as the positive reference product of template |
| CN107090511A (en) * | 2017-06-27 | 2017-08-25 | 踏石生物科技（苏州）有限公司 | Using the site AA types of people CYP2C19*2 genes 681 as the positive reference product of template |
| CN107227361A (en) * | 2017-06-29 | 2017-10-03 | 庄江兴 | Primer, probe and detection kit for detecting CYP2C19 gene pleiomorphisms |
| CN109295195A (en) * | 2018-08-08 | 2019-02-01 | 济南齐鲁医学检验有限公司 | A kind of fluorescent quantitative PCR detection method based on CYP2C19 gene |
| CN109811043A (en) * | 2019-03-14 | 2019-05-28 | 武汉明德生物科技股份有限公司 | Gene pleiomorphism detecting method and detection kit based on Taqman-MGB probe |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102534005A (en) * | 2012-01-14 | 2012-07-04 | 长沙三济生物科技有限公司 | Fluorescent polymerase chain reaction (PCR) kit for detecting CYP2C19 genotypes |
| CN103757106A (en) * | 2014-01-07 | 2014-04-30 | 河南科技大学 | Kit and method for detecting gene polymorphism of human MTHFR (methylene tetrahydrofolate reductase) based on Taqman-MGB (Minor Groove Binder) probe |
-
2016
- 2016-03-11 CN CN201610141328.4A patent/CN106319040A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102534005A (en) * | 2012-01-14 | 2012-07-04 | 长沙三济生物科技有限公司 | Fluorescent polymerase chain reaction (PCR) kit for detecting CYP2C19 genotypes |
| CN103757106A (en) * | 2014-01-07 | 2014-04-30 | 河南科技大学 | Kit and method for detecting gene polymorphism of human MTHFR (methylene tetrahydrofolate reductase) based on Taqman-MGB (Minor Groove Binder) probe |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107083445A (en) * | 2017-06-20 | 2017-08-22 | 嘉兴雅康博贝南生物科技有限公司 | A kind of multiple fluorescence PCR method detects the kit of CYP2C19 gene pleiomorphisms |
| CN107083447A (en) * | 2017-06-27 | 2017-08-22 | 踏石生物科技（苏州）有限公司 | Using the site GG types of people CYP2C19*2 genes 681 as the positive reference product of template |
| CN107090511A (en) * | 2017-06-27 | 2017-08-25 | 踏石生物科技（苏州）有限公司 | Using the site AA types of people CYP2C19*2 genes 681 as the positive reference product of template |
| CN107227361A (en) * | 2017-06-29 | 2017-10-03 | 庄江兴 | Primer, probe and detection kit for detecting CYP2C19 gene pleiomorphisms |
| CN109295195A (en) * | 2018-08-08 | 2019-02-01 | 济南齐鲁医学检验有限公司 | A kind of fluorescent quantitative PCR detection method based on CYP2C19 gene |
| CN109811043A (en) * | 2019-03-14 | 2019-05-28 | 武汉明德生物科技股份有限公司 | Gene pleiomorphism detecting method and detection kit based on Taqman-MGB probe |
| CN113512585A (en) * | 2021-06-17 | 2021-10-19 | 湖南菲思特精准医疗科技有限公司 | Rapid reaction kit for aspergillin dosage prediction and detection method and application thereof |
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