CN106480177A - The test kit of detection people's CYP3A5 gene type and detection method - Google Patents
The test kit of detection people's CYP3A5 gene type and detection method Download PDFInfo
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Abstract
The invention provides a kind of test kit of detection people's CYP3A5 gene type, described test kit includes reactant liquor, described reactant liquor includes primer for CYP3A5 gene 6986 site, probe, 5 ' ends of described probe, 3 ' ends are all marked with fluorophor, and described 5 ' ends are different with the fluorophor that 3 ' hold；Described test kit also includes archaeal dna polymerase, and described archaeal dna polymerase is the archaeal dna polymerase with flap endonuclease activity.Carry out, by real-time PCR reactions, the gene pleiomorphism that SNP detects 6986 sites of CYP3A5 using archaeal dna polymerase, specific dual labelled fluorogeilic probe and the specific primer with flap endonuclease activity, and testing result has high resolution, rapid, accurate advantage.
Description
Technical field
The invention belongs to biomedical sector, it is related to PCR fluorescent probe technique, more particularly, to a kind of detection people's CYP3A5 base
Test kit and its detection method because of typing.
Background technology
Cytochrome P450 (Cytochrome P450proteins, CYP) is a class heme-thiolate proteins
The 26S Proteasome Structure and Function of composition is similar, by the isozyme of superfamily gene code.CYP is that a class is located at people's cell microsome, line grain
Monooxygenase on body and endoplasmic reticulum, participates in catalyzing endogenous material, such as steroidal, bile acid, fatty acid, biogenic Ammonia etc.
Metabolism, also metabolism allogenic material include most drug.Cytochrome P450 is distributed widely in each organ of human body, main point
Cloth is in the liver and intestinal of people.Its catalytic mechanism is that active compound is oxidized in liver, reduction, hydrolysis slough or increase by one
A little functional groups so that it becomes the higher metabolite of polarity, be expelled directly out with urine or further with glucuronic acid, amino
Acid, acetyl sulfate base, peptide or methyl etc. combine discharges with urine again.
In Cytochrome P450 superfamily, the metabolic enzyme related to drug metabolism is mainly CYP1, CYP2, CYP3 family.
CYP3A5 is the important member of CYP3A subfamily, participates in the metabolism of multi-medicament.There is genetic polymorphism, there is individuality, race
Diversity, leads to Different Individual, the medicament metabolism ability of race to have differences.Wherein CYP3A5*3 (6986A>G) in intron3
Mutation cause variable sheer, create unstable protein so that saltant type homozygotic individual does not express CYP3A5,
Enzymatic activity can be caused to reduce even disappear.The different genotype of CYP3A5 directly affects the power of curative effect of medication and toxic and side effects,
Genotype therefore according to CYP3A5 come instruct accurately, safe medication there is important clinical meaning.
Method currently used for detection gene pleiomorphism mainly has sequencing, PCR-RFLP (polymerase chain reaction-restricted
Fragment length polymorphism), gene chips, fluorescence quantitative PCR method.Accurately, but high cost, time-consuming for sequencing result.PCR-
RFLP detects gene pleiomorphism, low cost, visual result, but operating process vulnerable to pollution by the method for restriction enzyme digestion and electrophoresis, leads to
Error result.Gene chips detect genotype by nucleic acid hybridization, and advantage is that flux is high, and shortcoming is relatively costly, and operation is numerous
Trivial, result hardly possible interpretation.Fluorescence quantitative PCR method has simple to operate, time-consuming short, result accurately and reliably advantage, and current fluorescence is fixed
Amount PCR is just increasingly widely applied in clinical examination.Fluorescent PCR sonde method and glimmering can be divided into according to its principle difference
Light PCR melting curve method.The former is directed to target SNP site and designs two kinds of probes, mates with saltant type with wild type respectively, utilizes
Amplification Ct value difference different come interpretation genotype.The latter is directed to target SNP site and designs a kind of probe, based on probe and target nucleic acid
The accuracy (i.e. in SNP site, different bases cause the difference of melting temperature) of intermolecular hybrid carry out interpretation genotype.
Content of the invention
It is an object of the invention to provide a kind of detection test kit of people's CYP3A5 gene type and its detection method it is intended to
Solve the problems, such as that prior art is difficult to CYP3A5 gene type.
The present invention is achieved in that a kind of test kit of detection people's CYP3A5 gene type, and described test kit includes instead
Answer liquid, described reactant liquor includes primer for CYP3A5 gene 6986 site, probe, wherein, described CYP3A5 gene 6986
The primer sequence in site is as follows：
Forward primer 6986FP：5’-ATTATGGAGAGTGGCATAGGAG-3’；
Downstream primer 6986RP：5’-GCAAGAGTCTCACACAGGAG-3’；
5 ' ends of described probe, 3 ' ends are all marked with fluorophor, and described 5 ' ends are different with the fluorophor that 3 ' hold；
Described test kit also includes archaeal dna polymerase, and described archaeal dna polymerase is to have flap endonuclease activity
Archaeal dna polymerase.
And, a kind of detection method of the test kit of detection people's CYP3A5 gene type, comprise the following steps：
Extract people's Whole Blood Genomic DNA and mentioned reagent box；
Add in the reaction system of described test kit by people's Whole Blood Genomic DNA extracting solution, wherein, described people's whole blood gene
The ratio of group DNA extraction liquid and described reaction system ratio is for 1：8；
PCR expands；
Result judgement.
The test kit of detection people's CYP3A5 gene type that the present invention provides, using having flap endonuclease activity
Archaeal dna polymerase, and cutting position when 5 ' ends in excision valvular structure are single-stranded using this polymerase is located at valvular structure
First base of double stranded section and second base between this characteristic, in combination with being modified with double fluorescent labelling and quench
Go out the dual labelled fluorogeilic DNA probe of group and specific primer, carries out SNP detection by real-time PCR reactions in single tube, determines
CYP3A5 gene, determines the gene pleiomorphism in site, realizes detecting two kinds in target nucleic acid SNP site with a fluorescent probe
Typing, and testing result has high resolution, rapid, accurate advantage.Detection people's CYP3A5 gene type that the present invention provides
Detection method, detection SNP specificity derive from have flap endonuclease activity archaeal dna polymerase excision valvular structure
When cutting position accuracy, the fluorescent PCR melting curve method before comparing is more accurate.Therefore the method is carrying out gene
Traditional quantitative fluorescent PCR is compared on typing and shows advantage.Additionally, the inventive method has simple to operate, rapid, result
Accurately and reliably advantage.
Brief description
Fig. 1 is the wild pattern detection result figure of 6986 site AA homozygosis of CYP3A5 in the embodiment of the present invention 8；
Fig. 2 is the 6986 site GG homozygous mutation pattern detection result figures of CYP3A5 in the embodiment of the present invention 8；
Fig. 3 is the 6986 site AG heterozygosis pattern detection result figures of CYP3A5 in the embodiment of the present invention 8.
Specific embodiment
In order that the technical problem to be solved in the present invention, technical scheme and beneficial effect become more apparent, below in conjunction with
Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only in order to explain
The present invention, is not intended to limit the present invention.
The test kit of people's CYP3A5 gene type is detected, the know-why of employing is based on fluorescence described in the embodiment of the present invention
PCR sonde method, using the archaeal dna polymerase with flap endonuclease activity, designs special primer, spy according to site to be detected
The dual labelled fluorogeilic probe of the opposite sex.
Embodiments provide a kind of test kit of detection people's CYP3A5 gene type, described test kit includes reacting
Liquid, described reactant liquor includes primer for CYP3A5 gene 6986 site, probe, wherein, described CYP3A5 gene 6986
The primer sequence of point is as follows：
Forward primer 6986FP：5’-ATTATGGAGAGTGGCATAGGAG-3’；
Downstream primer 6986RP：5’-GCAAGAGTCTCACACAGGAG-3’；
5 ' ends of described probe, 3 ' ends are all marked with fluorophor, and described 5 ' ends are different with the fluorophor that 3 ' hold；
Described test kit also includes archaeal dna polymerase, and described archaeal dna polymerase is to have flap endonuclease activity
Archaeal dna polymerase.
5 ' ends of probe described in the embodiment of the present invention, 3 ' ends are all marked with fluorophor, and 5 ' ends, the 3 ' fluorophors held
Different.As being modified with one respectively and sending green fluorescence (as fluorescein when 5 ' ends of double fluorescent labeled DNA probe and 3 ' ends
FAM) and red fluorescence (as VIC) group, and the next base (holding the 3 ' directions held along 5 ') in SNP site upper is modified
There is a quenching group (as the ZENTM Internal Quencher of Integrated Device Technology, Inc.), this quenching group can be quenched green simultaneously
Fluorophor and the fluorescence of red fluorescence group, therefore, before entering performing PCR reaction, this DNA probe is not send any fluorescence
's.It is complementary with corresponding sequence on target nucleic acid by the sequence SNP site to its 3 ' end on double fluorescent labeled DNA probe,
And not complementary with corresponding sequence on target nucleic acid by the sequence SNP site to its 5 ' end, therefore form valvular structure, i.e. portion
It is divided into double-strand, be partly single-stranded Y-shaped structure.Because described archaeal dna polymerase has flap endonuclease activity, work as forward direction
When primer is extended at fluorescent probe, on fluorescent probe, the sequence complementary with target nucleic acid can not excised by polymerase, that is, have
The described archaeal dna polymerase having flap endonuclease activity can be by 5 ' end single stranded portion excisions in valvular structure, cleavage
Put between first base of double stranded section and second base of valvular structure.Base when SNP site on target nucleic acid
During with the corresponding base complementrity on fluorescent probe, then excise between SNP site and next base.Therefore, green fluorescence base
Group is separated with quenching group, thus producing green fluorescence.Continue complementary with target nucleic acid on cutting fluorescent probe with polymerase
Sequence, quenching group is also separated with the red fluorescence group of the other end, thus producing red fluorescence.Therefore, in green fluorescence
In the case that the luminous efficiency of group and red fluorescence group is substantially the same, if the base of the SNP site on target nucleic acid with
Corresponding base complementrity on fluorescent probe, the intensity of the green fluorescence recording is generally equal with the intensity of red fluorescence.Work as mesh
The base of the relevant position not mutual added time on base fluorescent probe in the SNP site of mark nucleic acid, polymerase is enterprising in fluorescent probe
The position of row cutting is after the base being modified with quenching group.Therefore, not complementary with target nucleic acid sequence on fluorescent probe
After row are removed, green fluorescence group and quenching group remain in this and are removed in the sequence got off, therefore green
The fluorescence of fluorophor is still quenched group and is quenched, thus not producing green fluorescence.But quenching group and red fluorescence group
Separate, thus producing red fluorescence.Therefore, if the base of the SNP site on target nucleic acid and the corresponding base on fluorescent probe
Not complementary, the intensity of the green fluorescence recording is negligible compared with the intensity of red fluorescence.When containing in sample of nucleic acid simultaneously
There are the target nucleic acid of the base of SNP site of half and the corresponding base complementrity on fluorescent probe and the alkali of the SNP site of half
The base not target nucleic acid with the corresponding base complementrity on fluorescent probe.In this case, the base of SNP site and fluorescent probe
On the target nucleic acid of corresponding base complementrity produce green fluorescence and red fluorescence simultaneously, and the base of SNP site not with fluorescence
The target nucleic acid of the corresponding base complementrity on probe only produces red fluorescence.Because the content of two kinds of target nucleic acids is equal, green
In the case that the luminous efficiency of color fluorophor and red fluorescence group is substantially the same, the intensity of the described green fluorescence recording
It is about the half of the intensity of red fluorescence.
It can be seen that, the embodiment of the present invention is combined dual glimmering using the described archaeal dna polymerase with flap endonuclease activity
Signal DNA probe carries out Fluorescence PCR it is achieved that detect two kinds in target nucleic acid SNP site with a fluorescent probe
Typing, simplifies operation, has saved cost.The specificity detecting SNP additionally, due to the method derives to be had in flap nucleic acid
The archaeal dna polymerase of enzyme cutting activity excises the accuracy of cutting position during valvular structure, rather than based on DNA probe and target core
The accuracy (i.e. in SNP site, different bases cause the difference of melting temperature) of the hybridization between acid, therefore, the method detects
The accuracy of SNP and reliability are significantly higher than prior art.
Further, carefully study through inventor, preferably following have specific probe sets, specifically, described
The probe sequence in CYP3A5 gene 6986 site is as follows：
Probe 6986A：5’-TCTTTCAATATCTCTTCCCTGTTTGGA-3’；
Described probe 5 ' end, 3 ' end be respectively adopted FAM, HEX, TET, VIC, ROX, CY5, CY3, JOE, ALEX, CAL,
One of Fluor fluorophor is marked, the 9th T site-tag fluorescent quenching group of described probe 6986A.
Double fluorescent labeled DNA probe used by the embodiment of the present invention, its alkali with target sequence SNP site correspondence position
Base (be set to A and C, that is, homozygous with wild type) identical, the next kilobase marker fluorescent quenching group of SNP site.For example
5 ' ends of FAM label probe, 3 ' ends of VIC label probe.When target nucleic acid is wild type, as AA type, then can lead in FAM
Road and VIC passage are detected simultaneously by the roughly equal fluorescence signal of intensity.When target nucleic acid is saltant type, as GG type, then
Can be in VIC Air conduct measurement to fluorescence signal, the intensity of the green fluorescence of FAM passage is very faint, compared with the intensity of red fluorescence
Negligible.When target nucleic acid is heterozygous, as AG type, then the green fluorescence intensity of FAM passage is the redness of VIC passage
The half of fluorescence intensity.Achieve two kinds of genotype in probe in detecting CYP3A5 gene 6986 site, compare traditional
Fluorescent PCR sonde method (corresponds to two kinds of genotype respectively using two kinds of probes), simplifies operation, has saved cost.It is additionally based on
The archaeal dna polymerase with flap endonuclease activity excises the accuracy of cutting position during valvular structure so that this method
Detection accuracy be higher than conventional fluorescent PCR sonde method.
As a particular preferred embodiment, described test kit also includes UNG enzyme (uracil glycosylase enzyme), described UNG
Enzyme and archaeal dna polymerase are mixed to form enzymatic mixture.Test kit reaction system described in the embodiment of the present invention adopts UNG-dUTP antifouling
Dye system, wherein, the uracil glycosidic bond that described UNG enzyme can be ruptured in the double-strand containing dU or single stranded DNA with selective hydrolysis,
The DNA having disappearance base being formed, the further hydrolytic cleavage of meeting under alkaline medium and high temperature, therefore in reaction system
Add appropriate dUTP, before PCR amplification, can effectively prevent from testing the pollution that indoor amplified production leads to using UNG enzyme effect, from
And whole detection system is included with the concentration of sample, interference factor carries out quality control.And UNG enzyme can be inactivated when 95 DEG C,
Do not affect subsequent PCR amplification effect.Wherein, described archaeal dna polymerase is from the DNA polymerization with flap endonuclease activity
Enzyme, the Taq archaeal dna polymerase of the concrete preferred New England Biolabs company of the embodiment of the present invention.This preferred mixed enzyme
System, enters performing PCR amplification not only by archaeal dna polymerase, at the same time it can also pollution is eliminated by described UNG enzyme, thus more
Ensure that well 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 include buffer, dNTPs (containing dUTP),
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 negative controls,
Described positive reference substance comprises 2 kinds of plasmids, and described negative controls are TE buffer；Wherein, described 2 kinds of plasmids are respectively
CYP3A5 gene 6986 site A and G detection site recombiant plasmid.More specifically, described CYP3A5 positive reference substance is by CYP3A5
Gene 6986 site A and G detection site recombiant plasmid press 1：1 ratio composition.
The test kit of detection people's CYP3A5 gene type provided in an embodiment of the present invention, using having flap Cobra venom endonuclease
The archaeal dna polymerase of activity, and cutting position when 5 ' ends in excision valvular structure are single-stranded using this polymerase is located at lobe
This characteristic between first base of the double stranded section of shape structure and second base, in combination with being modified with double fluorescent mark
The dual labelled fluorogeilic DNA probe of note and quenching group and specific primer, carry out SNP inspection by real-time PCR reactions in single tube
Survey, determine CYP3A5 gene, determine the gene pleiomorphism in site, realize detecting target nucleic acid SNP site with a fluorescent probe
On two kinds of typings, and testing result has high resolution, rapid, accurate advantage.
And, the embodiment of the present invention additionally provides a kind of detection method of the test kit of detection people's CYP3A5 gene type,
Comprise the following steps：
S01. people's Whole Blood Genomic DNA and mentioned reagent box are extracted；
S02. press in the reaction system that people's Whole Blood Genomic DNA extracting solution adds described test kit, wherein, described people's whole blood
The ratio of extracting genome DNA liquid and described reaction system is than for 1：8；
S03.PCR expands；
S04. result judgement.
Specifically, in above-mentioned steps S01, described test kit is mentioned reagent box, the extraction of described people's Whole Blood Genomic DNA
Method can refer to following specific embodiments and carries out, it is of course possible to extract the method obtaining people's Whole Blood Genomic DNA, all at this
In bright scope of embodiments.
In above-mentioned steps S02, the ratio of described people's Whole Blood Genomic DNA extracting solution and described reaction system is than for 1：8 add
Plus, as a specific embodiment, in proportion 5 μ l people's Whole Blood Genomic DNA extracting solution are added the anti-of test kit described in 40 μ l
Answer in system, wherein, test kit described in 40 μ l includes 34.5 μ lPCR reactant liquors and 0.5 μ l enzymatic mixture, certainly, concrete volume
Can be adjusted accordingly according to this ratio.The pipe number entering performing PCR reaction is not particularly limited, and usually sample number+2 is (negative right
According to, positive control).Therefore, this step at least includes being separately added into sample, sun in the reaction system of different described test kits
Property comparison and negative control.
In above-mentioned steps S03, the reaction condition of described PCR amplification is：
50℃2min；
95 DEG C of denaturations 2min；
95℃15s；62℃45s；35 circulations,
Collect fluorescence in 62 DEG C of 45s.
In above-mentioned steps S04, by being analyzed to result judging, described CYP3A4 gene C YP3A4*1B position can be learned
Point, the concrete gene type in CYP3A4*3 site, described analysis decision method, it is described in following specific embodiments.
It should be noted that the detection method of detection people's CYP3A4 gene type, for determining the typing of CYP3A4 gene,
This typing can but not only can be instructed with adjuvant clinical, it may also be used for analysis hereditism, gene pleiomorphism etc..And this
Detect the detection method of people's CYP3A4 gene type described in bright embodiment, be used only for diagnosis and the therapy field of non-diseases.
The detection method of detection people's CYP3A5 gene type provided in an embodiment of the present invention, the specificity source of detection SNP
The accuracy of the cutting position when archaeal dna polymerase with flap endonuclease activity excises valvular structure, before comparing
Fluorescent PCR melting curve method more accurate.Therefore the method is compared traditional quantitative fluorescent PCR on carrying out gene type and is shown
Show advantage.Additionally, present invention method has simple to operate, rapid, result accurately and reliably advantage.
Illustrate with reference to specific embodiment.
Embodiment 1 design of primers synthesizes
Embodiment of the present invention CYP3A5 gene 6986 single nucleotide polymorphisms design of primers, according to NCBI people's CYP3A5 gene
Sequential design is as follows：
Forward primer 6986FP：5’-ATTATGGAGAGTGGCATAGGAG-3’；
Downstream primer 6986RP：5’-GCAAGAGTCTCACACAGGAG-3’.
The design synthesis of embodiment 2 probe
Corresponding probe is designed according to NCBI gene bank people's CYP3A5 gene pleiomorphism as follows：
Probe 6986A：5’-TCTTTCAATATCTCTTCCCTGTTTGGA-3’；
Wherein, probe 5 ' end and 3 ' ends can use multiple fluorophor labellings, and the embodiment of the present invention selects 5 ' ends to be marked with FAM
Note；VIC labelling, the adjacent base in SNP site downstream, i.e. the 9th T labelling quenching group ZEN are used in probe 3 ' endTMInternal
Quencher.
The preparation of embodiment 3 CYP3A5PCR reactant liquor
CYP3A5PCR reactant liquor is CYP3A5PCR reactant liquor 1, its composition be buffer, dNTPs (containing dUTP), MgCl2,
Primer, probe, concentration of component is as follows：
CYP3A5PCR reactant liquor 1：
Example 4 enzymatic mixture
Enzymatic mixture described in the embodiment of the present invention is mixed by uracil glycosylase enzyme (UNG enzyme) and archaeal dna polymerase, its
In, described archaeal dna polymerase is the archaeal dna polymerase with flap endonuclease activity, the embodiment of the present invention specifically preferably New
The Taq archaeal dna polymerase of England Biolabs company.
Embodiment 5 positive reference substance and the preparation of blank
Design positive reference substance sequence according to ncbi database CYP3A5 gene 6986 single nucleotide polymorphisms, synthesize sequence
Row, constructed dna recombiant plasmid.CYP3A5 positive reference substance includes 2 kinds of plasmids and is respectively 6986A wild type, 6986G saltant type.
Specifically press 1 by 6986A wild type, 6986G saltant type recombiant plasmid:1 mixes.
The outfit of embodiment 6 reaction system
The reaction system of 40 μ l is adopted, its concrete reactive component is as shown in table 1 below in the present invention.
Table 1
The setting of embodiment 7 response procedures
In the embodiment of the present invention, preferred response procedures are 50 DEG C, 2min；95 DEG C, 2min；95 DEG C, 15s, 62 DEG C, 45s,
35 circulations.
8 test kits of embodiment detect the detection method of clinical sample
This test kit detects the detection method of clinical sample, comprises the following steps：
1. the extraction of human blood sample genomic dna, can divide public affairs using Shenzhen HYK Gene Technology Co., Ltd. Bao'an
The poba gene group extraction purification test kit that department produces, concrete operations are as follows：
(1) from extracts kit take out 1.5ml centrifuge tube several, each pipe add anticoagulation 200 μ l.
(2) add 500 μ l buffer CLG solution in anticoagulation, cover tightly centrifugation lid, whirlpool shakes more than 20s, fully
Reverse mixing is it is necessary to abundant mixing or whirlpool concussion are to guarantee to discharge genomic DNA.
(3) 100 μ l buffer PP solution, whirlpool concussion or back and forth reverse 10s are added.
(4) 12,000g centrifugation 5min.
(5) from extracts kit take out DNA purification column, the supernatant in step 4 is transferred to purification column, 8000g from
Heart 1min.For improving DNA extraction amount, after centrifugation, filtrate can rewind be centrifuged once again.
(6) abandon filtrate after post, DNA purification column was put in same collecting pipe, add 750 μ l bufferings toward in purification column
Liquid W I, after room temperature places 2min, 8000g centrifugation 1min (confirms that buffer W I has added dehydrated alcohol).
(7) abandoned filtrate after post, added 750 μ l Buffer buffer W II toward in purification column, 8000g was centrifuged 1min
(confirming that buffer W II has added dehydrated alcohol).
(8) abandoned filtrate after post, by purification column 12,000g is centrifuged 2min.
(9) purification column is transferred in new 1.5ml centrifuge tube, room temperature is placed 5min and fully volatilized ethanol.Plus 70～100
μ l collection liquid EB (collection liquid EB is preheating to 65 DEG C and is conducive to improving the elution efficiency of DNA), room temperature places 1min, 12,000 centrifugations
2min eluting genomic DNA.Can be then added to crossing the eluent sucking-off after post on purification column film, room temperature placement 1min, 12,000g
Centrifugation 1min can improve the elution amount of DNA.
(10) concentration of genomic DNA and quality should measure OD260 and OD280 extinction by micro ultraviolet spectrophotometer
(the NanoPhotometerTMP-Clas micro-spectrophotometer of conventional Germany Implen, the development in science and technology of the triumphant Austria in Beijing are limited for value
The K2800 nucleic acids instrument of company), DNA concentration should be in 2ng/ μ l≤DNA concentration≤100ng/ μ l scope OD260/OD280
Value is between 1.5～2.0.Purification DNA sample preserves at 4 DEG C and is less than 7 days；The DNA sample failing in time detection is protected in -20 DEG C
Deposit, the holding time is less than 3 months；Purification DNA sample multigelation is less than 5 times.
2. the fluorescence quantitative PCR detection of sample
(1) reagent prepares
Take out CYP3A5PCR reactant liquor from test kit, thaw on ice, calculate the PCR required for each reaction system
Reaction tube number, pipe number is n (n=sample number+2 (negative control, positive control)).According to the form below 2 takes the PCR of amount of calculation to react respectively
Liquid and enzymatic mixture, in suitable centrifuge tube, overturn and mix 10 times, prepare reaction system, and 2000g is centrifuged 10 seconds.
Table 2
Take 35 μ l reaction systems respectively to PCR reaction tube, the different reaction system of labelling.
(2) it is loaded
Positive control and negative control take out when receiving, and are put into sample treatment room/sample addition zone.Positive control and feminine gender are right
Impinge upon and thaw on ice, mix in turbula shaker, 2000g is centrifuged 10 seconds.It is separately added into in n set PCR reaction tube
The each 5 μ l of sample, positive control, negative control, cover tightly lid, and make a record.Mix the PCR reactant in each PCR reaction tube
System and added sample.PCR reaction tube 2000g is centrifuged 10 seconds.It is transferred to detection zone, is placed in PCR instrument, record sample is put
Sequentially.
(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) is set to None, threshold
Value line is set to 50000.
PCR amplification program：
50℃2min；
95 DEG C of denaturations 2min；
95℃15s；62℃45s；35 circulations,
Collect fluorescence in 62 DEG C of 45s, that is, phosphor collection is arranged on " step 3：62 DEG C, 45s ".
3. the Analysis of test results of sample
(1) as sample CYP3A5PCR reactant liquor 1 is more than 37.5 in VIC sense channel no Ct or Ct, then the base of judgment sample
Because group DNA concentration is less than test limit.
(2) in the case that CYP3A5PCR reactant liquor 1 is less than or equal to 37.5 in VIC sense channel Ct, sentence by following
Disconnected.
Comparative sample FAM passage and the fluorescence intensity of VIC passage, according to the form below 3 result of determination：
Table 3
CYP3A5 gene type is as shown in table 5 below：
Table 4
In embodiment of the present invention test kit, the testing result in CYP3A5 gene 6986 site has 3 kinds, and Fig. 1 show
6986 sites are the testing result of AA wild type, and Fig. 2 show the testing result that 6986 sites are GG saltant type, and Fig. 3 show
The testing result of 6986 site AG heterozygous.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (8)
1. a kind of test kit of detection people's CYP3A5 gene type is it is characterised in that described test kit includes reactant liquor, described anti-
Liquid is answered to include primer for CYP3A5 gene 6986 site, probe, wherein, the primer sequence in described CYP3A5 gene 6986 site
Row are as follows：
Forward primer 6986FP：5’-ATTATGGAGAGTGGCATAGGAG-3’；
Downstream primer 6986RP：5’-GCAAGAGTCTCACACAGGAG-3’；
5 ' ends of described probe, 3 ' ends are all marked with fluorophor, and described 5 ' ends are different with the fluorophor that 3 ' hold；
Described test kit also includes archaeal dna polymerase, and described archaeal dna polymerase is that the DNA with flap endonuclease activity gathers
Synthase.
2. the test kit of detection people's CYP3A5 gene type as claimed in claim 1 is it is characterised in that described CYP3A5 gene
The probe sequence in 6986 sites is as follows：
Probe 6986A：5’-TCTTTCAATATCTCTTCCCTGTTTGGA-3’；
5 ' ends of described probe, 3 ' ends are respectively adopted FAM, HEX, TET, VIC, ROX, CY5, CY3, JOE, ALEX, CAL, Fluor
One of fluorophor is marked, the 9th T site-tag fluorescent quenching group of described probe 6986A.
3. the test kit of detection people's CYP3A5 gene type as claimed in claim 1 is it is characterised in that described test kit also wraps
Include UNG enzyme, described UNG enzyme and archaeal dna polymerase are mixed to form enzymatic mixture.
4. the test kit of described detection people's CYP3A5 gene type as arbitrary in claim 1-3 is it is characterised in that described reagent
Box is provided with positive reference substance and negative controls, and described positive reference substance comprises 2 kinds of plasmids, and described negative controls delay for TE
Rush liquid；Wherein, described 2 kinds of plasmids are respectively CYP3A5 gene 6986 site A and G detection site recombiant plasmid.
5. the test kit of detection people's CYP3A5 gene type as claimed in claim 4 is it is characterised in that described positive reference substance
Press 1 by CYP3A5 gene 6986 site A and G detection site recombiant plasmid：1 ratio composition.
6. the test kit of described detection people's CYP3A5 gene type as arbitrary in claim 1-3 is it is characterised in that described reaction
Liquid includes buffer, dNTPs (containing dUTP), MgCl2.
7. a kind of detection method of the test kit of detection people's CYP3A5 gene type, comprises the following steps：
Extract people's Whole Blood Genomic DNA and the arbitrary described test kit of claim 1-6；
Add in the reaction system of described test kit by people's Whole Blood Genomic DNA extracting solution, wherein, described people's whole blood genome
The ratio of DNA extraction liquid and described reaction system is than for 1：8；
PCR expands；
Result judgement.
8. the detection method of the test kit of detection people's CYP3A5 gene type as claimed in claim 7 is it is characterised in that described
PCR amplification reaction condition be：
50℃2min；
95 DEG C of denaturations 2min；
95℃15s；62℃45s；35 circulations.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110093415A (en) * | 2019-04-30 | 2019-08-06 | 上海百傲科技股份有限公司 | Method, kit, primer pair and probe for detecting CYP3A5 gene |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436631A (en) * | 2013-09-22 | 2013-12-11 | 刘辉 | Kit and method for detecting CYP3A5 gene polymorphism |
| CN104164506A (en) * | 2014-08-13 | 2014-11-26 | 深圳联合医学科技有限公司 | Method for real-time PCR (polymerase chain reaction) detection of single nucleotide polymorphism and application of method |
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2016
- 2016-09-21 CN CN201610840080.0A patent/CN106480177A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436631A (en) * | 2013-09-22 | 2013-12-11 | 刘辉 | Kit and method for detecting CYP3A5 gene polymorphism |
| CN104164506A (en) * | 2014-08-13 | 2014-11-26 | 深圳联合医学科技有限公司 | Method for real-time PCR (polymerase chain reaction) detection of single nucleotide polymorphism and application of method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110093415A (en) * | 2019-04-30 | 2019-08-06 | 上海百傲科技股份有限公司 | Method, kit, primer pair and probe for detecting CYP3A5 gene |
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