CN106319073A - Primer, probe, kit and method for detecting subtypes of human gene CYP1A2 - Google Patents
Primer, probe, kit and method for detecting subtypes of human gene CYP1A2 Download PDFInfo
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Abstract
The invention belongs to the technical field of genotyping and particularly relates to a primer, probe, kit and method for detecting subtypes of a human gene CYP1A2. The primer and the probe comprise the following nucleotide sequences: the nucleotide sequence of a forward primer is SEQ ID NO: 1: 5'-AAACTGAGATGATGTGTGGAGG-3'; the nucleotide sequence of a reverse primer is SEQ ID NO: 2: 5'-CACGCATCAGTGTTTATCAAA-3'; the nucleotide sequence of the probe is SEQ ID NO: 3: 5'-GTGGGCCCAGGACGCATGGTAGATGGA-3'. The kit comprises the primer, the probe, dNTPs, MgCl2, a mixed enzyme and positive and negative reference substances, wherein the mixed enzyme is prepared from a UNG enzyme and DNA polymerase with the activity of flap endonuclease. According to the method for detecting the subtypes of the gene CYP1A2 by using the kit, different subtypes of a target nucleic acid SNP locus can be detected by one fluorescent probe, so that the operating process is simplified, the cost is reduced, and the detection result is high in resolution and is rapid and accurate.
Description
Technical field
The invention belongs to genotyping technique field, be specifically related to a kind of primer detecting mankind's CYP1A2 gene type and
Probe, test kit, method.
Background technology
In Cytochrome P450 superfamily, the metabolic enzyme relevant to drug metabolism is mainly CYP1, CYP2, CYP3 family.
CYP1A2 is the important member of CYP1A subfamily, is one of most important CYP enzyme in people's liver, accounts for the 13 of liver CYP enzyme total amount
～15%.CYP1A2 has specific expressed in hepatic tissue, and arylamine, heterocyclic amine and some Halogen hydrocarbonylation things are all its important ends
Thing.There is genetic polymorphism, there is individuality, racial diversity, cause Different Individual, the existence of ethnic medicament metabolism ability poor
Different.CYP1A2*1F (-163C > A) is positioned at No. 1 intron, and sudden change can increase the activity of CYP1A2.The different genotype of CYP1A2
Directly affect the power of curative effect of medication and toxic and side effects, therefore instruct accurately according to the genotype of CYP1A2, safe medication tool
There is important clinical meaning.
Sequencing, PCR-RFLP (polymerase chain reaction-restriction fragment is mainly had for detecting the method for gene pleiomorphism
Length polymorphism), gene chips, fluorescence quantitative PCR method.Sequencing result is accurate, but cost is high, the longest.PCR-RFLP
Detect gene pleiomorphism, low cost, visual result, but operating process vulnerable to pollution by the method for restriction enzyme digestion and electrophoresis, cause mistake
Result.Gene chips detects genotype by nucleic acid hybridization, and advantage is that flux is high, and shortcoming is relatively costly, complex operation, knot
The difficult interpretation of fruit.Conventional fluorescent quantitative PCR method can be divided into fluorescent PCR sonde method and fluorescent PCR melting curve according to its principle difference
Method.The former designs two kinds of probes for target SNP site, mates with saltant type with wild type respectively, utilizes the Ct value difference of amplification
Different come interpretation genotype.The latter is for a kind of probe of target SNP site design, standard based on probe Yu the intermolecular hybrid of target nucleic acid
Really property (i.e. in SNP site, different bases cause the difference of melting temperature) carrys out interpretation genotype.Traditional fluorescence quantitative PCR method inspection
The accuracy surveyed depends on the difference of the affine degree between probe and corresponding allele, the i.e. difference of its solution temperature (Tm)
Not, when this difference is not the biggest, often leading to final testing result and there is deviation, accuracy is the highest.Along with technology
Constantly bring forth new ideas, a kind of novel SNP detection technique based on flap Cobra venom endonuclease and double fluorescence labeling probe is developed.
The method uses the archaeal dna polymerase with flap endonuclease activity, and utilizes this polymerase in excision valvular structure
5 ' end strand time cutting position between first base and second base of the double stranded section of valvular structure this
Characteristic, in conjunction with the DNA probe being modified with double fluorescent labelling and quenching group, it is achieved that with a fluorescent probe detection target core
Typing in acid SNP site.
Detection method currently for CYP1A2 gene type limits to said gene chip method, the spy of conventional fluorescent quantitative PCR
The skill of handling needles, its shortcoming is increasingly difficult to meet the requirement of Clinical Laboratory.Utilizing the novel SNP detection technique of double fluorescence labeling probe
The research of detection CYP1A2 gene type, also nobody's report.
Summary of the invention
Present invention aim to overcome that the deficiencies in the prior art, by autonomous Design primer and probe, and combine based on lobe
Shape Cobra venom endonuclease and the novel SNP detection technique of double fluorescence labeling probe, it is provided that a kind of detection mankind's CYP1A2 gene type
Primer and probe, test kit, method, with solve currently for CYP1A2 gene type detection confinement problems.
In order to realize foregoing invention purpose, the technical solution used in the present invention is as follows:
For detecting primer and the probe of mankind's CYP1A2 gene type, comprise following nucleotide sequence:
Forward primer SEQ ID NO:1:5 '-AAACTGAGATGATGTGTGGAGG-3 '；
Downstream primer SEQ ID NO:2:5 '-CACGCATCAGTGTTTATCAAA-3 '；
Probe SEQ ID NO:3:5 '-GTGGGCCCAGGACGCATGGTAGATGGA-3 '.
Primer and probe that the present invention provides can accurately detect CYP1A2 gene mutation site region.
The present invention also proposes a kind of test kit detecting mankind's CYP1A2 gene type, and described test kit comprises PCR reaction
Liquid, described PCR reactant liquor includes above-mentioned primer and probe.
The test kit low cost that the present invention provides, uses simple, can accurately detect CYP1A2 gene type.
The present invention also provides for a kind of method utilizing mentioned reagent box detection mankind's CYP1A2 gene type, and it includes as follows
Step:
Extract human gene group DNA；
Described DNA is made into reaction system with described test kit, carries out fluorescent quantitative PCR；
Described CYP1A2 gene type is analyzed according to described amplification.
The method of the detection CYP1A2 gene type that the present invention provides, can detect target nucleic acid with a fluorescent probe
Multiple typing in SNP site, simplifies operating process, has saved cost, and testing result resolution is high, the most accurate.
It addition, the present invention also provides for the purposes utilizing mentioned reagent box in detection CYP1A2 gene type.This purposes can
Abolish the limitation of existing CYP1A2 gene type detection, hinge structure, be more easy to meet the requirement of Clinical Laboratory.
Accompanying drawing explanation
Fig. 1 is CYP1A2 gene-1 63 site wild type testing result figure；
Fig. 2 is CYP1A2 gene-1 63 site homozygous mutant testing result figure；
Fig. 3 is CYP1A2 gene-1 63 site heterozygous testing result figure；
Fig. 4 is CYP1A2 gene-1 63 site homozygous mutant contrast test testing result figure.
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, below in conjunction with
Drawings and Examples, are further elaborated to the present invention.Should be appreciated that specific embodiment described herein is only used
To explain the present invention, it is not intended to limit the present invention.
The embodiment of the present invention provides a kind of primer for detecting mankind's CYP1A2 gene type and probe, comprises following core
Nucleotide sequence:
Forward primer SEQ ID NO:1:5 '-AAACTGAGATGATGTGTGGAGG-3 '；
Downstream primer SEQ ID NO:2:5 '-CACGCATCAGTGTTTATCAAA-3 '；
Probe SEQ ID NO:3:5 '-GTGGGCCCAGGACGCATGGTAGATGGA-3’。
The 7th nucleotide of this probe is complementary with corresponding sequence on target nucleic acid to the sequence between 3 ' ends, this CYP1A2 base
Cause sport c.-163C > A.The primer of the present embodiment and probe can accurately detect CYP1A2 gene mutation site region.
Specifically, hold respectively with two kinds of different fluorescent reporter group labellings with 3 ' at probe 5 ' end, it is preferable that fluorescence report
Announcement group is selected from: FAM, HEX, TET, VIC, ROX, CY5, CY3, JOE, ALEX, CAL；The 8th nucleotide site at probe is used
Fluorescent quenching group labelling.In the present embodiment, 5 ' ends and the 3 ' ends of DNA probe have one to send green fluorescence (FAM) respectively
And the group double labeling of red fluorescence (VIC), and SNP site next base (along 5 ' end to 3 ' hold directions ground 8
Base) on be modified with a quenching group (ZEN of Integrated Device Technology, Inc.TMInternal Quencher), this quenching group can be simultaneously
Cancellation green fluorescence group and the fluorescence of red fluorescence group.3 ' it is held by SNP site to it on double fluorescent labeled DNA probe
Between sequence and target nucleic acid on corresponding sequence complementary, and by SNP site to its 5 ' hold sequence and phase on target nucleic acid
Answer sequence the most complementary, therefore form valvular structure.
The embodiment of the present invention also provides for a kind of test kit detecting mankind's CYP1A2 gene type, and this test kit comprises by upper
State primer and the PCR reactant liquor of probe composition, wherein: forward primer concentration is: 0.1-0.5 μM；Downstream primer concentration is: 0.1-
0.5μM；Concentration and probe concentration is: 0.1-0.5 μM.The most in the present embodiment, forward primer 0.5 μM；Downstream primer 0.5 μM；Probe
0.5μM.This test kit low cost, uses simple, can accurately detect CYP1A2 gene type.
Specifically, this test kit is possibly together with dNTPs, MgCl2.Wherein dNTPs concentration is: 0.2-0.5mM；MgCl2Concentration
For: 0.5-2.5mM；In the present embodiment, preferably dNTPs 0.5mM；MgCl22.5mM.Specifically, this test kit also comprises UNG
Enzyme and archaeal dna polymerase, this archaeal dna polymerase has flap endonuclease activity.
Owing to archaeal dna polymerase has flap endonuclease activity, when forward primer is extended at fluorescent probe, glimmering
Sequence not complementary with target nucleic acid on light probe can be excised by polymerase.Cutting position valvular structure double stranded section
Between one base and second base.When the base of SNP site and the corresponding base complementrity on fluorescent probe on target nucleic acid
Time, then excise between SNP site and next base.Therefore, green fluorescence group separates with quenching group, thus produces green
Color fluorescence.Along with polymerase continues sequence complementary with target nucleic acid on cutting fluorescent probe, quenching group also with the other end
Red fluorescence group separates, thus produces red fluorescence.Therefore, in the luminous efficiency of green fluorescence group Yu red fluorescence group
In the case of being substantially the same, if the base of the SNP site on target nucleic acid and the corresponding base complementrity on fluorescent probe, record
The intensity of green fluorescence the most equal with the intensity of red fluorescence.When the base in the SNP site of target nucleic acid and fluorescence
The base of the relevant position not mutual added time on probe, the position that polymerase carries out cutting on fluorescent probe is being modified with quenching group
Base after.Therefore, sequence not complementary with target nucleic acid on fluorescent probe is cut get off after, green fluorescence group
Remaining in this cut sequence got off with quenching group, therefore the fluorescence of green fluorescence group is still quenched group and quenches
Go out, thus do not produce green fluorescence, but quenching group separates with red fluorescence group, thus produce red fluorescence, now record
The intensity of green fluorescence negligible compared with the intensity of red fluorescence.When the SNP containing half in sample of nucleic acid simultaneously
The target nucleic acid of the base in site and the corresponding base complementrity on fluorescent probe and the base of second half SNP site not with fluorescence
The target nucleic acid of the corresponding base complementrity on probe, in this case, the base of SNP site and the corresponding alkali on fluorescent probe
The complementary target nucleic acid of base produces green fluorescence and red fluorescence simultaneously, and the base of SNP site not right with on fluorescent probe
The target nucleic acid answering base complementrity only produces red fluorescence.Owing to the content of two kinds of target nucleic acids is equal, at green fluorescence group
In the case of being substantially the same with the luminous efficiency of red fluorescence group, the intensity of the described green fluorescence recorded is about red glimmering
The half of the intensity of light.
The specificity of the test kit detection SNP of the present embodiment derives from the DNA polymerization with flap endonuclease activity
Enzyme action is except the accuracy of cutting position during valvular structure rather than standard based on the hybridization between DNA probe and target nucleic acid
Really property (i.e. in SNP site, different bases cause the difference of melting temperature), therefore, the accuracy of this test kit detection SNP and can
It is significantly higher than prior art by property.
The test kit reaction system of the embodiment of the present invention uses UNG-dUTP decontamination system, and wherein, affiliated UNG enzyme is permissible
Selective hydrolysis fracture containing the uracil glycosidic bond in the double-strand of dU or single stranded DNA, the DNA having disappearance base of formation,
Under alkaline medium and high temperature, the further hydrolytic cleavage of meeting, therefore adds appropriate dUTP in reaction system, expands at PCR
Before, utilize UNG enzyme effect can effectively prevent the pollution that amplified production in laboratory causes, thus whole detection system is included sample
The concentration of product, interference factor carry out quality control.And when 95 DEG C, UNG enzyme can be inactivated, and does not affect subsequent PCR amplification effect.
More specifically, embodiment of the present invention test kit is provided with positive reference substance and negative controls, positive reference substance bag
Containing two kinds of plasmids, negative controls is TE buffer.These two kinds of plasmids comprise the-163C wild-type nucleic acid of CYP1A2 gene respectively
Sequence and-163A mutant nucleic acid sequence.Two kinds of plasmid molar concentration ratio composition than 1:1.By positive, negative right
According to, make the present embodiment technical method more accurately true.
The embodiment of the present invention also provides for a kind of method detecting mankind's CYP1A2 gene type, and it comprises the steps:
Extract human gene group DNA；
DNA is made into reaction system with mentioned reagent box, carries out fluorescent quantitative PCR；
CYP1A2 gene type is analyzed according to amplification.
Specifically, this reaction system is 40ul.Wherein, DNA mass: 10-500ng, preferably 100ng；PCR reactant liquor
34.5ul, mixed enzyme 0.5ul.The response procedures of fluorescent quantitative PCR is: 50 DEG C of 2min；95 DEG C of denaturations 2min；95 DEG C of changes
Property 15s；62 DEG C extend 45s；35 circulations.
This method utilizes flap Cobra venom endonuclease to combine double fluorescent labeled DNA probe to carry out Fluorescence PCR and achieve
With the multiple typing in a fluorescent probe detection target nucleic acid SNP site, simplify operation, saved cost.Based on the party
Method, accurately detection CYP1A2 gene type.
Finally, according to embodiments of the invention, the present inventor can naturally expect that mentioned reagent box is at detection CYP1A2
Purposes in gene type.This purposes can abolish the limitation of existing CYP1A2 gene type detection, hinge structure, is more easy to
Meet the requirement of Clinical Laboratory.
The present invention successively carried out test of many times, now lifted A partial experiment result as with reference to carrying out invention the most in detail
Thin description, is described in detail below in conjunction with specific embodiment.
Embodiment 1 detects the test kit of mankind's CYP1A2 gene type
Primer and probe design
Inventor can detect primer and the spy of-163 single nucleotide polymorphisms according to NCBI people's CYP1A2 gene order, design
Pin nucleotide sequence is as follows:
Forward primer SEQ ID NO:1:5 '-AAACTGAGATGATGTGTGGAGG-3 '；
Downstream primer SEQ ID NO:2:5 '-CACGCATCAGTGTTTATCAAA-3 '；
Probe SEQ ID NO:3:5 '-GTGGGCCCAGGACGCATGGTAGATGGA-3’。
Wherein, the sequence between the 7th nucleotide of probe to 3 ' end is complementary with corresponding sequence on target nucleic acid, and probe is with double
Fluorescent reporter group labelling, its 5 ' end use FAM labelling, and 3 ' hold and use VIC labelling, the 8th T labelling quenching group ZEN of probeTM
Internal Quencher。
The preparation of CYP1A2 fluorescence quantitative PCR reaction solution
The configuration of the present embodiment PCR reaction solution concentration is as follows: buffer, dNTPs (containing dUTP) 0.5mM；MgCl22.5mM；
Forward primer 0.5 μM；Downstream primer 0.5 μM；Probe 0.5 μM.
Mixed enzyme
In test kit, mixed enzyme used is mixed by uracil glycosylase enzyme (UNG enzyme) and archaeal dna polymerase, wherein,
Archaeal dna polymerase is the archaeal dna polymerase with flap endonuclease activity, selected from the Taq of New England Biolabs company
Archaeal dna polymerase.The mixed enzyme active concentration of this composition is 2-4U.
Reference substance
Design positive reference substance sequence according to ncbi database CYP1A2 gene-1 63 single nucleotide polymorphisms, synthesize sequence
Row, constructed dna recombiant plasmid.CYP1A2 positive reference substance includes that 2 kinds of plasmids are respectively-163C wild type ,-163A saltant type.
Specifically mixed by 1:1 by-163C wild type ,-163A saltant type recombiant plasmid.And negative controls is TE buffer.
According to above-mentioned PCR reactant liquor, mixed enzyme, reference substance i.e. form that the present embodiment provides for detecting the mankind
The test kit of CYP1A2 gene type.
It should be understood that the content of each component is not limited to above-mentioned concentration in PCR reactant liquor, all can use as follows as long as meeting
In follow-up test, it may be assumed that dNTPs concentration 0.2-0.5mM；MgCl2Concentration 0.5-2.5mM；Forward primer concentration 0.1-0.5 μM；Under
Trip primer concentration 0.1-0.5 μM；Concentration and probe concentration 0.1-0.5 μM.
The fluorescence quantitative PCR detection of embodiment 2CYP1A2 gene type and analysis
(1) extraction of human blood sample genomic dna
The poba gene group that the present embodiment utilizes Bao'an branch company of Shenzhen HYK Gene Technology Co., Ltd. to produce is extracted
Purification kit extracts sample DNA, and 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) add 500 μ l buffer CLG solution in anticoagulation, cover tightly centrifugal lid, whirlpool concussion more than 20s, fully
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 column, 8000g
Centrifugal 1min.For improving DNA extraction amount, after being centrifuged, filtrate can rewind be centrifuged once again.
(6) abandon filtrate after post, DNA purification column was put in same collecting pipe, in purification column, add 750 μ l bufferings
Liquid W I, after room temperature places 2min, 8000g is centrifuged 1min (confirming that buffer W I has added dehydrated alcohol).
(7) abandoning filtrate after post, and added 750 μ l Buffer buffer W II in purification column, 8000g is centrifuged 1min
(confirming 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 collects liquid EB (collect liquid EB and be preheating to 65 DEG C of elution efficiencies being conducive to improving DNA), and room temperature places 1min, and 12,000 are centrifuged
2min eluting genomic DNA.Eluent sucking-off after crossing post can be then added on purification column film, room temperature placement 1min, 12,000g
Centrifugal 1min can improve the elution amount of DNA.
Finally, concentration and the quality of genomic DNA measures OD by trace ultraviolet spectrophotometer260And OD280Light absorption value
(Germany's NanoPhotometerTMP-Clas micro-spectrophotometer of Implen, Beijing triumphant AudioCodes skill Development Co., Ltd
K2800 foranalysis of nucleic acids instrument), DNA concentration is at 2ng/ μ l～100ng/ μ l；OD260/OD280Value, between 1.5～2.0, meets follow-up
Test requirements document.
The DNA sample of purification preserves less than 7 days at 4 DEG C；The DNA sample failing to detect in time, in-20 DEG C of preservations, preserves
Time is less than 3 months；Purification DNA sample multigelation is less than 5 times.
(2) reagent prepares
From test kit, take out PCR reactant liquor, thaw on ice, calculate the PCR reaction tube required for each reaction system
Number, pipe number is N (N=n sample number+1 negative control+1 positive control, N >=3).According to the form below 1 takes the PCR reaction of amount of calculation respectively
Liquid and mixed enzyme are in suitable centrifuge tube, and reverse mixing 10 times, 2000g is centrifuged 10 seconds, the reaction system that labelling is different.
Table 1
(3) sample-adding
Positive control and negative control take out, and thaw on ice, mix at turbula shaker, and 2000g is centrifuged 10 seconds.To institute
The PCR reaction tube set is separately added into sample, positive control, each 5ul of negative control, covers tightly lid, and make a record.Mixing
PCR reaction system in each PCR reaction tube and added sample.PCR reaction tube 2000g is centrifuged 10 seconds.It is transferred to detection zone,
Being placed in PCR instrument, order put by record sample.
It should be noted that above-mentioned sample, positive control dna mass concentration are between 2ng/ μ l～100ng/ μ l, the most often
Individual reaction system is that 10-500ng can meet test requirements document.
(4) 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 DEG C of 2min；95 DEG C of denaturations 2min；95 DEG C of degeneration 15s；62 DEG C extend 45s；35 circulations,
Collect fluorescence, i.e. phosphor collection when 62 DEG C of 45s and be arranged on " step 3:62 DEG C, 45s ".
(5) Analysis of test results
As reaction system testing result is more than 37.5 at VIC sense channel without Ct or Ct, then the genomic DNA of judgment sample
Concentration is less than detection limit.When reaction system testing result is in the case of VIC sense channel Ct is less than or equal to 37.5, by following
Table 2 judges (i.e. comparative sample FAM passage and the fluorescence intensity of VIC passage), and CYP1A2 gene type is as shown in table 3 below.
Table 2
Table 3
In the present embodiment testing result, the testing result in CYP1A2 gene-1 63 site has 3 kinds.As shown in Figure 1: FAM
Fluorescence intensity and VIC channel fluorescence intensity are close, and i.e.-163 sites are CC wild type；As shown in Figure 2: FAM channel fluorescence intensity
Comparing VIC channel fluorescence intensity negligible, i.e.-163 sites are AA homozygous mutant；As shown in Figure 3: FAM fluorescence intensity
For VIC channel fluorescence intensity half, i.e.-163 sites are CA heterozygous.
The present embodiment devises general T aqMan probe and carries out contrast test, for wild (FAM labelling) and mutational site
(VIC labelling) separately designs general T aqMan probe.Detect the result of homozygous mutant sample as shown in Figure 4, the open country of FAM labelling
Raw type coupling probe has obvious amplified signal (the most now should not having amplified signal or negligible), and its result is relative
Dual labelled probe accuracy designed by the present invention substantially reduces.
It follows that this CYP1A2 gene type detection method, have simple to operate, rapid, resolution is high, result is more accurate
True feature.
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 amendment, equivalent and the improvement etc. made within god and principle, should be included within the scope of the present invention.
Claims (10)
1., for detecting primer and the probe of mankind's CYP1A2 gene type, comprise following nucleotide sequence:
Forward primer SEQ ID NO:1:5 '-AAACTGAGATGATGTGTGGAGG-3 '；
Downstream primer SEQ ID NO:2:5 '-CACGCATCAGTGTTTATCAAA-3 '；
Probe SEQ ID NO:3:5 '-GTGGGCCCAGGACGCATGGTAGATGGA-3 '.
2. primer as claimed in claim 1 and probe, it is characterised in that described probe 5 ' end and 3 ' hold respectively with two kinds different
Fluorescent reporter group labelling, described fluorescent reporter group is selected from: FAM, HEX, TET, VIC, ROX, CY5, CY3, JOE, ALEX,
CAL；
8th nucleotide site fluorescent quenching group labelling of described probe.
3. primer as claimed in claim 1 or 2 and probe, it is characterised in that described CYP1A2 gene sport c.-163C
>A。
4. the test kit detecting mankind's CYP1A2 gene type, it is characterised in that described test kit comprises PCR reactant liquor,
Described PCR reactant liquor includes the primer as described in claim 1-3 is arbitrary and probe.
5. test kit as claimed in claim 4, it is characterised in that in described PCR reactant liquor, forward primer concentration is: 0.1-
0.5μM；Downstream primer concentration is: 0.1-0.5 μM；Concentration and probe concentration is: 0.1-0.5 μM.
6. test kit as claimed in claim 4, it is characterised in that described test kit also includes by UNG enzyme and archaeal dna polymerase group
The mixed enzyme become, described archaeal dna polymerase has flap endonuclease activity；And/or
Described test kit also includes positive reference substance and negative controls；Described positive reference substance includes two kinds of plasmids, described two
Kind of plasmid comprises-163C wild type nucleic acid sequence and the-163A mutant nucleic acid sequence of described CYP1A2 gene respectively, and described two
Plant plasmid molar concentration to mix than 1:1；Described negative controls is TE buffer；And/or
Described test kit also includes dNTPs, MgCl2, described dNTPs concentration is: 0.2-0.5mM；MgCl2Concentration is: 0.5-
2.5mM。
7. the method detecting mankind's CYP1A2 gene type, it comprises the steps:
Extract human gene group DNA；
Arbitrary with claim 4-6 for described DNA described test kit is made into reaction system, carries out fluorescent quantitative PCR；
Described CYP1A2 gene type is analyzed according to described amplification.
8. method as claimed in claim 7, it is characterised in that described reaction system is 40ul, wherein:
PCR reactant liquor 34.5ul, mixed enzyme 0.5ul, DNA mass 10-500ng.
9. method as claimed in claim 7 or 8, it is characterised in that the response procedures of described fluorescent quantitative PCR is: 50
℃2min；95 DEG C of denaturations 2min；95 DEG C of degeneration 15s；62 DEG C extend 45s；35 circulations.
10. the test kit of claim 4-6 purposes in detection CYP1A2 gene type.
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| CN112301096A (en) * | 2019-07-26 | 2021-02-02 | 杭州丹威生物科技有限公司 | Novel nucleic acid probe labeling method |
| CN112553349A (en) * | 2021-01-15 | 2021-03-26 | 内蒙古农业大学 | Identification primer, probe, kit and method for homozygote and heterozygote of Hulunbel short-tailed sheep |
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