CN107586838A

CN107586838A - For detecting the primer pair and kit of fentanyl medication related gene polymorphism

Info

Publication number: CN107586838A
Application number: CN201711037623.6A
Authority: CN
Inventors: 滕祥云; 曾好; 黄少亚
Original assignee: CHANGSHA 3G BIOTECH CO LTD
Current assignee: CHANGSHA 3G BIOTECH CO LTD
Priority date: 2017-10-25
Filing date: 2017-10-25
Publication date: 2018-01-16

Abstract

The present invention relates to a kind of primer pair and kit for being used to detect fentanyl medication related gene polymorphism, belong to beyond body nucleic acid detection technique field.The primer pair includes positive amplimer, reverse amplimer and sequencing primer, and 5 ' ends of the positive amplimer carry out biotin labeling；The kit includes positive amplimer, PCR reaction solutions, sequencing primer, uracil dna glycosylase, Taq polymerase and positive reference substance containing reverse amplimer.The kit provided by the invention has that testing result is accurate, specificity is high, detection cycle is short, simple to operate and the advantages of can effectively meet clinical examination requirement；In addition, also tool can monitor that reaction process, reaction time is short, PCR primer simple process can go up pyrophosphoric acid sequencer in real time, and than goldstandard method, i.e. Capillary Electrophoresis PCR sequencing method sensitivity is higher, more applicable mutation analysis.

Description

For detecting the primer pair and kit of fentanyl medication related gene polymorphism

Technical field

The present invention relates to beyond body nucleic acid detection technique field, more particularly to one kind to be used to detect fentanyl medication related gene The primer pair and kit of polymorphism.

Background technology

It is elimination pain when performing a surgical operation when anesthesia is or carries out diagnostic test operation, ensures that patient safety, creation are good Good surgical condition and the various methods taken.Anesthesia is also used for control pain, is performed the operation or when diagnostic test operates, sick People can feel pain, it is necessary to be allowed to swound with arcotic or other modes.Common Anesthesia medicine includes sedative Thing：Such as midazolam, Propofol；Central analgesicses：Morphine, fentanyl etc.；Muscle relaxant：Atracuium, along Ah Bent storehouse amine etc.；Local anesthetic：Lidocaine, Bupivacaine etc..

Fentanyl is artificial synthesized potent narcotic antalgesic, and Analgesic Mechanism is similar to morphine, is opiate receptor Activator, action intensity are 60~80 times of morphine.Compared with morphine and pethidine, fentanyl effect is rapid, holds time short, Do not discharge histamine, on cardiovascular function influence it is small, stress reaction during trachea cannula can be suppressed.Gradually taken after fentanyl listing For the status of morphine main flow analgesic in surgery anesthesia, turn into one of Narcotic analgesic drug being most often selected.Then Constantly there is new fentanyl class product to emerge, clinical alternative product is also more and more.The product that the country uses at present is main It is fentanyl, Remifentanil and sufentanil.

Fentanyl is as artificial synthesized opiates potent narcotic antalgesic, and in clinical anesthesia section of China, application is very wide It is general.But due to the presence of individual difference, Isodose fentanyl effect of drugs and drug metabolism in different patients have aobvious Difference is write, so as to influence its analgesic effect and adverse reaction.

Fentanyl is mainly metabolized in liver by CYP3A4 enzymes, and CYP3A4 enzymes belong to Cytochrome P450 oxidase system (CYP), it is most important drug metabolic enzyme.Wherein CYP3A contents in hepatomicrosome P450 are most, account for the half of total amount with On, the clinical medicine metabolism of participation about 60%.CYP3A4 is one of CYP3A families important member, controls some local anaesthetics, steroid The metabolism of body class analgesics anti-inflammatory, fentanyl class etc..Research display：Use the fentanyl of same dose, CYP3A4 gene mutations CYP3A4 enzyme systems can be caused to produce function to sexually revise, reduce fentanyl metabolism, corresponding blood concentration increase, acted on most strong； Heterozygous mutation is placed in the middle；Wild homozygote blood concentration is minimum, and effect is also most weak.

CYP3A4 is widely present genetic polymorphism phenomenon in crowd, is caused between individual between race to same substrate One of the reason for metabolic capability difference.The website of P450 allele name at present discloses 55 gene pleiomorphisms.CYP3A4 etc. Significant race and ethnic origin difference be present in position gene frequency.In Chinese population, gene frequency highest is CYP3A4* 1G, up to 27%.CYP3A4*1G types allelic mutation can cause the activity of CYP3A4 metabolic enzymes to change, and influence correlation The metabolic capability of medicine, so as to influence its blood concentration.

Researcher is when to Abdominal hysterectomy and myomectomy patient postoperative fentanyl anesthesia analgesia research It was found that CYP3A4*1G mutation are relevant with fentanyl Postoperative Patient-controlled Intravenous Analgesia effect, postoperative fentanyl consumption can be reduced.Min Yuyuan Found Deng when being worth to CYP3A4 gene pleiomorphisms to the personalized medicine of Patients with Big Area Burn application Analgesia with Fentanyl： CYP3A4*1G mutation patient's fentanyl dosages are significantly lower than the patient of control group, thus it is speculated that fentanyl is in CYP3A4*1G gene mutations Difference caused by individual analgesic effect is due to gene mutation and causes enzyme system expression to change, and drops CYP3A4 enzymatic activitys It is low.

Department of anesthesia clinician has a strict limitation when using analgesics such as fentanyls, and drug effect deficiency does not reach fiber crops in art Liquor-saturated standard, surgical effect will be influenceed；Postoperative drug effect deficiency can increase patient suffering；Drug dose can excessively cause respiratory system tight Adverse reaction again；Chronic pain patient dosage is improper to cause pharmacological dependence.Personalized medicine is rationally adjusted according to Patient genotype Whole therapeutic regimen, have great advantage improving curative effect with drug safety tool.

This kit is exactly the genotype for distinguishing the CYP3A4*1G sites of different patients, is that doctor's Clinical practice is fragrant Too Buddhist nun's class Narcotic analgesic drug provides reference, and avoids the generation of the serious adverse reactions such as postoperative pain, respiration inhibition.

Pyrosequencing (Pyrosequencing) is that a kind of DNA sequencing based on polymerization principle (determines nucleosides in DNA The order of acid) method, belong to DNA sequence analysis technology of new generation, possess while a large amount of samples are carried out with the ability of sequencing analysis, And there is the advantages of high flux, specificity is high, quick, directly perceived and inexpensive.Its general principle is, by the same of 4 kinds of enzymatics Enzyme cascade chemiluminescence reaction in reaction system, in each round sequencing reaction, only adds a kind of dNTP, if the dNTP and mould Plate matches, and polymerase can is incorporated into primer strand and discharges the pyrophosphoric acid group (PPi) of equimolar number, and PPi can Visible light signal is eventually converted into, and a peak value is converted into by PyrogramTM, is mixed in the height of each peak value and reaction Nucleotide number it is directly proportional；Then a kind of dNTP under adding, continues the synthesis of DNA；Finally by the feelings for analyzing peak value Condition, reach the purpose of measure DNA sequence dna.But, CYP3A4 bases also are detected without using pyrosequencing techniques in the prior art Because of the product of parting.

" goldstandard " that hospital is used for the detection of CYP3A4 Genotypings is PCR- direct sequencings, but direct sequencing is quick Perception it is not high, tumor tissues of the mutant cell ratio in more than 10-20% can only be detected, for containing<10% mutant cell Tumor tissues and peripheral blood, Standard PCR-direct sequencing are almost helpless.Compared to direct sequencing, pyrosequencing Sensitivity is higher, cost is lower.

At present, in the document of existing disclosed report, only apllied China when 2015 of this research team is special Sharp CN201510672628.0 is used to enter in " goldstandard " (the PCR- direct sequencings) of the detection of CYP3A4 Genotypings to hospital Innovation of having gone improves；But the technical scheme involved by patent CN201510672628.0, after product puts into clinical market, Especially during hospital's large-area applications, it is found that designed primer and kit specificity are not also especially high, detections As a result accuracy can also fluctuate once in a while, thus it is speculated that be probably the environment of design of primers and hospital's actual molecules diagnostic test room Adaptability problem be present；Therefore, design of primers optimization and corresponding PCR system stability still have larger room for improvement；We In many ways after investigating each hospital clinical molecular diagnostic laboratories environment and the problematic examining report result of statistical analysis, comprehensively to this Item purpose primer has carried out re-optimization design, and optimizes PCR reaction systems, significantly improves reagent kit product.

The content of the invention

In order to solve testing result be present during above method detection fentanyl medication correlation CYP3A4 gene pleiomorphisms Accuracy is not high, detection cycle is long, cumbersome and be difficult to the technical problem for meeting clinical examination requirement, and the present invention provides a kind of Testing result is accurate, specificity is high, detection cycle is short, cost is low, meets quantitatively and effectively the detection sweet smell of clinical examination requirement too The primer pair and kit of Buddhist nun's medication correlation CYP3A4*1G gene pleiomorphism；Using PCR combinations pyrosequencing techniques come real It is existing.

The invention provides a kind of primer pair for being used to detect fentanyl medication related gene polymorphism, the fentanyl is used The pleomorphism site of medicine phases correlation gene detection is CYP3A4*1G, and the primer pair is Pyrosequencing primer pair, and it includes：

Positive amplimer：5’-CCTTCCTACATAGAGTCA-3’(SEQID NO.1)；

Reverse amplimer：5’-ATTGATGCAGTTTTACCC-3’(SEQID NO.2)；

Sequencing primer：5’-CACGATTCTAGCTATGT-3’(SEQID NO.3)；

Wherein, 5 ' ends of the positive amplimer carry out biotin labeling.

Present invention also offers a kind of kit for being used to detect fentanyl medication related gene polymorphism, the fentanyl The pleomorphism site of medication related gene detection is CYP3A4*1G, and the kit includes：

Positive amplimer：5’-CCTTCCTACATAGAGTCA-3’；

PCR reaction solutions, the PCR reaction solutions contain reverse amplimer：

5’-ATTGATGCAGTTTTACCC-3’；

Sequencing primer：5’-CACGATTCTAGCTATGT-3’；

Wherein, 5 ' ends of the positive amplimer carry out biotin labeling.

In a kind of preferred embodiment of the kit provided by the invention, the kit also includes：CYP3A4*1G Positive reference substance 1, it is the wild homozygote plasmids of CYP3A4*1G for being inserted with nucleotide sequence shown in SEQID NO.4；

Nucleotide sequence shown in the SEQID NO.4 is as follows:

CTGCCAGTAGCAACCATTTGCTATGTTTCTTTCTTTTTCTTTTCAGAGCCTTCCTACATAGAGTCAGTGAAAGAATC AGTGATTATGCTTTTTATAAAAATTCTCCTGGGAAGTGGTGAGGAGGCATTTTTGCTAAGGTTTCACCTCCTCCCTC CTTCTCCATGTACCATCCACTCACCTTATTGGGTAAAACTGCATCAATTTCCTCCTGCAGTTTCTGCTGGACATCAG GGTGAGTGGCCAGTTCATACATAATGAAGGAGAGAACACTGCTCGTGGTTTCATAGCCAGCAAAAATAAAGATAATT GATTGGGCCACGAGCTCCAGATCGGACAGAGCTGAAAGGAGAGGAAAGACATTTTAGGTAAATCAGATCAATGTAGG GCATCACAGTTTAGATGAAGAGAAATCTAAGTGAAGCCCTCAAATCCCTAAGGGAAAAGAATAGAAAAGCAATTCAG AGGTACACTGGGGGTGGTTTCATTCT；

CYP3A4*1G positive reference substances 2, it is the wild homozygote plasmids of the CYP3A4*1G and is inserted with SEQID NO.5 The plasmid mixture of the CYP3A4*1G no mutant homozygote plasmid composition of shown nucleotide sequence；

Nucleotide sequence shown in the SEQID NO.5 is as follows:

CTGCCAGTAGCAACCATTTGCTATGTTTCTTTCTTTTTCTTTTCAGAGCCTTCCTACATAGAGTCAGTGAAAGAATC AGTGATTATGCTTTTTATAAAAATTCTCCTGGGAAGTGGTGAGGAGGCATTTTTGCTAAGGTTTCACCTCCTCCCTC CTTCTCCATGTATCATCCACTCACCTTATTGGGTAAAACTGCATCAATTTCCTCCTGCAGTTTCTGCTGGACATCAG GGTGAGTGGCCAGTTCATACATAATGAAGGAGAGAACACTGCTCGTGGTTTCATAGCCAGCAAAAATAAAGATAATT GATTGGGCCACGAGCTCCAGATCGGACAGAGCTGAAAGGAGAGGAAAGACATTTTAGGTAAATCAGATCAATGTAGG GCATCACAGTTTAGATGAAGAGAAATCTAAGTGAAGCCCTCAAATCCCTAAGGGAAAAGAATAGAAAAGCAATTCAG AGGTACACTGGGGGTGGTTTCATTCT；

CYP3A4*1G positive reference substances 3, it is the CYP3A4*1G mutation for being inserted with nucleotide sequence shown in SEQID NO.5 Homozygote plasmid；

Wherein, plasmid vector is pMD18-T plasmids；CYP3A4*1G dashes forward described in the CYP3A4*1G positive reference substances 2 Become the quantity ratio of homozygote plasmid and the wild homozygote plasmids of the CYP3A4*1G as 1:1.

In a kind of preferred embodiment of the kit provided by the invention, the kit also includes：Blank control Product, the blank control product are sterilizing purified water.

The PCR reaction solutions also contain other components, are respectively conventional 10 × PCR Buffer, dNTPS and H2O, respectively The component routinely volume ratio configuration (volume ratio of reverse amplimer in 10 × PCRBuffer, dNTPs, H2O and PCR reaction solution For 5:3.375:37.5:1).

Other reagents and the conventional reagent that solution is PCR and DNA pyrosequencings, are such as polymerize by DNA in the kit The enzymatic mixture that enzyme, adenosine triphosphate sulfurylase, luciferase and bisphosphatase form, by 5'- phosphosulfates and fluorescein The substrate mixture of composition, uracil dna glycosylase, Taq polymerase etc..

Present invention also offers primer pair as described above to prepare for pyrosequencing method detection CYP3A4*1G genes Application in the reagent of polymorphism.

Present invention also offers kit as described above to prepare for pyrosequencing method detection CYP3A4*1G genes Application in the reagent of polymorphism.

Compared to prior art, provided by the present invention for detect fentanyl medication related gene polymorphism primer pair and Kit has the advantages that：

First, redesigned by using pyrosequencing law technology and optimize high sensitivity and the good primer pair of specificity And its kit so that for the kit when detecting CYP3A4*1G gene pleiomorphisms, it is qualitative accurate to have, high sensitivity and The advantages of high specificity；In addition, also having, sample treatment is simple, sequencing steps are simple, sequencing speed is fast, half an hour completes one Secondary upper machine reaction, directly give the advantages of detection site frequency analysis and visual result；

2nd, redesigned by using pyrosequencing law technology and optimize high sensitivity and the good primer pair of specificity And its kit so that the kit can monitor reaction process, reaction in real time when detecting CYP3A4*1G gene pleiomorphisms Time is short, PCR primer simple process can go up pyrophosphoric acid sequencer, easy to operate and high flux sample detection, and than gold Standard method, i.e. Capillary Electrophoresis PCR sequencing method sensitivity is higher, is particularly suited for the requirement of clinical examination；

3rd, by being provided with blank control product and positive reference substance in the kit so that the kit is being examined When surveying CYP3A4*1G gene pleiomorphisms, the accuracy of testing result can be preferably ensured.

Brief description of the drawings

Fig. 1 is the pyrosequencing figure of clinical sample CYP3A4*1G wild types；

Fig. 2 is the pyrosequencing figure that clinical sample CYP3A4*1G is mutated heterozygous；

Fig. 3 is the pyrosequencing figure of clinical sample CYP3A4*1G mutant homozygous types；

Fig. 4 is the pyrosequencing figure of clinical blank control product；

Fig. 5 to Fig. 7 is the pyrosequencing figure of multigroup design primer；Wherein Fig. 5 and Fig. 6 sequencing result is inaccurate, Only Fig. 7 sequencing result is true and reliable.

Embodiment

In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with the accompanying drawings and embodiment, it is right The present invention is described in further detail.It should be appreciated that specific embodiment described herein is only to explain the present invention, and It is not used in the restriction present invention.

Embodiment 1：The preparation (30 tests/box) of kit

1st, the design and synthesis of primer and probe

For the pleomorphism site CYP3A4*1G of people's CYP3A4 genetic tests, special mutational site is selected, is used PyroMark Assay Design2.0 softwares, design primer；Wherein positive amplimer, reverse amplimer and sequencing primer PAGE purifying is first passed through, then is purified through HPLC, wherein the 5 ' of positive amplimer be biotin labeling.

The mutational site of table 1. and type

Mutational site	Sequence change
		CYP3A4*1G(rs2242480)	C>T

Extension increasing sequence such as table 2：

The specificity amplification primer of table 2. and primer sequence

2nd, PCR reaction solutions form

Table 3.PCR reaction solutions form

3rd, the composition of enzyme mixation

The composition of the enzyme mixation of table 4.

Material name	Volume (μ L)	30 tests/box (μ L)
			Taq enzyme	0.5	15
UNG enzymes	0.25	7.5
			Cumulative volume	0.75	22.5

4th, the composition of sequencing primer

The composition of the sequencing primer of table 5.

Material name	Volume (μ L)	30 tests/box (μ L)
			CYP3A4*1G sequencing primers	1.2	36

5th, the composition of positive reference substance

The composition of the positive reference substance of table 6.

Material name	Volume (μ L)	30 tests/box (μ L)
			Positive reference substance 1	1	30
Positive reference substance 2	1	30
			Positive reference substance 3	1	30

6th, the composition of blank control product

The composition of the blank control product of table 7.

Material name	Volume (μ L)	30 tests/box (μ L)
			Blank control	1	30

Embodiment 2：The use of kit

1st, sample detection

Dissolve primer dry powder (term of validity is 1 month after primer dissolving).System is prepared according to template number：PCR reaction solutions are taken, Solvent primer, uracil dna glycosylase, Taq archaeal dna polymerases are added, dispenses system, adds sample DNA, blank control Product or positive reference substance are template, form PCR reaction systems.Enter performing PCR amplification according to PCR response procedures.

Each main component of CYP3A4*1G systems is as follows：

Each main component of table 8.CYP3A4*1G systems

The system response procedures are as follows：

Table 9.PCR response procedures

After amplification is completed, Ago-Gel examines PCR results, to carry out next step program.

2nd, pyrosequencing

Sequencing procedures are carried out according to pyrosequencing standard practice instructions, are mainly comprised the following steps：The preparation and purification of sample, so Sample after purification is added into upper machine sequencing in the MIX containing annealing liquid and sequencing primer afterwards.In pyrosequencing instrument agent bin Add the corresponding dATP, dTTP of operation program, dCTP, dGTP, enzymatic mixture, substrate mixture.

3rd, result judges

In CYP3A4*1G DNA sequencing peak value figure, frequency≤10% of C frequency >=90%, T, that is, CYP3A4* is thought 1G wild types；40%≤C frequency≤60%, 40%≤T frequency≤60%, that is, think that CYP3A4*1G is mutated heterozygous；T Frequency >=90%, C frequency≤10%, that is, think CYP3A4*1G mutant homozygous types；If result is shown in red, quality Analysis result is Failed or N/A, that is, thinks that result is invalid or does not detect, such as blank control.

4th, quality control standard

(1) all kinds of control quality-control product judged results are as follows：

Positive reference substance 1：As a result blueness is shown, quality analysis results are Passed, the frequency of C frequency >=90%, T Rate≤10%；

Positive reference substance 2：As a result blueness is shown, quality analysis results are Passed, 40%≤C frequency≤60%, 40%≤T frequency≤60%；

Positive reference substance 3：As a result blueness is shown, quality analysis results are Passed, the frequency of T frequency >=90%, C Rate≤10%；

Blank control：As a result shown in red, quality analysis results are Failed or N/A.

Conditions above must all meet that otherwise this experimental result is invalid in once experiment.

5th, result is reported：

As a result as shown in Figure 1, Figure 2 and Figure 3, the criterion of sample result is as follows：

Table 10. reports sample detection result

	Pattern detection result	Report result
			1	CYP3A4*1G (C >=90%, T≤10%)	Wild type
2	CYP3A4*1G (C≤10%, T >=90%)	Mutant homozygous type
			3	CYP3A4*1G (40%≤C≤60%, 40%≤T≤60%)	It is mutated heterozygous

The wild type of CYP3A4*1G in clinical sample testing result is shown in Fig. 1, and clinical sample inspection is shown in Fig. 2 The mutation heterozygous of CYP3A4*1G in result is surveyed, the mutation that CYP3A4*1G in clinical sample testing result is shown in Fig. 3 is pure Mould assembly；The pyrosequencing figure of the blank control product in clinical detection result is shown in Fig. 4, and design is shown in Fig. 5 to Fig. 7 Multigroup primer pyrosequencing design sketch, wherein Fig. 7 primer be optimal be in our products select primer.

Have provided by the present invention for the primer pair and kit for detecting fentanyl medication related gene polymorphism following Beneficial effect：

Embodiments of the invention are the foregoing is only, are not intended to limit the scope of the invention, it is every to utilize this hair The equivalent flow conversion that bright description is made, or other related technical fields are directly or indirectly used in, similarly wrap Include in the scope of patent protection of the present invention.

Sequence table

<110>The Ji bio tech ltd of Changsha three

<120>For detecting the primer pair and kit of fentanyl medication related gene polymorphism

<130> 2017

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

ccttcctaca tagagtca 18

<210> 2

<211> 18

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

attgatgcag ttttaccc 18

<210> 3

<211> 17

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

cacgattcta gctatgt 17

<210> 4

<211> 488

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

ctgccagtag caaccatttg ctatgtttct ttctttttct tttcagagcc ttcctacata 60

gagtcagtga aagaatcagt gattatgctt tttataaaaa ttctcctggg aagtggtgag 120

gaggcatttt tgctaaggtt tcacctcctc cctccttctc catgtaccat ccactcacct 180

tattgggtaa aactgcatca atttcctcct gcagtttctg ctggacatca gggtgagtgg 240

ccagttcata cataatgaag gagagaacac tgctcgtggt ttcatagcca gcaaaaataa 300

agataattga ttgggccacg agctccagat cggacagagc tgaaaggaga ggaaagacat 360

tttaggtaaa tcagatcaat gtagggcatc acagtttaga tgaagagaaa tctaagtgaa 420

gccctcaaat ccctaaggga aaagaataga aaagcaattc agaggtacac tgggggtggt 480

ttcattct 488

<210> 5

<211> 488

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

ctgccagtag caaccatttg ctatgtttct ttctttttct tttcagagcc ttcctacata 60

gagtcagtga aagaatcagt gattatgctt tttataaaaa ttctcctggg aagtggtgag 120

gaggcatttt tgctaaggtt tcacctcctc cctccttctc catgtatcat ccactcacct 180

tattgggtaa aactgcatca atttcctcct gcagtttctg ctggacatca gggtgagtgg 240

ccagttcata cataatgaag gagagaacac tgctcgtggt ttcatagcca gcaaaaataa 300

agataattga ttgggccacg agctccagat cggacagagc tgaaaggaga ggaaagacat 360

tttaggtaaa tcagatcaat gtagggcatc acagtttaga tgaagagaaa tctaagtgaa 420

gccctcaaat ccctaaggga aaagaataga aaagcaattc agaggtacac tgggggtggt 480

ttcattct 488

Claims

A kind of 1. primer pair for being used to detect fentanyl medication related gene polymorphism, it is characterised in that the fentanyl medication The pleomorphism site of related gene detection is CYP3A4*1G, and the primer pair is Pyrosequencing primer pair, and it includes：

Positive amplimer：5’-CCTTCCTACATAGAGTCA-3’；

Reverse amplimer：5’-ATTGATGCAGTTTTACCC-3’；

Sequencing primer：5’-CACGATTCTAGCTATGT-3’；

Wherein, 5 ' ends of the positive amplimer carry out biotin labeling.
A kind of 2. kit for being used to detect fentanyl medication related gene polymorphism, it is characterised in that the fentanyl medication The pleomorphism site of related gene detection is CYP3A4*1G, and the kit includes：

Positive amplimer：5’-CCTTCCTACATAGAGTCA-3’；

PCR reaction solutions, the PCR reaction solutions contain reverse amplimer：

5’-ATTGATGCAGTTTTACCC-3’；

Sequencing primer：5’-CACGATTCTAGCTATGT-3’；

Wherein, 5 ' ends of the positive amplimer carry out biotin labeling.
3. kit according to claim 2, it is characterised in that the kit also includes：CYP3A4*1G positive controls Product 1, it is the wild homozygote plasmids of CYP3A4*1G for being inserted with nucleotide sequence shown in SEQIDNO.4；

CYP3A4*1G positive reference substances 2, it is the wild homozygote plasmids of the CYP3A4*1G and is inserted with core shown in SEQIDNO.5 The plasmid mixture of the CYP3A4*1G no mutant homozygote plasmid composition of nucleotide sequence；

CYP3A4*1G positive reference substances 3, it is the CYP3A4*1G no mutant homozygote for being inserted with nucleotide sequence shown in SEQIDNO.5 Plasmid；

Wherein, plasmid vector is pMD18-T plasmids.
4. kit according to claim 3, it is characterised in that described in the CYP3A4*1G positive reference substances 2 The quantity ratio of CYP3A4*1G no mutant homozygote plasmid and the wild homozygote plasmids of the CYP3A4*1G is 1:1.
5. kit according to claim 3, it is characterised in that the kit also includes blank control product, the sky White reference substance is sterilizing purified water.
6. the primer pair described in claim 1 is preparing the examination for pyrosequencing method detection CYP3A4*1G gene pleiomorphisms Application in agent.
7. the kit described in claim 2 is preparing the examination for pyrosequencing method detection CYP3A4*1G gene pleiomorphisms Application in agent.