CN109439738A - ABCB1, CYP3A5 genetic test primer sets, kit and detection method - Google Patents
ABCB1, CYP3A5 genetic test primer sets, kit and detection method Download PDFInfo
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Abstract
The present invention relates to genetic engineering and molecular biology fields, a kind of ABCB1, CYP3A5 genetic test primer sets are provided, for carrying out specific amplification to the nucleic acid fragment containing site to be measured, the site to be measured includes the site rs776746 on the site rs1045642 and CYP3A5 gene on ABCB1 gene, and the primer sets include rs1045642 primer sets, rs776746 primer.The present invention also provides a kind of gene detecting kit and detection methods.Genetic test primer sets of the invention can carry out specific amplification to the nucleic acid fragment containing above-mentioned site to be measured, so that low to ABCB1, CYP3A5 detection in Gene Mutation high sensitivity, false positive rate；The present invention is detected using the genotype that second generation high throughput gene sequencing technology treats location point, so that detection cycle is short, flux is high.
Description
Technical field
The present invention relates to genetic engineering and molecular biology fields, more specifically to a kind of ABCB1, CYP3A5 base
Because of detection primer group, kit and detection method.
Background technique
Taxol is a kind of tricyclic diterpene class compound, have unique antitumor mechanism, extensive anticancer spectrum and really
The clinical efficacy cut has obtained the attention of people.Afterwards it has been investigated that taxol is a kind of specific stabilizer of micro-pipe, can promote
Assembly and holding microtubule stabilization into micro-pipe.When cell and after paclitaxel exposure, a large amount of micro-pipe can be accumulated in the cell, and this
The accumulation of the pipe various normal functions that will interfere cell slightly, especially make cell division stop at m period, block
The proper splitting of cell.Based on above-mentioned mechanism, taxol is to study the only one kind reported at present to can control growth of cancer cells
Botanical medicine.Scholars speculate that the generation of the adverse reaction of taxol is primarily due to taxol and acts on intracellular canaliculus system
System, and the system is dispersed throughout cells all in vivo, therefore the effect of taxol fails specifically target cancer cell, but generally make
For internal various cells.
Research shows that people ABCB1 gene, which will hydrolyze offer power by ATP, to be drained into extracellularly outside substrate, it can be by tumour medicine
Inverse concentration gradient is pumped out of endochylema to outside endochylema, to reduce the Apoptosis of induced by chemotherapeutic agents and cause tumour cell resistance to
Medicine.There is research to think, people's CYP3A5 gene mutation is the major regulatory mode for causing its expression to change, while also can
Different degrees of change occurs for the activity for influencing its metabolic enzyme, the ability and rate for being eventually exhibited as metabolism substrate, is to cause to face
There is the most important reason of difference between personal, race in bed medication.
It has been found that ABCB1 gene pleiomorphism such as ABCB1 C3435T(rs1045642) mutation lead to extracellular pump capacity
It reduces, shows in tumour cell, then make that curative effect is better.CYP3A5*3(rs776746) genotype is the 3 of CYP3A5 gene
What 6986A > G mutation of introne generated, the expression of CYP3A5 and its activity of enzyme are closely related.CYP3A5*3 genotype
Cause the montage of mRNA to change, terminate translation ahead of time, protein truncates, and causes CYP3A5 enzymatic activity to reduce and even disappears
It loses, is the most important gene pleiomorphism of CYP3A5 gene in crowd.
Detected by two kinds of genes to patient, obtain the genotype of specific site, using genetic test result as
The method of the information of intermediate result is not belonging to the diagnostic method of disease；The selection of drug is determined with the testing result of genotype,
It can realize personalized medicine, avoid to immunosuppressor adverse reaction.
At present to the detection of gene mutation and gene pleiomorphism, it is common have direct sequencing, gene chip hybridization method,
Taqman fluorescence probe method, allele specific amplification method (Amplification Refractory Mutation
System Real Time PCR, ARMS-RT PCR), allele specific amplification method (Amplification
Refractory Mutation System Real Time PCR, ARMS-RT PCR) etc..Above method sensitivity is low, false sun
Property rate is high, takes a long time, flux is small.
Therefore, it is necessary to the kits and detection method of a kind of new detection ABCB1, CYP3A5 gene mutation, so that right
The high sensitivity of ABCB1, CYP3A5 gene mutation, false positive rate is low, and detection cycle is short.
Summary of the invention
The purpose of the present invention is to provide a kind of ABCB1, CYP3A5 genetic test primer sets, kit and detection method,
Aim to solve the problem that ABCB1, CYP3A5 genetic test sensitivity are low in the prior art, false positive rate is high, the period is long, the small technology of flux
Problem.
The present invention provides a kind of ABCB1, CYP3A5 genetic test primer sets, for the nucleic acid fragment containing site to be measured
Specific amplification is carried out, the site to be measured includes on the site rs1045642 and CYP3A5 gene on ABCB1 gene
The site rs776746, the primer sets include rs1045642 primer sets, rs776746 primer sets；
The rs1045642 primer sets include rs1045642 outer primer group and rs1045642 inner primer group, the rs1045642
Outer primer group includes the outer downstream primer SEQ ID NO:3 of the outer upstream primer SEQ ID NO:1 and rs1045642 of rs1045642；
The rs1045642 inner primer group includes that downstream is drawn in upstream primer SEQ ID NO:2 and rs1045642 in rs1045642
Object SEQ ID NO:4；
The rs776746 primer sets include rs776746 outer primer group and rs776746 inner primer group, are drawn outside the rs776746
Object group includes the outer downstream primer SEQ ID NO:7 of the outer upstream primer SEQ ID NO:5 and rs776746 of rs776746；It is described
Rs776746 inner primer group includes downstream primer SEQ ID in upstream primer SEQ ID NO:6 and rs776746 in rs776746
NO:8。
A kind of ABCB1, CYP3A5 gene detecting kit, the kit include ABCB1, CYP3A5 gene inspection
Survey primer sets.
Preferably, contain sequence label on the connector, the connector for directly with the site containing rs1045642 and
The amplified production in the site rs776746 connects.
Preferably, the kit further includes the oligonucleotide probe of fluorescent marker and the oligonucleotides of unstressed configuration label
Acid probe.
Preferably, the kit further includes sequencing primer, and the sequencing primer includes the text to the site containing rs1045642
Rs1045642 sequencing primer SEQ ID NO:9 that library molecule is sequenced, the library molecule in the site containing rs776746 is surveyed
The rs776746 sequencing primer SEQ ID NO:10 of sequence.
A kind of ABCB1, CYP3A5 gene tester, comprising the following steps:
A, using primer sets, the sample in the site containing rs1045642 and the site rs776746 is expanded respectively, production must be expanded
Object；The primer sets include rs1045642 primer sets, rs776746 primer；
The rs1045642 primer sets include rs1045642 outer primer group and rs1045642 inner primer group, the rs1045642
Outer primer group includes the outer downstream primer SEQ ID NO:3 of the outer upstream primer SEQ ID NO:1 and rs1045642 of rs1045642；
The rs1045642 inner primer group includes that downstream is drawn in upstream primer SEQ ID NO:2 and rs1045642 in rs1045642
Object SEQ ID NO:4；
The rs776746 primer sets include rs776746 outer primer group and rs776746 inner primer group, are drawn outside the rs776746
Object group includes the outer downstream primer SEQ ID NO:7 of the outer upstream primer SEQ ID NO:5 and rs776746 of rs776746；It is described
Rs776746 inner primer group includes downstream primer SEQ ID in upstream primer SEQ ID NO:6 and rs776746 in rs776746
NO:8；
B, the jointing on the amplified production, obtains library molecule；
C, the library molecule is sequenced using second generation high throughput gene sequencing technology, determines the gene in site to be measured
Type.
Preferably, the step A the following steps are included:
A1, ABCB1 gene is expanded using rs1045642 outer primer group, obtains the first rs1045642 amplified production；Using
Rs776746 outer primer group expands CYP3A5 gene, obtains the first rs776746 amplified production；
The rs1045642 outer primer group includes under the outer upstream primer SEQ ID NO:1 and rs1045642 of rs1045642 is outer
Swim primer SEQ ID NO:3；The rs776746 outer primer group include the outer upstream primer SEQ ID NO:5 of rs776746 and
The outer downstream primer SEQ ID NO:7 of rs776746；
A2, the first rs1045642 amplified production is expanded using rs1045642 inner primer group, obtains the 2nd rs1045642 expansion
Increase production object；Rs776746 inner primer group is used, to expand, to obtain the 2nd rs776746 expansion to the first rs776746 amplified production
Increase production object；
The rs1045642 inner primer group include in rs1045642 in upstream primer SEQ ID NO:2 and rs1045642 under
Swim primer SEQ ID NO:4；The rs776746 inner primer group include in rs776746 upstream primer SEQ ID NO:6 and
Downstream primer SEQ ID NO:8 in rs776746.
It preferably, further include to the 2nd rs1045642 amplified production, the 2nd rs776746 amplified production in the step B
On uracil carry out specificity cutting the step of, the 2nd rs1045642 amplified production, the 2nd rs776746 amplified production
The first cohesive end is respectively formed through cutting.
Preferably, the connector includes the second cohesive end, for connecting with the first cohesive end.
Preferably, step C is attached sequencing using sequencing primer, and the sequencing primer includes to containing rs1045642
The rs1045642 sequencing primer SEQ ID NO:9 that the library molecule of point is sequenced and the library to the site containing rs776746
The rs776746 sequencing primer SEQ ID NO:10 that molecule is sequenced.
ABCB1, CYP3A5 genetic test primer sets provided by the invention can carry out the segment containing site to be measured special
Property amplification, reduce expand multiple target sites a possibility that, improve treat location point amplification specificity and sensitivity, reduce
False positive rate；Meanwhile using second generation high throughput gene sequencing technology, so that the detection cycle for treating location point is short, flux
It is high.
Detailed description of the invention
Fig. 1 is the PAGE gel electrophoresis figure of secondary amplified production in fourth embodiment.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.
The present invention proposes first embodiment, a kind of ABCB1, CYP3A5 genetic test primer sets, for containing site to be measured
Nucleic acid fragment carry out specific amplification, the site to be measured includes the site rs1045642 and CYP3A5 base on ABCB1 gene
Because of the upper site rs776746, the primer sets include rs1045642 primer sets, rs776746 primer；
The rs1045642 primer sets include rs1045642 outer primer group and rs1045642 inner primer group, the rs1045642
Outer primer group includes the outer downstream primer SEQ ID NO:3 of the outer upstream primer SEQ ID NO:1 and rs1045642 of rs1045642；
The rs1045642 inner primer group includes that downstream is drawn in upstream primer SEQ ID NO:2 and rs1045642 in rs1045642
Object SEQ ID NO:4；
The rs776746 primer sets include rs776746 outer primer group and rs776746 inner primer group, are drawn outside the rs776746
Object group includes the outer downstream primer SEQ ID NO:7 of the outer upstream primer SEQ ID NO:5 and rs776746 of rs776746；It is described
Rs776746 inner primer group includes downstream primer SEQ ID in upstream primer SEQ ID NO:6 and rs776746 in rs776746
NO:8。
ABCB1, CYP3A5 genetic test primer sets that this programme provides, being capable of the specific amplification site containing rs1045642
And/or the nucleic acid fragment of rs776746, a possibility that expanding non-target fragment is reduced, so as to the site rs1045642
And/or the high sensitivity of rs776746 site primer, false positive rate are low.Two groups of ABCB1, CYP3A5 genes that this programme provides
Detection primer group treats location point and carries out two-wheeled nested amplification respectively, very due to sequence all complementary with two groups of primer sets simultaneously
It is few, to increase the susceptibility treated and survey site primer a possibility that reducing non-specific amplification.In the present solution, SEQ
It include cggy sequence on ID NO:2, SEQ ID NO:6, symbol y is uracil or the like in the present embodiment, using we
ABCB1, CYP3A5 gene primer group of case expand the nucleic acid fragment containing site to be measured, include on obtained amplified production
Cggy sequence cuts the phosphodiester bond between amplified production g and y using the enzyme of specificity cutting uracil, 5 '
End will form cohesive end, and cohesive end joint efficiency during subsequent jointing is higher, be conducive to subsequent
Jointing in reaction process.
The present invention proposes second embodiment, and a kind of ABCB1, CYP3A5 gene detecting kit is implemented containing above-mentioned first
ABCB1, CYP3A5 genetic test primer sets of example.
Preferably, the kit further includes connector, contains sequence label on the connector, the connector for directly with
The site containing rs1045642 is connected with the amplified production in the site rs776746.The sequence label be nnnn, wherein n be A, G, C or
T.Connector of the invention can take various forms, including but not limited to flat end fitting, protruding terminus connector.
Preferably, the joint sequence also includes the sequence of sequencing primer sequence complementation and the sequence of amplimer.
Preferably, the kit further includes the enzyme of specificity cutting uracil, the enzyme of the specificity cutting uracil
For USER enzyme, RNase H or UDG enzyme.
Preferably, the connector is protruding terminus connector, due to SEQ ID NO:2, the SEQ ID NO:6 of the second wheel amplification
Include cggy sequence in sequence, therefore also include cggy sequence on obtained amplified production, uracil is cut using specificity
Enzyme amplified production is cut, formed cohesive end, cohesive end joint efficiency during subsequent jointing
It is higher.Preferably, contain biotin labeling on the connector, for making it be fixed on the magnetic bead containing Avidin.Of the invention
Connector can take various forms, including but not limited to flat end fitting, can also be protruding terminus connector, Y connection or
Connector containing loop-stem structure.
Preferably, contain biotin labeling on 5 ' ends of the joint sequence, and biotin labeling is fixed in advance
On magnetic bead containing Streptavidin or Avidin.Using the biotin labeling, it can connect after reaction, very easily will
Nucleic acid fragment containing site to be measured and connector is separated.
Preferably, the kit further includes the oligonucleotide probe of fluorescent marker and the oligonucleotides of unstressed configuration label
Acid probe.
It should be noted that the oligonucleotide probe of unstressed configuration label with connect fluorescence used in sequencing technologies
Probe is compared, and sequence is identical, and difference is only that unstressed configuration marks.The kit of this programme, is added nothing in sequencing procedure
The oligonucleotide probe of fluorescent marker can effectively reduce the error signal occurred in connection sequencing, improve and treat location point
The accuracy of detection.
Preferably, the kit further includes sequencing primer, and the sequencing primer includes the text to the site containing rs1045642
Rs1045642 sequencing primer SEQ ID NO:9 that library molecule is sequenced, the library molecule in the site containing rs776746 is surveyed
The rs776746 sequencing primer SEQ ID NO:10 of sequence.The sequencing primer of this programme design, is connected to be measured in sequencing procedure
Location proximate region, so that in sequencing procedure, it is only necessary to which the sequencing of fewer number can determine the genotype in site to be measured.
Preferably, the phosphorylated modification in 5 ' ends of the sequencing primer.The sequencing primer of this programme is suitable for using company
It connects PCR sequencing PCR and treats location point and detected, this programme makes that the 5 ' ends that probe is attached to sequencing primer are sequenced.
Preferably, the kit further includes ligase.Under the action of ligase, between the amplified production and connector
Connection that can be stable.
Preferably, the kit further includes connection buffer.
It should be noted that kit provided by the invention, is carried out separately the amplification step in different sites to be measured,
Multiple amplified productions containing different sites to be measured may be mixed together while be sequenced.When using kit of the invention
When being detected simultaneously in same system to the site rs1045642 of same sample, the site rs776746, and containing respectively to location
The connector of the amplified production connection of point, sequence label thereon may be the same or different.When the sequence label on connector not
Meanwhile site to be measured can be distinguished by the detection of butt joint sequence label, it, can be with when the sequence label on connector is identical
Site to be measured is distinguished by the difference of used sequencing primer.
When using kit of the invention to being detected in same system to the same site to be measured of different samples,
Since the sequencing primer used in sequencing procedure is identical, it is therefore desirable to be connected respectively using the connector containing different sequence labels
Different samples is connect, by the way that sequence label is sequenced, can effectively be distinguished not without carrying out repeatedly sequencing to sample
With the sequencing result of sample.
The present invention proposes 3rd embodiment, a kind of ABCB1, CYP3A5 gene tester, comprising the following steps:
A, using primer sets, the sample in the site containing rs1045642 and the site rs776746 is expanded respectively, production must be expanded
Object；The primer sets include rs1045642 primer sets, rs776746 primer；
The rs1045642 primer sets include rs1045642 outer primer group and rs1045642 inner primer group, the rs1045642
Outer primer group includes the outer downstream primer SEQ ID NO:3 of the outer upstream primer SEQ ID NO:1 and rs1045642 of rs1045642；
The rs1045642 inner primer group includes that downstream is drawn in upstream primer SEQ ID NO:2 and rs1045642 in rs1045642
Object SEQ ID NO:4；
The rs776746 primer sets include rs776746 outer primer group and rs776746 inner primer group, are drawn outside the rs776746
Object group includes the outer downstream primer SEQ ID NO:7 of the outer upstream primer SEQ ID NO:5 and rs776746 of rs776746；It is described
Rs776746 inner primer group includes downstream primer SEQ ID in upstream primer SEQ ID NO:6 and rs776746 in rs776746
NO:8；
B, the jointing on the amplified production, obtains library molecule；
C, the library molecule is sequenced using second generation high throughput gene sequencing technology, determines the gene in site to be measured
Type.
ABCB1, CYP3A5 gene tester provided by the invention carries out drawing for specific amplification using location point is treated
Object group expands the nucleic acid fragment containing site to be measured, improves the sensitivity treated and survey site primer, reduces the false positive rate of detection.
Preferably, the step A the following steps are included:
A1, ABCB1 gene is expanded using rs1045642 outer primer group, obtains the first rs1045642 amplified production；Using
Rs776746 outer primer group expands CYP3A5 gene, obtains the first rs776746 amplified production；
The rs1045642 outer primer group includes under the outer upstream primer SEQ ID NO:1 and rs1045642 of rs1045642 is outer
Swim primer SEQ ID NO:3；The rs776746 outer primer group include the outer upstream primer SEQ ID NO:5 of rs776746 and
The outer downstream primer SEQ ID NO:7 of rs776746；
A2, the first rs1045642 amplified production is expanded using rs1045642 inner primer group, obtains the 2nd rs1045642 expansion
Increase production object；Rs776746 inner primer group is used, to expand, to obtain the 2nd rs776746 expansion to the first rs776746 amplified production
Increase production object；
The rs1045642 inner primer group include in rs1045642 in upstream primer SEQ ID NO:2 and rs1045642 under
Swim primer SEQ ID NO:4；The rs776746 inner primer group include in rs776746 upstream primer SEQ ID NO:6 and
Downstream primer SEQ ID NO:8 in rs776746.
This programme carries out two-wheeled specificity by two pairs of PCR amplification primer sets of design, to the nucleic acid fragment containing site to be measured
Amplification, can effectively ensure in the second amplified production not because primer pairing specificity it is not strong caused by non-specific amplification dirt
Dye reduces a possibility that expanding multiple target sites, increases the specificity and sensitivity of amplification.
It should be noted that first round nested amplification can be separated and be carried out when expanding to different sites to be measured,
It can be carried out in same system；Second wheel nested amplification separately carries out.This programme can be distinguished by the second wheel nested primer
Sample.
When expanding to different sites to be measured, the first round and the second wheel nested amplification are both needed to separate progress.
It preferably, further include to the 2nd rs1045642 amplified production, the 2nd rs776746 amplified production in the step B
On uracil carry out specificity cutting the step of, the 2nd rs1045642 amplified production, the 2nd rs776746 amplified production
The first cohesive end is respectively formed through cutting.
Due to including cggy sequence on SEQ ID NO:2, SEQ ID NO:6, the 2nd rs1045642 is obtained by amplification
After amplified production and/or the 2nd rs776746 amplified production, the 2nd rs1045642 amplification is cut in the digestion of specificity cutting uracil
Phosphodiester bond between product and/or the 2nd rs776746 amplified production g and y forms phosphate group is contained in 5 ' ends the
One cohesive end, the cohesive end are conducive to jointing during subsequent reactions.
Preferably, the connector contains the second cohesive end, the first viscosity of second cohesive end and amplified production
The pairing of end complete complementary.Connector is connected by the first cohesive end complementary pairing of the second cohesive end and amplified production, is mentioned
High joint efficiency.
Preferably, contain biotin labeling on the connector end opposite with the second cohesive end, and fixed in advance
On the magnetic bead containing Streptavidin or Avidin.Using the biotin labeling, very easily can will after step B
Nucleic acid fragment containing site to be measured and connector is separated.
Preferably, the second generation high throughput gene sequencing technology includes but are not limited to connection PCR sequencing PCR or synthesis order-checking
Method.
Preferably, the step C the following steps are included:
C1, the oligonucleotide probe marked using unstressed configuration, to rs1045642 library molecule and/or rs776746 library molecule
Carry out primary connection sequencing；
C2, sequencing is attached to rs1045642 library molecule and/or rs776746 library molecule using fluorescence probe, determined
The genotype in the site rs1045642 and/or rs776746.
It should be noted that the oligonucleotide probe of unstressed configuration label with connect fluorescence used in sequencing technologies
Probe is compared, and sequence is identical, and difference is only that unstressed configuration marks.The detection method of this programme, is added in sequencing procedure
The oligonucleotide probe of unstressed configuration label can effectively reduce the error signal occurred in connection sequencing, improve and treat location
The accuracy of point detection.
The following steps are included: sequencing primer is anchored, flushing (removes the extra sequencing not being anchored for connection sequencing each time
Primer), linking probe rinses and (removes excess probes, ligase etc.), adopts figure and (obtains position corresponding to fluorescent marker on probe
Sequence information), denaturation elution connection product (to connect the anchoring of sequencing primer in sequencing reaction next time).In step C1
Connection sequencing reaction, using unstressed configuration mark oligonucleotide probe, can without adopting figure step, with reduce experiment step
Suddenly, conventional efficient is improved.
Preferably, the rs1045642 sequencing primer library molecule in the site containing rs1045642 being sequenced in step C
For SEQ ID NO:9；The rs776746 sequencing primer that the library molecule in the site containing rs776746 is sequenced is SEQ ID
NO:10.The sequencing primer of this programme design, is connected to location proximate region to be measured, so that in sequencing procedure in sequencing procedure
In, it is only necessary to the sequencing of fewer number can determine the genotype in site to be measured.
Preferably, the phosphorylated modification in 5 ' ends of the sequencing primer.The sequencing primer of this programme is particularly suitable for adopting
With connection PCR sequencing PCR, in sequencing procedure, sequencing probe is connected to the 5 ' end of sequencing primer.
Compared with the existing technology, ABCB1, CYP3A5 gene tester provided by the invention, to different sites to be measured
Amplification step is carried out separately, and multiple amplified productions containing different sites to be measured may be mixed together while be surveyed
Sequence.When using kit of the invention in same system to the site rs1045642 of same sample, the site rs776746 simultaneously
When being detected, the connector connecting with the amplified production containing each site to be measured, sequence label thereon can be identical, can also not
Together.When the sequence label difference on connector, site to be measured can be distinguished by the detection of butt joint sequence label, works as connector
On sequence label it is identical when, site to be measured can be distinguished by the difference of used sequencing primer.
When using ABCB1, CYP3A5 gene tester of the invention in same system to the same of different samples
When site to be measured is detected, since the sequencing primer used in sequencing procedure is identical, it is therefore desirable to using containing difference
The connector of sequence label is separately connected different samples, more without carrying out to sample by the way that sequence label is sequenced
Secondary sequencing can effectively distinguish the sequencing result of different samples.
The present invention provides fourth embodiment, a kind of ABCB1, CYP3A5 gene tester, with the poba gene of two people
Group DNA is template, is detected on the site rs1045642 and CYP3A5 genetic fragment in each template ABCB1 genetic fragment respectively
The site rs776746.Reaction system is established according to the following steps:
A1, it is directed to above-mentioned two sample, respectively prepares totally 4 reactions of two amplification ABCB1 genetic fragments and CYP3A5 genetic fragment
System: the poba gene group DNA1 μ L of 50ng；The production of 2 × Hot Taq Mix(Shenzhen HYK Gene Technology Co., Ltd.) 10 μ
L；0.4 μ L of the outer upstream primer of 10 μM of RS1045642/RS776746；The outer downstream primer of 10 μM of RS1045642/RS776746
0.4μL；Add deionized water to 20 μ L；It mixes and is centrifuged.Then centrifuge tube is placed in PCR instrument, response procedures: 95 DEG C of items is set
Continue 10 minutes under part；It is for 20 seconds under the conditions of 95 DEG C, it is for 20 seconds under the conditions of 58 DEG C, it is for 20 seconds under the conditions of 72 DEG C, altogether
20 circulations；Continue 5 minutes under the conditions of 72 DEG C；After the completion of amplified reaction, the first amplified production is obtained.Wherein, RS1045642/
The outer upstream primer of RS776746 includes that the outer upstream primer sets of the outer upstream primer SEQ ID NO:1 and RS776746 of RS1045642 are
SEQ ID NO:5；The outer downstream primer of RS1045642/RS776746 include the outer downstream primer SEQ ID NO:3 of RS1045642 and
The outer downstream primer SEQ ID NO:7 of RS776746.Downstream in upstream primer SEQ ID NO:2 and rs1045642 in rs1045642
Primer SEQ ID NO:4 is used for amplification system 1 and system 3；Upstream primer SEQ ID NO:6 and rs776746 in rs776746
Interior downstream primer SEQ ID NO:8 is used for amplification system 2 and system 4.
A2, using the first amplified production of above-mentioned two sample as template, each one amplification site containing rs1045642 of configuration with
The reaction system of the segment in the site rs776746, the system in one site rs1045642 of amplified sample are amplification system 1, expand sample
The system in this site rs776746 is amplification system 2, and the system in two site rs1045642 of amplified sample is amplification system 3, is expanded
The system for increasing two site rs776746 of sample is amplification system 4, and reaction system component is as follows: the first amplification of 100 times of dilution produces
1 μ L of object；10 μM of RS1045642/RS776746 inner primer group totally 0.8 μ L；2 × Hot Taq Mix, 10 μ L(Shenzhen Hua Yinkang
Gene Tech. Company Limited's production)；Add deionized water to 20 μ L；It mixes and is centrifuged.Centrifuge tube is placed in PCR instrument, setting is anti-
It answers program: continuing 10 minutes under the conditions of 95 DEG C；It is for 20 seconds under the conditions of 95 DEG C, it is for 20 seconds under the conditions of 60 DEG C, under the conditions of 72 DEG C
For 20 seconds, 20 recycle altogether；Continue 5 minutes under the conditions of 72 DEG C；PCR after the reaction was completed, obtains the second amplified production.Wherein,
In RS1045642/RS776746 upstream primer include in RS1045642 in upstream primer SEQ ID NO:2 and RS776746 on
Trip primer sets are SEQ ID NO:6；Downstream primer includes downstream primer SEQ in RS1045642 in RS1045642/RS776746
Downstream primer SEQ ID NO:8 in ID NO:4 and RS776746.In rs1045642 upstream primer SEQ ID NO:2 and
Downstream primer SEQ ID NO:4 is used for amplification system 1 and system 3 in rs1045642；Upstream primer SEQ ID in rs776746
Downstream primer SEQ ID NO:8 is used for amplification system 2 and system 4 in NO:6 and rs776746.
After the completion of second expands, is detected through Page gel electrophoresis, contain target in the second amplified production of two samples
Molecule, and electrophoretogram shows no miscellaneous band, band is single；Illustrate that required amplified production has successfully been obtained in second of amplification.Such as Fig. 1
It is shown, the detection of PAGE gel electrophoresis figure is carried out to the second amplification group product, swimming lane 1,4 is respectively 100bp, 20bp in figure
Marker, swimming lane 2,5 are the amplified production in the site containing rs1045642, and swimming lane 3,6 is the amplified production in the site containing rs776746,
There is purpose band near 62bp, 98bp respectively in it, illustrates that amplimer group of the invention can be very good amplification containing to be measured
The nucleic acid fragment in site.
B1, for the second amplified production obtained in amplification system 1-4, prepare cutting-coupled reaction system 1-4 respectively:
The 10 μ L of USER enzyme of second amplified production, 20 μ L, T4 ligase buffer solution, 8 μ L, T4 DNA ligase, 2 μ L, 1U/ μ L, 10 μM
1 μ L of connector, 25 DEG C are reacted 2 hours, obtain connection product, i.e., to sequencing library molecule；
B2, by library molecule respectively with Avidin modification magnetic bead in conjunction with so that library molecule is fixed on magnetic bead surfaces.
The load sample piece (slide) that above-mentioned product point sample to isothiocyano is modified is completed to be fixed on magnetic bead in 45 DEG C of fixed 1h
Addressable to sequencing library molecule is fixed；
Wherein buffer is Tris containing 400mM, 100mM MgCl2,100mM DTT, 5mM ATP, the solution of pH value 7.8.It cuts
Cut-coupled reaction system 1 used in connector sequence label be AGTC；Connector used in cutting-coupled reaction system 2
Sequence label is ACAC；The sequence label of connector used in cutting-coupled reaction system 3 is CAGT；Cutting-connection reactant
The sequence label for being connector used in 4 is GCCG.
C, the high-throughput gene sequencer of Shenzhen HYK Gene Technology Co., Ltd. is used in this embodiment
Pstar II A sequenator, and be sequenced using connection PCR sequencing PCR.In sequencing procedure, for the site rs1045642, used
Sequencing primer be SEQ ID NO:9；For the site rs776746, used sequencing primer is SEQ ID NO:10.Sequencing
The result shows that the genotype in the site rs1045642 of sample one is wild-type homozygote GG, the site rs776746 of sample one
Genotype is wild-type homozygote CC, and the genotype in the site rs1045642 of sample two is saltant type heterozygote AG；Sample two
Rs776746 loci gene type is mutant homozygote CT.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Shenzhen HYK Gene Technology Co., Ltd.
<120>ABCB1, CYP3A5 genetic test primer sets, kit and detection method
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tcccaggctg tttatttgaa g 21
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cgggatgaag gcatgtatgt tggc 24
<210> 3
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tgaatgttca gtggctccg 19
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gggtggtgtc acaggaagag a 21
<210> 5
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
agcaagagtc tcacacagga gc 22
<210> 6
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
cggttcatat gatgaagggt aatgtgg 27
<210> 7
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ggagagtggc ataggagata cc 22
<210> 8
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tgctctactg tcatttctaa ccata 25
<210> 9
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gtgagggcag caaagga 17
<210> 10
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
tatctcttcc ctgtttg 17
Claims (10)
1. a kind of ABCB1, CYP3A5 genetic test primer sets, for carrying out specific expansion to the nucleic acid fragment containing site to be measured
Increase, which is characterized in that the site to be measured includes on the site rs1045642 and CYP3A5 gene on ABCB1 gene
The site rs776746, the primer sets include rs1045642 primer sets, rs776746 primer；
The rs1045642 primer sets include rs1045642 outer primer group and rs1045642 inner primer group, the rs1045642
Outer primer group includes the outer downstream primer SEQ ID NO:3 of the outer upstream primer SEQ ID NO:1 and rs1045642 of rs1045642；
The rs1045642 inner primer group includes that downstream is drawn in upstream primer SEQ ID NO:2 and rs1045642 in rs1045642
Object SEQ ID NO:4；
The rs776746 primer sets include rs776746 outer primer group and rs776746 inner primer group, are drawn outside the rs776746
Object group includes the outer downstream primer SEQ ID NO:7 of the outer upstream primer SEQ ID NO:5 and rs776746 of rs776746；It is described
Rs776746 inner primer group includes downstream primer SEQ ID in upstream primer SEQ ID NO:6 and rs776746 in rs776746
NO:8。
2. a kind of ABCB1, CYP3A5 gene detecting kit, which is characterized in that the kit includes described in claim 1
ABCB1, CYP3A5 genetic test primer sets.
3. gene detecting kit according to claim 2, which is characterized in that the kit further includes connector, described
Contain sequence label on connector, the connector is for the amplified production directly with the site containing rs1045642 and the site rs776746
Connection.
4. gene detecting kit according to claim 2, which is characterized in that the kit further includes fluorescent marker
The oligonucleotide probe of oligonucleotide probe and unstressed configuration label.
5. gene detecting kit according to claim 2, which is characterized in that the kit further includes sequencing primer,
The sequencing primer includes the rs1045642 sequencing primer SEQ ID that the library molecule in the site containing rs1045642 is sequenced
NO:9, the rs776746 sequencing primer SEQ ID NO:10 that the library molecule in the site containing rs776746 is sequenced.
6. a kind of ABCB1, CYP3A5 gene tester, which comprises the following steps:
A, using primer sets, the sample in the site containing rs1045642 and the site rs776746 is expanded respectively, production must be expanded
Object；The primer sets include rs1045642 primer sets, rs776746 primer；
The rs1045642 primer sets include rs1045642 outer primer group and rs1045642 inner primer group, the rs1045642
Outer primer group includes the outer downstream primer SEQ ID NO:3 of the outer upstream primer SEQ ID NO:1 and rs1045642 of rs1045642；
The rs1045642 inner primer group includes that downstream is drawn in upstream primer SEQ ID NO:2 and rs1045642 in rs1045642
Object SEQ ID NO:4；
The rs776746 primer sets include rs776746 outer primer group and rs776746 inner primer group, are drawn outside the rs776746
Object group includes the outer downstream primer SEQ ID NO:7 of the outer upstream primer SEQ ID NO:5 and rs776746 of rs776746；It is described
Rs776746 inner primer group includes downstream primer SEQ ID in upstream primer SEQ ID NO:6 and rs776746 in rs776746
NO:8；
B, the jointing on the amplified production, obtains library molecule；
C, the library molecule is sequenced using second generation high throughput gene sequencing technology, determines the gene in site to be measured
Type.
7. ABCB1, CYP3A5 gene tester according to claim 6, which is characterized in that the step A include with
Lower step:
A1, ABCB1 gene is expanded using rs1045642 outer primer group, obtains the first rs1045642 amplified production；Using
Rs776746 outer primer group expands CYP3A5 gene, obtains the first rs776746 amplified production；
The rs1045642 outer primer group includes under the outer upstream primer SEQ ID NO:1 and rs1045642 of rs1045642 is outer
Swim primer SEQ ID NO:3；The rs776746 outer primer group include the outer upstream primer SEQ ID NO:5 of rs776746 and
The outer downstream primer SEQ ID NO:7 of rs776746；
A2, the first rs1045642 amplified production is expanded using rs1045642 inner primer group, obtains the 2nd rs1045642 expansion
Increase production object；Rs776746 inner primer group is used, to expand, to obtain the 2nd rs776746 expansion to the first rs776746 amplified production
Increase production object；
The rs1045642 inner primer group include in rs1045642 in upstream primer SEQ ID NO:2 and rs1045642 under
Swim primer SEQ ID NO:4；The rs776746 inner primer group include in rs776746 upstream primer SEQ ID NO:6 and
Downstream primer SEQ ID NO:8 in rs776746.
8. gene tester according to claim 7, which is characterized in that further include to second in the step B
Uracil on rs1045642 amplified production, the 2nd rs776746 amplified production carries out the step of specificity cutting, and described second
Rs1045642 amplified production, the 2nd rs776746 amplified production are respectively formed the first cohesive end through cutting.
9. gene tester according to claim 8, which is characterized in that the connector includes the second cohesive end, is used
It is connect in the first cohesive end.
10. gene tester according to claim 6, which is characterized in that step C is attached survey using sequencing primer
Sequence, the sequencing primer include the rs1045642 sequencing primer SEQ that the library molecule in the site containing rs1045642 is sequenced
The ID NO:9 and rs776746 sequencing primer SEQ ID NO:10 that the library molecule in the site containing rs776746 is sequenced.
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