CN104988224A - Primer and method for simultaneously detecting XRCC1, ERCC1 and GSTP1 gene polymorphisms - Google Patents

Primer and method for simultaneously detecting XRCC1, ERCC1 and GSTP1 gene polymorphisms Download PDF

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CN104988224A
CN104988224A CN201510384466.0A CN201510384466A CN104988224A CN 104988224 A CN104988224 A CN 104988224A CN 201510384466 A CN201510384466 A CN 201510384466A CN 104988224 A CN104988224 A CN 104988224A
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primer
xrcc1
ercc1
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gstp1
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赵薇薇
胡昌明
燕启江
郭周萍
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Guangzhou Kingmed Diagnostics Central Co Ltd
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Abstract

The invention belongs to the technical field of biological detection, and provides a primer for simultaneously detecting XRCC1, ERCC1 and GSTP1 gene polymorphisms. The primer comprises a PCR amplification primer and a SNaPshot PCR primer. The primer can achieve specific detection on XRCC1, ERCC1 and GSTP1 gene polymorphisms, causes no cross reaction and is good in accuracy.

Description

Detect primer and the method for XRCC1, ERCC1 and GSTP1 gene pleiomorphism simultaneously
Technical field
The invention belongs to technical field of biological, particularly relate to a kind of primer and the method that detect XRCC1, ERCC1 and GSTP1 gene pleiomorphism simultaneously.
Background technology
Platinum-containing anticancer drug, comprising cis-platinum, carboplatin and oxaliplatin etc., is cancer therapy drug kinds cancer in current clinical application to greater activity, mainly through causing in cell DNA damage to remove cancer cells.
X-ray repairs cross complementary group 1 (XRCC1), and be positioned at human chromosomal 19q13.2-13.3, size is 33 kb.Excision repair cross-completion 1 gene (ERCC1), is mankind's DNA damage revision points, is positioned at karyomit(e) 19q13.2-13.3.Glutathione S transferase M1 (GSTP1), glutathione s-transferase (GST) is most important II phase metabolic enzyme in body, by directly playing function of detoxification in conjunction with chemical carcinogen or by being combined with gsh, degradable exogenous compounds toxicity.
Existing data display, XRCC1_R399Q R/R, R/Q and Q/Q genotype frequency is about 55%, 37%, 8% respectively; ERCC1_C118T C/C, C/T, T/T genotype frequency is about 52.6% respectively, and 43.1%, 4.2%; GSTP1_I105V Ile/Ile(I/I), Ile/Val(I/V), Val/Val(V/V) and genotype frequency is respectively 57.4%, 35.9%, 6.7%.
Therefore, by the detection of XRCC1/ERCC1/GSTP1 gene pleiomorphism, contribute to the prognosis predicting platinum-based chemotherapy, significant in help direction of medication usage and personalized treatment.Prior art lack a species specificity good, highly sensitive, the XRCC1/ERCC1/GSTP1 genetic polymorphism detection primer that simultaneously detects in multiple site and method thereof can be realized.
Summary of the invention
For solving problems of the prior art, the invention provides a kind of primer and the method that detect XRCC1/ERCC1/GSTP1 gene pleiomorphism simultaneously, there is the advantages such as specificity is good, highly sensitive, accuracy is good, detect while achieving XRCC1/ERCC1/GSTP1 gene pleiomorphism.The site that the present invention detects simultaneously comprises XRCC1_R399Q:c.1196A>G (p.Gln399Arg) (SNP:rs25487), ERCC1_C118T:c.354T>C (p.Asn118=) (SNP:rs11615), GSTP1_I105V:c.313A>G (p.Ile105Val) (SNP:rs1695).
The invention provides a kind of primer simultaneously detecting XRCC1/ERCC1/GSTP1 gene pleiomorphism, comprise pcr amplification primer and SNaPshot PCR primer, described pcr amplification primer comprises: for upstream primer 5 '-TCTGACTCCCCTCCAGATT-3 ' (SEQ ID NO.1) and the downstream primer 5 '-GCCCCTCAGATCACACCTA-3 ' (SEQ ID NO.2) of XRCC1_R399Q, for upstream primer 5 '-TCCAGAACACTGGGACATGA-3 ' (SEQ ID NO.3) and the downstream primer 5 '-TCCCTATTGATGGCTTCTGC-3 ' (SEQ ID NO.4) of ERCC1_C118T, for upstream primer 5 '-ACCCCAGGGCTCTATGGGAA-3 ' (SEQ ID NO.5) and the downstream primer 5 '-TGAGGGCACAAGAAGCCCCT-3 ' (SEQ ID NO.6) of GSTP1_I105V, described SNaPshot PCR primer comprises: for SNaPshot the PCR primer 5 '-CGTCGGCGGCTGCCCTCCC-3 ' (SEQ ID NO.7) of XRCC1_R399Q, for SNaPshot the PCR primer 5 '-TTTTTTTTAGGGGCAATCCCGTACTGAAGTTCGTGCGCAA-3 ' (SEQ ID NO.8) of ERCC1_C118T, for SNaPshot the PCR primer 5 '-TTTTTTTTTTTTTTTTTTTTTGCGTGGAGGACCTCCGCTGCAAATAC-3 ' (SEQ ID NO.9) of GSTP1_I105V.
Adopt technique scheme, the primer simultaneously detecting XRCC1/ERCC1/GSTP1 gene pleiomorphism provided by the invention, can realize the specific detection of XRCC1/ERCC1/GSTP1 gene pleiomorphism, cross reaction can not occur, accuracy is good.
Preferably, in described pcr amplification primer, the primer concentration of XRCC1_R399Q, ERCC1_C118T and GSTP1_I105V is than being 1:1:1; In described SNaPshot PCR primer, the primer concentration of XRCC1_R399Q, ERCC1_C118T and GSTP1_I105V is than being 1:1:1.
Correspondingly, the present invention also provides a kind of method simultaneously detecting XRCC1/ERCC1/GSTP1 gene pleiomorphism, comprises the steps: A) extract DNA sample; B) adopt the pcr amplification primer described in claim 1 or 2 to carry out multiplexed PCR amplification, purifying is carried out to amplified production; C) adopt the SNaPshot PCR primer described in claim 1 or 2 to carry out SNaPshot pcr amplification, purifying is carried out to amplified production; D) capillary electrophoresis technique detects, and analyzes detected result, determines SNP site and genotype thereof.
Preferably, described steps A) in DNA sample be the DNA sample of EDTA anticoagulation cirumferential blood.
Preferably, described step B) in multiplexed PCR amplification reaction conditions comprise: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:58 DEG C 30s; Stage 4:72 DEG C 1min; Stage 5: turn back to stage 2,29 circulation; Stage 6:72 DEG C 5min; Stage 7:25 DEG C of insulation.That is, Step 1:98 ° of C for 3minutes; Step 2:98 ° of C for 10 seconds; Step 3:58 ° of C for 30 seconds; Step 4:72 ° of C for 1 minute; Step 5:Go to step 2,29 times; Step 6:72 ° of C for 5 minutes; Step 7:25 ° of C forever.
Described step C) in SNaPshot pcr amplification reaction condition comprise: stage 1:96 DEG C 10s; Stage 2:55 DEG C 5s; Stage 3:60 DEG C 30s; Stage 4: turn back to stage 1,25 circulation; Stage 5:4 DEG C of insulation.That is, Step 1:96 ° of C for10seconds; Step 2:55 ° of C for 5 seconds; Step 3:60 ° of C for 30seconds; Step 4:Go to step 1,25 times; Step 5:4 ° of C forever.
Preferably, described step D) in, adopt GENEMAPPERID V4.1 software to analyze detected result.
In addition, the present invention also provides the purposes of the primer simultaneously detecting XRCC1/ERCC1/GSTP1 gene pleiomorphism in preparation detection XRCC1/ERCC1/GSTP1 gene pleiomorphism reagent.
Compared with prior art, the invention has the beneficial effects as follows: the invention provides a kind of primer simultaneously detecting XRCC1/ERCC1/GSTP1 gene pleiomorphism, the specificity of primer is good, cross reaction can not be there is, accuracy is good, detect while achieving XRCC1/ERCC1/GSTP1 gene pleiomorphism, improve detection efficiency.In addition, present invention also offers a kind of SNaPshot method simultaneously detecting XRCC1/ERCC1/GSTP1 gene pleiomorphism, by using specific pcr amplification primer and SNaPshot PCR primer, there is the advantages such as specificity is good, highly sensitive, accuracy is good, guidance can be provided for the clinical application of platinum medicine.
Accompanying drawing explanation
The amplified fragments of Fig. 1 XRCC1/ERCC1 gene primer provided by the invention.
The amplified fragments of Fig. 2 GSTP1 gene primer provided by the invention.
The part sequencer map of Fig. 3 XRCC1/ERCC1 gene primer amplified production.
The part sequencer map of Fig. 4 GSTP1 gene primer amplified production.
The XRCC1/ERCC1/GSTP1 genetic polymorphism detection result figure of Fig. 5 sample.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further details.
embodiment one primer
Contriver, for the gene polymorphism sites of XRCC1/ERCC1/GSTP1, devises a large amount of primer, by the optimization of primer reaction conditions with compare, filtered out specificity good, can not cross reaction be there is and PCR reaction conditions primer closely.Primer provided by the invention comprises pcr amplification primer and SNaPshot PCR primer, and pcr amplification primer and SNaPshot PCR primer are corresponding.All primer sequences provided by the invention all by the comparison of UCSC database, without known SNP site.
the specificity of embodiment two primer
Primer provided by the invention is carried out in UCSC Blasting, XRCC1_R399Q primer amplification fragment and be positioned at chr19:44055651-44055888, length is 238bp; ERCC1_C118T primer amplification fragment is positioned at chr19:45923504-45923722, and length is 219bp; GSTP1_I105V primer amplification fragment is positioned at chr11:67352602-67352777, and length is 176bp; As shown in Figure 1 to Figure 2, the amplified fragments of all primers all covers corresponding detection site to result, without other homologous gene.
In use table 1, pcr amplification primer increases and Sanger order-checking to detection sample respectively, and sequencing result shows, and each primer amplification fragment and XRCC1/ERCC1/GSTP1 gene reference sequence coincide, and result as shown in Figure 3 to Figure 4.Use SNaPshot PCR primer in table 1, SNaPshot method detects, and result as shown in Figure 5.As can be seen from Figure 5, the base that the relative position at each product peak and sequencing reaction mix conforms to expection, and without other Interference Peaks.
the detection of embodiment two XRCC1/ERCC1/GSTP1 gene pleiomorphism
Extract the DNA sample of EDTA anticoagulation cirumferential blood, extracting method with reference to TIANamp Blood DNA Kit(purchased from Tiagen, article No.: DP318) specification sheets, DNA sample is diluted to 100ng/ μ L, for subsequent use.
Preparation pcr amplification primer mixture, the primer concentration of XRCC1_R399Q, ERCC1_C118T and GSTP1_I105V is 5pmol/uL than the primer concentration for 1:1:1, XRCC1_R399Q, and concussion mixing, pcr amplification adopts Q5 ?warm start surpasses fidelity 2X Master Mix(purchased from NEB company, article No.: M0494L), reaction system is as shown in table 2.Multiplexed PCR amplification reaction conditions comprises: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:58 DEG C 30s; Stage 4:72 DEG C 1min; Stage 5: turn back to stage 2,29 circulation; Stage 6:72 DEG C 5min; Stage 7:25 DEG C of insulation.After pcr amplification terminates, get 2.0 μ L ExoSAP-IT(purchased from Affymetrix, article No.: 78205) and 3.0 μ L ddH 2o, mixing, adds pcr amplification product 2.0 μ L, mixes micro-centrifugal; Endonuclease reaction is carried out, program: 37 DEG C, 15 min in PCR instrument; 80 DEG C, 15min; 4 DEG C, insulation.
Configuration SNaPshot PCR primer mixture, the primer concentration of XRCC1_R399Q, ERCC1_C118T and GSTP1_I105V is 5pmol/uL than the primer concentration for 1:1:1:1:1, XRCC1_R399Q, and concussion mixing, adopts SNaPshot multiplex Kit(purchased from ABI company, article No. 4323151) increase, reaction system is as shown in table 3.SNaPshot PCR reaction conditions comprises: stage 1:96 DEG C 10s; Stage 2:55 DEG C 5s; Stage 3:60 DEG C 30s; Stage 4: turn back to stage 1,25 circulation; Stage 5:4 DEG C of insulation.
In SNaPshot PCR primer, add 1.0 μ L SAP enzymes, react according to following program: 37 DEG C, 60min; 75 DEG C, 15min; 4 DEG C, insulation.After completion of the reaction, capillary electrophoresis technique detects, and adopt GENEMAPPERID V4.1 software to analyze to detected result, determine SNP site and genotype thereof, result as shown in Figure 5.
embodiment three detects the specificity of the method for XRCC1/ERCC1/GSTP1 gene pleiomorphism
The specificity of this detection method is defined as negative match-rate.Genotypic for XRCC1_R399Q G/G detecting is defined as negative findings by this detection, and genotypic the detecting of ERCC1_C118T C/C is defined as negative findings, and genotypic the detecting of GSTP1_I105V A/A is defined as negative findings.
Adopt SNaPshot method provided by the invention to detect 19 routine samples, adopt ARMS method or Sanger sequencing to verify simultaneously.The detected result of SNaPshot method conforms to the result of ARMS method or Sanger sequencing, as shown in table 4.The specificity of this detection method is 100%.
embodiment four detects sensitivity and the accuracy of the method for XRCC1/ERCC1/GSTP1 gene pleiomorphism
The sensitivity definition of this detection method is positive coincidence rate.Adopt SNaPshot method provided by the invention to detect 19 routine samples, adopt ARMS method or Sanger sequencing to verify simultaneously.The detected result of SNaPshot sequencing conforms to the result of ARMS method or Sanger sequencing, as shown in table 5.Sensitivity and the accuracy of this detection method are high.
This detection verifies the Monitoring lower-cut of this detection for XRCC1_R399Q pleomorphism site.Sample A and sample B presses 1:9, and detect after the mixing of 2:8,3:7,4:6,5:5 ratio, wherein, sample A XRCC1 R399Q genotype is A/A (saltant type), and sample B XRCC1 R399Q genotype is G/G (wild-type).Result is as shown in table 6, and when XRCC1 genic mutation type and WT ratios are 1:9 (mutatant ratio is 10%), this detection method also can detect saltant type.XRCC1_R399Q polymorphism is germline mutation, and in heterozygous genotypes G/A, mutation allele is 50%, therefore, and the detection sensitivity 100% of this detection.
embodiment five detects the precision of the method for XRCC1/ERCC1/GSTP1 gene pleiomorphism
The precision of this detection method is defined as carries out to sample the ability that duplicate detection obtains same result.This detection has been carried out between personnel, contrast experiment between the different hole of different time, different instrument, same sample, and result is as shown in table 7a and 7b, and all results all show unanimously, and this detection precision is 100%.Wherein, site 1:XRCC1_R399Q (G>A); Site 2:ERCC1_C118T (C>T); Site 3:GSTP1_I105V (A>G).
Therefore, primer provided by the present invention and detection method can be used as one independently, widely used authentication method, solve XRCC1/ERCC1/GSTP1 and carry out accurate Classification Identification problem, play the feature that PCR-SNaPshot PCR is accurate to described gene locus genotypic results, high-throughput operates, contribute to the prognosis predicting platinum-based chemotherapy, reduce untoward reaction, significant in help direction of medication usage and personalized treatment.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.
SEQUENCE LISTING
<110> Guangzhou Kingmed Center for Clinical Laboratory Co., Ltd.
<120> detects primer and the method for XRCC1, ERCC1 and GSTP1 gene pleiomorphism simultaneously
<130>
<160> 9
<170> PatentIn version 3.3
 
<210> 1
<211> 19
<212> DNA
<213> artificial sequence
<400> XRCC1_R399Q_F
tctgactccc ctccagatt 19
 
<210> 2
<211> 19
<212> DNA
<213> artificial sequence
<400> XRCC1_R399Q_R
gcccctcaga tcacaccta 19
 
<210> 3
<211> 20
<212> DNA
<213> artificial sequence
<400> ERCC1_C118T_F
tccagaacac tgggacatga 20
 
<210> 4
<211> 20
<212> DNA
<213> artificial sequence
<400> ERCC1_C118T_R
tccctattga tggcttctgc 20
 
<210> 5
<211> 20
<212> DNA
<213> artificial sequence
<400> GSTP1_I105V_F
accccagggc tctatgggaa 20
 
<210> 6
<211> 20
<212> DNA
<213> artificial sequence
<400> GSTP1_I105V_R
tgagggcaca agaagcccct 20
 
<210> 7
<211> 19
<212> DNA
<213> artificial sequence
<400> XRCC1_R399Q
cgtcggcggc tgccctccc 19
 
<210> 8
<211> 40
<212> DNA
<213> artificial sequence
<400> ERCC1_C118T
ttttttttag gggcaatccc gtactgaagt tcgtgcgcaa 40
 
<210> 9
<211> 47
<212> DNA
<213> artificial sequence
<400> GSTP1_I105V
tttttttttt tttttttttt tgcgtggagg acctccgctg caaatac 47
 

Claims (8)

1. detect a primer for XRCC1, ERCC1 and GSTP1 gene pleiomorphism simultaneously, it is characterized in that: comprise pcr amplification primer and SNaPshot PCR primer; Described pcr amplification primer comprises: for upstream primer 5 '-TCTGACTCCCCTCCAGATT-3 ' and the downstream primer 5 '-GCCCCTCAGATCACACCTA-3 ' of XRCC1_R399Q, for upstream primer 5 '-TCCAGAACACTGGGACATGA-3 ' and the downstream primer 5 '-TCCCTATTGATGGCTTCTGC-3 ' of ERCC1_C118T, for upstream primer 5 '-ACCCCAGGGCTCTATGGGAA-3 ' and the downstream primer 5 '-TGAGGGCACAAGAAGCCCCT-3 ' of GSTP1_I105V; Described SNaPshot PCR primer comprises: for SNaPshot the PCR primer 5 '-CGTCGGCGGCTGCCCTCCC-3 ' of XRCC1_R399Q, for SNaPshot the PCR primer 5 '-TTTTTTTTAGGGGCAATCCCGTACTGAAGTTCGTGCGCAA-3 ' of ERCC1_C118T, for SNaPshot the PCR primer 5 '-TTTTTTTTTTTTTTTTTTTTTGCGTGGAGGACCTCCGCTGCAAATAC-3 ' of GSTP1_I105V.
2. the primer simultaneously detecting XRCC1, ERCC1 and GSTP1 gene pleiomorphism according to claim 1, is characterized in that: in described pcr amplification primer, and the primer concentration of XRCC1_R399Q, ERCC1_C118T and GSTP1_I105V is than being 1:1:1; In described SNaPshot PCR primer, the primer concentration of XRCC1_R399Q, ERCC1_C118T and GSTP1_I105V is than being 1:1:1.
3. detect a method for XRCC1, ERCC1 and GSTP1 gene pleiomorphism simultaneously, it is characterized in that: comprise the steps: A) extract DNA sample; B) adopt the pcr amplification primer described in claim 1 or 2 to carry out multiplexed PCR amplification, purifying is carried out to amplified production; C) adopt the SNaPshot PCR primer described in claim 1 or 2 to carry out SNaPshot pcr amplification, purifying is carried out to amplified production; D) capillary electrophoresis technique detects, and analyzes detected result, determines SNP site and genotype thereof.
4. the method simultaneously detecting XRCC1, ERCC1 and GSTP1 gene pleiomorphism according to claim 3, is characterized in that: described steps A) in DNA sample be the DNA sample of EDTA anticoagulation cirumferential blood.
5. the method simultaneously detecting XRCC1, ERCC1 and GSTP1 gene pleiomorphism according to claim 3, is characterized in that: described step B) in multiplexed PCR amplification reaction conditions comprise: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:58 DEG C 30s; Stage 4:72 DEG C 1min; Stage 5: turn back to stage 2,29 circulation; Stage 6:72 DEG C 5min; Stage 7:25 DEG C of insulation.
6. the method simultaneously detecting XRCC1, ERCC1 and GSTP1 gene pleiomorphism according to claim 3, is characterized in that: described step C) in SNaPshot pcr amplification reaction condition comprise: stage 1:96 DEG C 10s; Stage 2:55 DEG C 5s; Stage 3:60 DEG C 30s; Stage 4: turn back to stage 1,25 circulation; Stage 5:4 DEG C of insulation.
7. the method simultaneously detecting XRCC1, ERCC1 and GSTP1 gene pleiomorphism according to claim 3, is characterized in that: described step D) in, adopt GENEMAPPERID V4.1 software to analyze detected result.
8. the primer simultaneously detecting XRCC1, ERCC1 and GSTP1 gene pleiomorphism according to claim 1 and 2 detects the purposes in XRCC1, ERCC1 and GSTP1 gene pleiomorphism reagent in preparation.
CN201510384466.0A 2015-06-30 2015-06-30 Primer and method for simultaneously detecting XRCC1, ERCC1 and GSTP1 gene polymorphisms Pending CN104988224A (en)

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CN108949977A (en) * 2018-07-10 2018-12-07 昆明理工大学 One group is detected primer and the application of ERCC4 and XRCC1 gene pleiomorphism simultaneously

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CN107227363A (en) * 2017-07-05 2017-10-03 上海赛安生物医药科技股份有限公司 ERCC1 genetic polymorphism detections system and its kit
CN108949977A (en) * 2018-07-10 2018-12-07 昆明理工大学 One group is detected primer and the application of ERCC4 and XRCC1 gene pleiomorphism simultaneously

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Application publication date: 20151021