CN105506100A - Primer and method for simultaneously detecting gene polymorphisms of IL28B and ITPA (inosineadenosine triphatase) - Google Patents
Primer and method for simultaneously detecting gene polymorphisms of IL28B and ITPA (inosineadenosine triphatase) Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, in particular relates to a primer and a method for simultaneously detecting gene polymorphisms of IL28B and ITPA (inosineadenosine triphatase). The primer for simultaneously detecting gene polymorphisms of IL28B and ITPA, provided by the invention, comprises a PCR (polymerase chain reaction) amplification primer and SNaPshot PCR primer. The primer for simultaneously detecting gene polymorphisms of IL28B and ITPA, provided by the invention, has the advantages of high specificity, high detection sensitivity and high accuracy, and detection on the gene polymorphisms of IL28B and ITPA is realized; the primer can be used for guiding dosage adjustment of ribavirin so as to increase reaction of antiviral therapy; meanwhile, the primer can also be used for providing a basis for clinically predicting antiviral individualized treatment effect on hepatitis c, and has great social significance.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of primer and the method thereof that detect IL28B and ITPA gene pleiomorphism simultaneously.
Background technology
Hepatitis C, referred to as hepatitis C, is that one infects the viral hepatitis caused, mainly through blood born by hepatitis C virus (HepatitisCvirus, HCV).Because major part infects the acute phase non-evident sympton of patient in infection of hepatitis C virus, therefore usually ignored by people, generally just discover to some extent when hepatitis C is converted into the intractable diseases such as chronic hepatitis C or liver cirrhosis, so often miss the optimal treatment phase of hepatitis C.According to World Health Organization's statistics, about there is 1.4-1.7 hundred million HCV infection person in the whole world, and China's HCV infection rate is about 3.2%, about has 3,800 ten thousand HCV infection persons, and the annual trend in rising, the health of the serious harm mankind.
2009, the Ge of Duke University etc. have delivered one section of full-length genome dependency (genomewideassociationstudy relevant to HCV infection on " nature ", GWAS) treatise is studied, reporting IL28B gene and be positioned at No. 19 the short arm of a chromosome (19q13), its coded interference element λ 3(IL28B) the single nucleotide polymorphism rs12979860 that is about near 3kb of upstream is relevant to the Be very effective of polyethylene glycol Interferon, rabbit, ribavirin combination therapy.In 1 type HCV infection person, the SVR of C/C genotype patient is the twice of C/T and T/T type patient.The distribution of C/C genotype in crowd presents obvious racial difference, accounts for 90%, accounts for 51% in the white race, in black race, account for 13% in the anthous.Rs8099917 is the most relevant to the NVR of standard treatment.In 1 type HCV infection person, the mortality of G/G genotype patient to standard regimens is 2 times of G/T or T/T type patient.
The same study group of Duke University is finding that IL28B genotype has on the basis of prediction antiviral therapy response; found by GWAS research; inosine adenosine triphosphatase (in0sineadenosinetriphatase; ITPA) genovariation can alleviate the hemolytic anemia that RBV causes, as rs1127354AA and rs7270101CC genotype is the protectiveness factor of anaemia.Therefore, be conducive to predicting the curative effect of HCV patient by detecting IL28B and ITPA gene pleiomorphism.
Chinese patent application 201510007333.1 discloses the IL28B genetic polymorphism detection test kit based on real-time fluorescence PCR, described test kit comprises IL28Brs8099917 type PCR reaction solution, IL28Brs12979860 type PCR reaction solution, it is by extracting DNA, join in the reaction solution based on the IL28BSNP detection kit of real-time fluorescence PCR, utilize real-time fluorescence PCR instrument to detect, carry out interpretation site mutation type by amplified fluorescence curve.
Chinese patent application 201210353290.9 discloses the method for the sudden change detecting IL28B (rs8099917) and ITPA (rs1127354), its polymorphic detection probe comprises the oligonucleotide of the oligonucleotide of P1 or P1 ', the oligonucleotide of P2 or P2 ' and P3 or P3 ', it carries out Tm analysis after probe is carried out pcr amplification, carries out somatotype to the rs1127354 of rs8099917 and the ITPA gene pleiomorphism of IL28B gene pleiomorphism.
Although current detection method can carry out somatotype to IL28B and ITPA, it is comparatively large to there is detected result error in aforesaid method, and repeatability is lower, and result judges more complicated defect.Therefore, research and develop out a kind of simple to operate, detected result error is little, and the detection method of the result high IL28B gene of repeatability and ITPA gene is still the focus of research at present.
Summary of the invention
In order to solve the defect existed in prior art, the invention provides a kind of primer and the method thereof that detect IL28B and ITPA gene pleiomorphism simultaneously, this primer is mainly for detection of IL28B_rs8099917, IL28B_rs12979860, ITPA_rs7270101 and ITPA_rs1127354 tetra-sites, there is the advantages such as specificity is good, highly sensitive, accuracy is good, for IL28B and ITPA gene pleiomorphism provides a kind of simple and effective detection method.
The invention provides a kind of primer simultaneously detecting IL28B and ITPA gene pleiomorphism, comprise pcr amplification primer and SNaPshotPCR primer, described pcr amplification primer comprises: for upstream primer 5 '-AGTAAGTCTTGTATTTCACCTCCTG-3 ' (SEQIDNO.1) and the downstream primer 5 '-AAAGCCAGCTACCAAACTGTAT-3 ' (SEQIDNO.2) of IL28B9917, for upstream primer 5 '-GTCGTGCCTGTCGTGTACT-3 ' (SEQIDNO.3) and the downstream primer 5 '-TCAGGGTCAATCACAGAAGGG-3 ' (SEQIDNO.4) of IL28B9860, for upstream primer 5 '-GGAGATGGGCAGCAGAGTTA-3 ' (SEQIDNO.5) and the downstream primer 5 '-CAGGTCACAGGAAGACAGAGA-3 ' (SEQIDNO.6) of ITPA, described SNaPshotPCR primer comprises: for SNaPshotPCR the primer 5 '-CATGGTTCCAATTTGGGTGA-3 ' (SEQIDNO.7) of IL28B9917, for SNaPshotPCR the primer 5 '-AAAAAAAAAAAAAATGCGGAGTGCAATTCAACCCTGGTTC-3 ' (SEQIDNO.8) of IL28B9860, for SNaPshotPCR the primer 5 '-AAAAAAAAAAAAGATCGATCGAGAAATCCAACCATCTTTTAAAAA-3 ' (SEQIDNO.9) of ITPA0101, for SNaPshotPCR primer 5 '-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATTTTCTGTGCCACCAAAGTGCA TG-3 ' (SEQIDNO.10) of ITPA7354.
Further, in described pcr amplification primer, the primer concentration of IL28B9917, IL28B9860 and ITPA is than being 1:1:1, and in described SNaPshotPCR primer, the primer concentration of IL28B9917, IL28B9860, ITPA0101 and ITPA7354 is than being 1:1:1:1.
In addition, the invention provides a kind of method simultaneously detecting IL28B and ITPA gene pleiomorphism, comprise the steps:
S1 extracts DNA sample;
The DNA sample that S2 extracts with step S1, for template, adopts the pcr amplification primer described in claim 1 to carry out multiplexed PCR amplification, and carries out purifying to amplified production;
S3 for template with the pcr amplification product after step S2 purifying, adopts the SNaPshotPCR primer described in claim 1 to carry out SNaPshotPCR amplification, and carries out purifying to SNaPshotPCR amplified production;
S4 adopts capillary electrophoresis technique to detect, and analyzes, determine SNP site and genotype thereof to detected result.
Further, the DNA sample in described step S1 is the DNA sample of EDTA anticoagulation cirumferential blood.
Further, the multiplexed PCR amplification reaction conditions in described step S2 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.
Further, the SNaPshotPCR amplification reaction condition in described step S3 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.
Further, GENEMAPPERIDV4.1 software is adopted to analyze detected result in described step S4.
In addition, the primer simultaneously detecting IL28B and ITPA gene pleiomorphism provided by the invention detects the purposes in IL28B and ITPA gene pleiomorphism reagent in preparation.
Compared with prior art, technical scheme provided by the invention has following advantage: it is good that the primer simultaneously detecting IL28B and ITPA gene pleiomorphism provided by the invention has specificity, cross reaction can not be there is, the advantage that accuracy is high, achieve the detection simultaneously detecting IL28B and ITPA gene pleiomorphism, can be used for instructing the consumption of ribavirin to adjust, to improve the reaction of antiviral therapy, also can provide foundation for the antiviral individualized treatment effect of dlinial prediction hepatitis C simultaneously, there is great social effect.
Accompanying drawing explanation
Fig. 1 is the amplified fragments of IL28B9917 gene primer provided by the invention;
Fig. 2 is the amplified fragments of IL28B9860 gene primer provided by the invention;
Fig. 3 is the amplified fragments of ITPA gene primer provided by the invention;
Fig. 4 is the detected result figure of IL28B and ITPA gene SNaPshotPCR primer provided by the invention.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further details.
Embodiment one, primer
IL28B and ITPA gene primer provided by the invention is as shown in table 1, comprises pcr amplification primer and SNaPshotPCR primer, and described pcr amplification primer and SNaPshotPCR primer are corresponding.All primer sequences provided by the invention all by the comparison of UCSC database, without known SNP site.
Table 1 primer provided by the invention
The specificity of embodiment two, primer
Primer provided by the invention is carried out Blasting in UCSC, and result is as follows:
1) IL28B and ITPA gene specific primer carries out Blast in UCSC, the amplified fragments of all primers all covers corresponding detection site, and without other homologous gene, IL28B9917 amplified fragments is positioned at chr19:39743077+39743211, length is 135bp, and extension increasing sequence figure is as Fig. 1; IL28B9860 amplified fragments is positioned at chr19:39738744+39738889, and length is 146bp, and extension increasing sequence figure is as Fig. 2; ITPA amplified fragments is positioned at chr20:3193710+3193935, and length is 226bp, and extension increasing sequence figure is as Fig. 3.
2) use SNaPshotPCR primer in table 1, SNaPshot method detects, and as shown in Figure 4, the base that the relative position at each product peak and sequencing reaction mix conforms to expection result.
The detection of embodiment three, IL28B and ITPA gene pleiomorphism
1) extract the DNA sample of EDTA anticoagulation cirumferential blood, extracting method with reference to TIANampBloodDNAKit(purchased from Tiagen, article No. DP318) specification sheets, DNA sample is diluted to 100ng/ μ L, for subsequent use.
2) primer mixture is prepared: by the primer concentration of IL28B9917, IL28B9860 and ITPA in pcr amplification primer than being 1:1:1 mixing, concussion mixing; Of short duration centrifugal rear for subsequent use; Pcr amplification adopts Q5
?warm start surpasses fidelity 2XMasterMix(purchased from NEB company, article No. M0494L), reaction system is as shown in table 2, concussion mixing, of short duration centrifugal after, packing 18.0 μ L is to the PCR reaction tubes that marked; Add primer mixture 5.0 μ L toward the PCR reaction tubes marked, carry out pcr amplification according to following program: 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 2,2:9 circulation; Stage 6:72 DEG C 5min; Stage 7:25 DEG C of insulation.
Table 2, PCR reagent preparation form
3) comparing by the primer concentration of IL28B9917, IL28B9860, ITPA0101 and ITPA7354 in SNaPshotPCR primer is 1:1:1:1 mixing, adds 1.0 μ LSAP enzymes, reacts according to following program: 37 DEG C, 15min in SNaPshotPCR product; 80 DEG C, 15min; 4 DEG C, insulation.After completion of the reaction, capillary electrophoresis technique detects, and adopt GENEMAPPERIDV4.1 software to analyze to detected result, determine SNP site and genotype thereof, result as shown in Figure 4.
The specificity of the method for embodiment four, detection IL28B and ITPA gene pleiomorphism
Detection specificity of the present invention is defined as negative match-rate.The present invention is optimized SNaPshot sequencing to 21 routine samples altogether and detects, and detected result adopts ARMS-PCR method to verify.The result that the negative findings that SNaPshot sequencing detects shows with ARMS-PCR method conforms to, without sample (feminine gender) totally 7 examples of sudden change, as shown in table 3.Detection specificity of the present invention is 100%.
Table 3, the specific testing data of IL28B and ITPA gene test
The sensitivity of the method for embodiment five, detection IL28B and ITPA gene pleiomorphism
Detection sensitivity of the present invention is defined as positive coincidence rate.The present invention is optimized SNaPshot sequencing to 21 routine samples altogether and detects, and detected result adopts ARMS-PCR method to verify.The result that the positive findings that SNaPshot sequencing detects shows with ARMS-PCR method conforms to, sample (positive) totally 14 examples of sudden change, as shown in table 4.Detection sensitivity of the present invention is 100%.
The testing data of table 4, IL28B and ITPA gene test sensitivity
The accuracy of the method for embodiment six, detection IL28B and ITPA gene pleiomorphism
Accuracy of the present invention is defined as the consistence of different methods detected result.The present invention carries out the detection of SNaPshot sequencing to 21 routine samples altogether, and detected result all adopts ARMS-PCR method to verify.Different methods detected result is consistent, as shown in table 5.Accuracy of the present invention is 100%.
The testing data of table 5, IL28B and ITPA gene 2SNPs site accuracy in detection
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.
SEQUENCELISTING
Science and Technology Co., Ltd. is detected in gold territory, <110> Guangzhou
<120> mono-kind detects primer and the method thereof of IL28B and ITPA gene pleiomorphism simultaneously
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Claims (8)
1. detect a primer for IL28B and ITPA gene pleiomorphism simultaneously, it is characterized in that: comprise pcr amplification primer and SNaPshotPCR primer; Described pcr amplification primer comprises: for upstream primer 5 '-AGTAAGTCTTGTATTTCACCTCCTG-3 ' and the downstream primer 5 '-AAAGCCAGCTACCAAACTGTAT-3 ' of IL28B9917, for upstream primer 5 '-GTCGTGCCTGTCGTGTACT-3 ' and the downstream primer 5 '-TCAGGGTCAATCACAGAAGGG-3 ' of IL28B9860, for upstream primer 5 '-GGAGATGGGCAGCAGAGTTA-3 ' and the downstream primer 5 '-CAGGTCACAGGAAGACAGAGA-3 ' of ITPA; Described SNaPshotPCR primer comprises: for SNaPshotPCR the primer 5 '-CATGGTTCCAATTTGGGTGA-3 ' of IL28B9917, for SNaPshotPCR the primer 5 '-AAAAAAAAAAAAAATGCGGAGTGCAATTCAACCCTGGTTC-3 ' of IL28B9860, for SNaPshotPCR the primer 5 '-AAAAAAAAAAAAGATCGATCGAGAAATCCAACCATCTTTTAAAAA-3 ' of ITPA0101, for SNaPshotPCR the primer 5 '-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATTTTCTGTGCCACCAAAGTGCA TG-3 ' of ITPA7354.
2. the primer simultaneously detecting IL28B and ITPA gene pleiomorphism according to claim 1, it is characterized in that: in described pcr amplification primer, the primer concentration of IL28B9917, IL28B9860 and ITPA is than being 1:1:1, and in described SNaPshotPCR primer, the primer concentration of IL28B9917, IL28B9860, ITPA0101 and ITPA7354 is than being 1:1:1:1.
3. detect a method for IL28B and ITPA gene pleiomorphism simultaneously, it is characterized in that: comprise the steps:
S1 extracts DNA sample;
The DNA sample that S2 extracts with step S1, for template, adopts the pcr amplification primer described in claim 1 to carry out multiplexed PCR amplification, and carries out purifying to amplified production;
S3 for template with the pcr amplification product after step S2 purifying, adopts the SNaPshotPCR primer described in claim 1 to carry out SNaPshotPCR amplification, and carries out purifying to SNaPshotPCR amplified production;
S4 adopts capillary electrophoresis technique to detect, and analyzes, determine SNP site and genotype thereof to detected result.
4. the method simultaneously detecting IL28B and ITPA gene pleiomorphism according to claim 3, is characterized in that: the DNA sample in described step S1 is the DNA sample of EDTA anticoagulation cirumferential blood.
5. the method simultaneously detecting IL28B and ITPA gene pleiomorphism according to claim 3, is characterized in that: the multiplexed PCR amplification reaction conditions in described step S2 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.
6. the method simultaneously detecting IL28B and ITPA gene pleiomorphism according to claim 3, is characterized in that: the SNaPshotPCR amplification reaction condition in described step S3 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.
7. the method simultaneously detecting IL28B and ITPA gene pleiomorphism according to claim 3, is characterized in that: adopt GENEMAPPERIDV4.1 software to analyze detected result in described step S4.
8. the primer simultaneously detecting IL28B and ITPA gene pleiomorphism according to claim 1 detects the purposes in IL28B and ITPA gene pleiomorphism reagent in preparation.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994629A (en) * | 2011-09-15 | 2013-03-27 | 爱科来株式会社 | Method for detecting mutations at genes IL28B (RS8099917) and ITPA (RS1127354) |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994629A (en) * | 2011-09-15 | 2013-03-27 | 爱科来株式会社 | Method for detecting mutations at genes IL28B (RS8099917) and ITPA (RS1127354) |
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| 1000GENOMES: "ss228224675", 《DBSNP DATABASE》 * |
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| ABI: "ss44192664", 《DBSNP DATABASE》 * |
| BCM_SSAHASNP: "ss10963718", 《DBSNP DATABASE》 * |
| FRÉDÉGONDE ABOUT等: "Impact of IL28B, APOH and ITPA Polymorphisms on Efficacy and Safety of TVR-or BOC-Based Triple Therapy in Treatment-Experienced HCV-1 Patients with Compensated Cirrhosis from the ANRS CO20-CUPIC Study", 《PLOS ONE》 * |
| J.萨姆布鲁克等: "《分子克隆实验指南（第三版）上册》", 31 August 2002, 科学出版社 * |
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Application publication date: 20160420 |