CN105483279A - Rs 3909184 detection genotyping kit based on AllGlo probe and genotyping method thereof - Google Patents
Rs 3909184 detection genotyping kit based on AllGlo probe and genotyping method thereof Download PDFInfo
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- CN105483279A CN105483279A CN201610083676.0A CN201610083676A CN105483279A CN 105483279 A CN105483279 A CN 105483279A CN 201610083676 A CN201610083676 A CN 201610083676A CN 105483279 A CN105483279 A CN 105483279A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention relates to single nucleotide polymorphism, in particular to an rs 3909184 detection genotyping kit based on an AllGlo probe and a genotyping method thereof. The kit comprises a real-time fluorescence quantification PCR (polymerase chain reaction) reagent, positive control and negative control, and rs 3909184 is an SNP (single nucleotide polymorphism) locus serial number provided by NCBI(National Center of Biotechnology Information). The detection genotyping method includes the steps that DNA in an EDTA anticoagulant whole blood sample is extracted by adopting a conventional method; the real-time fluorescence quantification PCR reagent provided by the rs 3909184 detection genotyping kit based on the AllGlo probe is used for conducting real-time fluorescence quantification PCR amplification on DNA; the rs 3909184 SNP locus is genotyped according to detected fluorescence signals. On the premise of keeping high specificity and sensibility of the AllGlo probe, the detection price is lower than that of a direct sequencing method, the process is simpler, and consumed time is shorter.
Description
Technical field
The present invention relates to single nucleotide polymorphism (SNP), especially relate to a kind of rs3909184 detection and genotyping test kit based on AllGlo probe and classifying method thereof.
Background technology
Single nucleotide polymorphism (singlenucleotidepolymorphism, SNP), mainly refers to the DNA sequence polymorphism caused by the variation of single core thuja acid in genomic level.It is modal one in the heritable variation of the mankind.SNPs extensively exists in human genome, on average just has 1 in every 500 ~ 1000 base pairs, estimate its sum can reach 3,000,000 even more.As third generation genetic marker, SNP and the many phenotypic differences of human body, drug sensitivity and disease susceptibility closely related.The a large amount of existence of SNP in genome, become a strong instrument, in disease location with clone, the design of medicine and test and biological fundamental research, play very important effect.
Individual Diagnosis treatment is the general orientation of future medicine development.The target spot of individuation is positioned certain gene, or even single core thuja acid, find the genetic marker of disease, the different genetic backgrounds contributed to according to each patient are carried out disease prevention, Diagnosis and Treat.The SNP of gene is the important genetic base forming interindividual variation, by the correlation analysis of SNP and disease and pharmacological agent, make doctor not only can predict, determine the gene relevant with disease by detected SNP, also can hold the reaction characteristics of individual patients for certain medicine in advance simultaneously, and selecting result for the treatment of best according to this feature, the dangerous minimum drugs on patients such as untoward reactions precisely treat.
SNP plays important role in the prevention of disease, diagnosis, treatment and individuality medicine development, therefore carries out SNP detection and genotyping quickly and accurately and is significant.At present, the method for SNP somatotype mainly contains direct Sequencing technical process, restriction fragment length polymorphism technology (RFLP), Tagman fluorescence probe method, amplification refractory mutation system (ARMS), high-resolution fusion curve analytical method (HRM) etc.Wherein, direct sequencing is at present directly perceived, the SNP classifying method that accuracy is the highest comparatively speaking, but its step is many and disperse, and cost is higher, and workload is large, and the cycle is long, expensive, is not suitable for the detection of large sample multidigit point; Restriction fragment length polymorphism technology (RFLP) is as a kind of DNA marker technology the earliest, low to instrument requirements, only need a PCR instrument and electrophoresis apparatus to test, but its experiment complex operation, sense cycle is long, is not suitable for large sample and detects; The accuracy of Tagman fluorescence probe method is better, but its design and synthesis program is complicated, and is not suitable for the somatotype of a small amount of sample of multidigit point, the site that especially frequency is on the low side; Amplification refractory mutation system (ARMS method) is quick, easy, but can not operate by stopped pipe, is difficult to realize high-throughput, automatization to Polymorphism Analysis; High-resolution fusion curve analytical method (HRM) has the advantages such as simple, quick, but the method specificity is not enough, and apparatus selection is few.
AllGlo probe, as the fluorescent probe of a new generation, on the basis possessing TaqMan-MGB probe advantage, improves signal to noise ratio greatly, reduces background fluorescence signal.At present, AllGlo probe has been successfully applied to and has detected simplexvirus, hepatitis B virogene sudden change (Feng, Z.L.Yu, X.Y.Lu, Z.M.Geng, D.Y.Zhang, L.Chen, S.J.RapiddetectionofthehepatitisBvirusYMDDmutantusingAll Glo
tMprobes [J] .ClinChimActa, 2011,412 (11-12): 1018-1021), invasive aspergillosis (Wu, D.S.Shen, J.Z.Zhou, X.Q.Shen, S.F.Wu, X.M.TheestablishmentandevaluationofdiagnosticaccuracyofA llGlo (TM) probe-basedtechniquesforinvasiveaspergillosis [J] .ZhonghuaNeiKeZaZhi, 2010,49 (2): 142-145).
The research of medicine aspect finds, human leucocyte antigen-B*15:02 can increase the risk that carbamazepine medicine untoward reaction occurs significantly, cause fatal skin reaction, such as stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) (GroverS, KukretiR (2014) HLAallelesandhypersensitivitytocarbamazepine:anupdatedsy stematicreviewwithmeta-analysis.PharmacogenetGenomics24: 94 – 112.doi:10.1097/FPC.0000000000000021).Research shows: HLA-B*15:02 almost exists only in asian population (CaudleKE, RettieAE, Whirl-CarrilloM, SmithLH, MintzerS, LeeMT, KleinTE, CallaghanJT (2014) ClinicalPharmacogeneticsImplementationConsortiumguidelin esforCYP2C9andHLA-Bgenotypesandphenytoindosing.ClinPharm acolTher96:542 – 548.doi:10.1038/clpt.2014.159), in China, Thailand, the patient of the subregion 10%-15% such as Taiwan or more may carry HLA-B*15:02.HapMap shows, in Chinese han population, mononucleotide polymorphism site rs3909184 can as of a HLA-B*15:02 Tag SNP, utilize the detection and genotyping of rs3909184 effectively can differentiate HLA-B*15:02, patient HLA-B*15:02 is detected before medication, identify adverse drug reaction individuality, avoid severe drug toxic side effects, for clinical treatment medication provides important reference frame.Under the background that Individual Diagnosis is fast-developing with treatment, rs3909184 is as the Tag SNP of HLA-B*15:02, become with the close ties of carbamazepine medicine untoward reaction the SNP site that has medical science potentiality, be therefore badly in need of the development that a kind of SNP classifying method more rapidly and efficiently carrys out satisfied accurate medical science.
Summary of the invention
An object of the present invention is the above-mentioned defect existed for the test kit used in existing detection SNP method and detection method, provides the rs3909184 detection and genotyping test kit based on AllGlo probe.
Two of object of the present invention is to provide the rs3909184 detection and genotyping method based on AllGlo probe.
Described based on AllGlo probe rs3909184 detection and genotyping test kit, comprise real-time fluorescence quantitative PCR reagent, positive control and negative control;
The SNP site numbering that described rs3909184 provides for American National Biotechnology Information center (NCBI).
Described real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid, 10mmol
2, the TaqHotstartDNA polysaccharase of 5u/ μ L, 10mmoldNTPs mixed solution, 50 × LowROX, 100mL nuclease free water, the specific forward primer of 10 μm of ol/Lrs3909184, the specific reverse primer of 10 μm of ol/Lrs3909184 and AllGlo probe; Described 10 × Taq damping fluid comprises 100mmolTris-HCl and 500mmolKCl.
The nucleotides sequence of the specific forward primer of described rs3909184 is classified as:
SEQIDNO.1：5＇-CTCTGATGAGCCCCCGTTT-3＇；
The nucleotides sequence of the specific reverse primer of described rs3909184 is classified as:
SEQIDNO.2：5＇-ATGTGCTCAGTGCCCTCAAG-3＇。
Described AllGlo probe is the specific probe of rs3909184, and its nucleotides sequence is classified as:
SEQIDNO.3：rs3909184-C:MAR-CCACCTGTGCCC-MAR；
SEQIDNO.4：rs3909184-G:JUP-CCACGTGTGCCC-JUP。
Described positive control comprises: isozygoty reference substance 1, the positive of the positive is isozygotied reference substance 2, positive heterozygous control product; The described positive isozygoty reference substance 1 for rs3909184 somatotype be the DNA sample of GG, the described positive isozygoty reference substance 2 for rs3909184 somatotype be the DNA sample of CC, the DNA sample of described positive heterozygous control product to be rs3909184 somatotype be GC.
Described negative control is the nuclease free water of 1mL.
The described rs3909184 detection and genotyping method based on AllGlo probe, its step is as follows:
1) DNA in ordinary method extraction EDTA anticoagulated whole blood sample is adopted;
2) adopt the described real-time fluorescence quantitative PCR reagent provided based on the rs3909184 detection and genotyping test kit of AllGlo probe that DNA is carried out real-time fluorescence quantitative PCR amplification;
3) according to the fluorescent signal detected, somatotype is carried out to rs3909184SNP site.
Described AllGlo probe can adopt the fluorescent quantitation probe of A1leLogicBiosciencesCorporation company of the U.S., AllGlo probe has general T aqman, Taqman-MGB and all advantages of molecular beacon (molecularbeacon) probe, overcome the maximum drawback of this several probe at present, it has broken the restriction of reporter group one end, traditional Taqman one end quenching group, make use of the special fluorescence dye of the several frequently seen wavelength of A1leLogicBiosciencesCorporation company of U.S. development, to be marked at above oligonucleotide reporter group and essence each other to go out group, and above containing the special chemical group that can improve Tm value (annealing temperature), improve probe specificity, hybrid specificities improves greatly, after hybridization hydrolysis, the dyestuff of two ends mark all becomes reporter group again, improve fluorescence increment.
Described AllGlo probe has following advantage: 1. improve Tm value (can reach more than 10 DEG C), probe is shorter can reach 15 ~ 16 bases, can be suitable for A, the sequences Design probe that T comparision contents is high; 2. increase the selection of multiple fluorescence quantitative, because not only often kind of dyestuff is reporter group but also be quenching group, the Taqman probe that breaks traditions, because of wavelength reason Marker selection difficulty, does not limit by quenching group wavelength; 3. greatly improve signal to noise ratio, without background signal, space length is near, better quenching effects; 4. cost is lower, and price is only equivalent to the half of Taqman-MGB probe, has MGB probe and had superiority.
Present invention employs AllGlo probe technique, devise Auele Specific Primer and corresponding AllGlo probe, probe marks 2 kinds of special fluorophors (MAR, JUP) respectively, and this technology reaction conditions is optimized, establish a kind of AllGlo probe SNP detection and genotyping method, have a extensive future.
Beneficial effect of the present invention is as follows:
The invention provides a kind of SNP detection and genotyping test kit based on AllGlo probe and detection method, its specificity and susceptibility high, quick and precisely can carry out SNP somatotype, compared with existing common technique, there is following advantage:
1., compared with direct sequencing, AllGlo probe, under the prerequisite keeping high specific and susceptibility, detect cost ratio direct sequencing cheaper, and process is simpler and easy consuming time shorter.
2. compared with taqman-MGB probe, AllGlo probe adopts identical fluorophor, each other reporter group and quenching group, and the fluorescent signal of release is on the one hand stronger, and background signal is lower on the other hand; And synthesis program is simple, price is only equivalent to the half of Taqman-MGB.
3. compared with restriction fragment length polymorphism technology (RFLP), reaction times shortens by SNP classifying method based on AllGlo probe greatly, flux is larger, and sensitivity and specificity will apparently higher than the SNP classifying method based on restriction enzyme site.
4. compared with amplification refractory mutation system (ARMS), stronger based on AllGlo probe SNP classifying method detection sensitivity, can realize " stopped pipe operation ", effectively prevent outside contamination, there is good specificity and accuracy.
5. compared with high-resolution fusion curve analytical method (HRM), higher based on AllGlo probe SNP classifying method specificity, and multiple instrument can be applied AllGlo probe and carries out SNP detection and genotyping, and available instrumentation is selected many, do not need particular technology human users, range of application is more wide.
6. the present invention establishes the detection and genotyping method of carrying out rs3909184 based on AllGlo probe, and applicable crowd carries out the SNP detection and genotyping of individuation, has promoted the development of SNP at clinical drug research and personalized medicine.
Embodiment
Embodiment 1: the rs3909184 detection and genotyping test kit that the present invention is based on AllGlo probe comprises following component: real-time fluorescence quantitative PCR reagent.
1. real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid (10 × Taq damping fluid comprises 100mmolTris-HCl and 500mmolKCl), 10mmol
2, the TaqHotstartDNA polysaccharase of 5u/ μ L, 10mmoldNTPs mixed solution, 50 × LowROX, 100mL nuclease free water, the specific forward primer of 10 μm of ol/Lrs3909184, the specific reverse primer of 10 μm of ol/Lrs3909184 and AllGlo probe.
2. positive control comprises: isozygoty reference substance 1, the positive of the positive is isozygotied reference substance 2, positive heterozygous control product.The described positive isozygoty reference substance 1 for rs3909184 somatotype be the DNA sample of GG, the described positive isozygoty reference substance 2 for rs3909184 somatotype be the DNA sample of CC, the DNA sample of described positive heterozygous control product to be rs3909184 somatotype be GC.
3. negative control is the nuclease free water of 1mL.
Embodiment 2
The detection and genotyping of peripheral blood rs3909184, comprises the following steps:
1. collect EDTA anticoagulation cirumferential blood: extract fresh blood sample 2mL and be loaded on EDTA anticoagulant tube and packing multitube, often pipe packing 400 ~ 500 μ L, get 200 μ L for DNA extraction, all the other whole blood samples be placed in-80 DEG C frozen.
2. extract DNA: the poba gene group DNA extraction kit (DP348) adopting the production of Tian Gen bio tech ltd, sample (whole blood) DNA extraction is carried out according to process specifications, 200 μ LEDTA anticoagulated whole bloods operate to specifications and carry out, finally use the elution buffer TB dissolving DNA of 50 μ L, concentration and purity is measured respectively on nucleic acid spectrophotometric instrument NanoDrop2000, get purity A260/A280 ratio between 1.7 ~ 1.9, the DNA extracted getting concentration 10 ~ 50ng/ μ L detects for SNP somatotype, remaining DNA is all frozen in-80 DEG C.
3. real-time fluorescence quantitative PCR amplification:
Each component of quantitative fluorescent PCR is placed in room-temperature dissolution by 3.1, and mixing is placed on ice.
3.2 preparation PCR reaction mixtures are as table 1.
Table 1
3.3 in 8 unions packing prepare PCR reaction mixture, often pipe adds the DNA of 1 μ L, fully mixes, and whole process is carried out on ice, avoids the generation of bubble.Each Setup Experiments negative control, arranges a positive and to isozygoty reference substance 1, and a positive is isozygotied reference substance 2 and positive heterozygous control product.
3.4 detections are carried out on real-time fluorescence quantitative PCR instrument, can use ABI7300, the multiple instruments such as 7500 (AppliedBiosystems companies of the U.S.).
3.5 arrange quantitative fluorescent PCR reaction conditions as table 2.
Table 2
4. interpretation of result and process: apply ABI instrument and carry software and carry out interpretation of result.According to contrast amplification, somatotype produces three kinds of genotype:
The first genotype: GG, contrasts on the same axis with GG type people's gene;
The second genotype: GC, contrasts on the same axis with GC type people's gene;
The third genotype: CC, contrasts on the same axis with CC type people's gene.
Embodiment 3. is organized or cell sample SNP detection and genotyping
1. sample preparation:
A. organize: animal tissues's (spleen organizes consumption to lack 10mg) should smash and be treated to cell suspension, then 10000rpm (~ 11200 × g) centrifugal 1min, use up supernatant, add 200ul damping fluid GA (sky root DNA extraction kit DP304), concussion is to thoroughly suspending;
B. the cell of adherent culture first should be treated to cell suspension, then 10000rpm (~ 11200 × g) centrifugal 1min, uses up supernatant, adds 200 μ L damping fluid GA (sky root DNA extraction kit DP304), and concussion is to thoroughly suspending;
2. organize, cell DNA extracts and enrichment:
Adopt the DNA extraction kit (DP304) of the production of Tian Gen bio tech ltd, carry out organizing or cell DNA extraction according to process specifications, finally use the elution buffer TE dissolving DNA of 50 μ L, on nucleic acid spectrophotometric instrument NanoDrop2000, measure concentration and purity respectively;
3. organize, the SNP detection and genotyping of cell sample.
Subsequent step is with reference to step 3 ~ 4 of embodiment 2.
Can range of application: the detection and genotyping that can be applicable to human tissue sample and hemocyte rs3909184 based on the rs3909184 detection and genotyping test kit of AllGlo probe and detection method thereof, above display and describe ultimate principle of the present invention, principal character and advantage of the present invention.
Claims (8)
1., based on the rs3909184 detection and genotyping test kit of AllGlo probe, it is characterized in that comprising real-time fluorescence quantitative PCR reagent, positive control and negative control;
The SNP site numbering that described rs3909184 provides for American National Biotechnology Information center.
2., if claim 1 is based on the rs3909184 detection and genotyping test kit of AllGlo probe, it is characterized in that described real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid, 10mmol
2, the TaqHotstartDNA polysaccharase of 5u/ μ L, 10mmoldNTPs mixed solution, 50 × LowROX, 100mL nuclease free water, the specific forward primer of 10 μm of ol/Lrs3909184, the specific reverse primer of 10 μm of ol/Lrs3909184 and AllGlo probe; Described 10 × Taq damping fluid comprises 100mmolTris-HCl and 500mmolKCl.
3., if claim 1 is based on the rs3909184 detection and genotyping test kit of AllGlo probe, it is characterized in that the nucleotides sequence of the specific forward primer of described rs3909184 is classified as:
SEQIDNO.1：5＇-CTCTGATGAGCCCCCGTTT-3＇；
The nucleotides sequence of the specific reverse primer of described rs3909184 is classified as:
SEQIDNO.2：5＇-ATGTGCTCAGTGCCCTCAAG-3＇。
4., if claim 1 is based on the rs3909184 detection and genotyping test kit of AllGlo probe, it is characterized in that described AllGlo probe is the specific probe of rs3909184, its nucleotides sequence is classified as:
SEQIDNO.3：rs3909184-C:MAR-CCACCTGTGCCC-MAR；
SEQIDNO.4：rs3909184-G:JUP-CCACGTGTGCCC-JUP。
5. if claim 1 is based on the rs3909184 detection and genotyping test kit of AllGlo probe, it is characterized in that described positive control comprises: isozygoty reference substance 1, the positive of the positive is isozygotied reference substance 2, positive heterozygous control product; The described positive isozygoty reference substance 1 for rs3909184 somatotype be the DNA sample of GG, the described positive isozygoty reference substance 2 for rs3909184 somatotype be the DNA sample of CC, the DNA sample of described positive heterozygous control product to be rs3909184 somatotype be GC.
6., if claim 1 is based on the rs3909184 detection and genotyping test kit of AllGlo probe, it is characterized in that described negative control is the nuclease free water of 1mL.
7., based on the rs3909184 detection and genotyping method of AllGlo probe, it is characterized in that its step is as follows:
1) DNA in ordinary method extraction EDTA anticoagulated whole blood sample is adopted;
2) adopt the real-time fluorescence quantitative PCR reagent provided as the rs3909184 detection and genotyping test kit based on AllGlo probe as described in arbitrary in claim 1 ~ 6 that DNA is carried out real-time fluorescence quantitative PCR amplification;
3) according to the fluorescent signal detected, somatotype is carried out to rs3909184SNP site.
8., if claim 1 is based on the rs3909184 detection and genotyping test kit of AllGlo probe, it is characterized in that described AllGlo probe adopts the fluorescent quantitation probe of A1leLogicBiosciencesCorporation company of the U.S..
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111304321A (en) * | 2020-04-17 | 2020-06-19 | 浙江迪谱诊断技术有限公司 | Primer combination sequence and kit for detecting child safety medication related gene mutation site |
| CN112501283A (en) * | 2020-12-29 | 2021-03-16 | 广东南芯医疗科技有限公司 | Guiding method and kit for carbamazepine personalized medicine gene |
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2016
- 2016-02-06 CN CN201610083676.0A patent/CN105483279A/en active Pending
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| CLARK ET AL.: "Accuracy of SNPs to predict risk of HLA alleles associated with drug-induced hypersensitivity events across racial groups", 《PHARMACOGENOMICS》 * |
| 俞立强: "HLA_B*1502/5801快速检测方法研究及苏南地区分布频率分析", 《苏州大学硕士学位论文》 * |
| 潘云燕: ""基于AllgloTM探针检测IL-1核苷酸多态性检测方法的建立", 《兰州大学硕士学位论文》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111304321A (en) * | 2020-04-17 | 2020-06-19 | 浙江迪谱诊断技术有限公司 | Primer combination sequence and kit for detecting child safety medication related gene mutation site |
| CN111304321B (en) * | 2020-04-17 | 2024-02-06 | 浙江迪谱诊断技术有限公司 | Primer combination sequence and kit for detecting mutation sites of children safety medication related genes |
| CN112501283A (en) * | 2020-12-29 | 2021-03-16 | 广东南芯医疗科技有限公司 | Guiding method and kit for carbamazepine personalized medicine gene |
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Application publication date: 20160413 |