CN106520950A - UGT1A1 gene polymorphism detection primer and probe and kit - Google Patents
UGT1A1 gene polymorphism detection primer and probe and kit Download PDFInfo
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- CN106520950A CN106520950A CN201611008201.1A CN201611008201A CN106520950A CN 106520950 A CN106520950 A CN 106520950A CN 201611008201 A CN201611008201 A CN 201611008201A CN 106520950 A CN106520950 A CN 106520950A
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a UGT1A1 gene polymorphism detection primer and probe and a kit. The detection primer and probe comprise a primer body and a probe body for detecting the UGT1A1*6SNP locus gene polymorphism and a primer body and a probe body for detecting UGT1A1*28SNP locus gene polymorphism. On the basis of the Taqman hydrolysis probe fluorogenic quantitative PCR method, high specificity is achieved, and the detection result coincides with the sequencing method by 100%; and various genotypes can be accurately distinguished, and the result is obvious and easy to indentify. According to the primer and probe sequence and the kit prepared through the primer and probe, the genome DNA lower to 1ng can be accurately detected, and sensitivity is high. According to the kit and a detection method, PCR subsequent operation is not needed, the pollution risk is reduced, meanwhile, detection can be finished within 90 min, the needed time is far shorter than that of the sequencing method, and the UGT1A1 gene polymorphism detection primer and probe and the kit are particularly suitable for the clinical medication diagnosis.
Description
Technical field
The present invention relates to technical field of gene detection, and in particular to UGT1A1 genetic polymorphism detection primers, probe and examination
Agent box.
Background technology
Irinotecan (irinotecan, CPT-11) is camptothecine semi-synthetic derivative, and it is to treat transitivity knot at present
One of maximally effective chemotherapeutics of rectal cancer.Multinomial clinical research shows that the chemotherapy regimen based on irinotecan significantly can be carried
High patient is without progression of disease life span and total life span.But irinotecan can cause the blood system of upper frequency and digestion
System untoward reaction.Late-onset diarrhea and Neutrophilic granulocytopenia are the common untoward reaction caused by irinotecan, clinical
Incidence rate is respectively 46% and 30%, can cause death when serious.Irinotecan main metabolic position in vivo is liver,
After intravenous administration, Jing carboxy-lesterases hydrolysis are SN38 (7-ethyl- to irinotecan in vivo
10-hydroxy-camptothecin, SN-38), SN-38 is DNA typeⅠtopoisomerase inhibitor, acts on the cell cycle S phases,
Suppress the reparation after DNA single-strand breaks, interference DNA replication dna and transcription, so as to play cellulotoxic effect.
In recent years, be improve irinotecan clinical practice safety and effectiveness, Chinese scholars to irinotecan not
The influence factor of good reaction has carried out numerous studies, and correlational study result shows that the polymorphism of drug metabolism related gene is to cause
One of major reason of individual untoward reaction difference.UGTlA1 is the key enzyme of irinotecan activated product metabolism, and its gene is more
State property can make the activity of enzyme change.SN-38 Jing uridine diphosphate glucuronatetransferases family (uridine
Diphosphate glucuronosyltransferases, UGTs) inactivation for after glucal acid product SN-38G, Jing bile
Intestinal is discharged into, and SN-38 is converted under the effect of intestinal bacteria GRD beta-glucuronidase, is caused intestinal mucosa injury and Delayed onset
Diarrhoea；And the UGTs enzymes in intestinal can be catalyzed SN-38 once again and be detoxified for SN-38G, the expression of UGTs enzymes and its activity with
The curative effect of irinotecan and untoward reaction are closely related.
Clinical research both at home and abroad shows that in the patient using irinotecan UGT1A1 gene promoter areas TATA repeats sequence
The change of row is that the heterozygous mutant TA6/7 and homozygous mutant TA7/7 of UGT1A1*28 dramatically increases the serious grain of patient's generation
The risk of Leukopenia and diarrhoea.In Chinese Han Population, UGT1A1*28 polymorphisms occurrence frequency about 15%～30%.In addition, near
The research in year shows the incidence rate up to 13%～23% that UGT1A1*6 is mutated in asian population, is controlled using irinotecan
In the patient for the treatment of, UGT1A1*6 heterozygous mutants G/A and homozygous mutant A/A dramatically increase patient that 3～4 grades of neutral grains occur are thin
The risk of born of the same parents' reduction, thrombocytopenia and diarrhoea.Therefore, comprehensive detection UGT1A1*6 and UGT1A1*28 single nucleotide polymorphisms
The generation of patient's irinotecan related reactions can be predicted, this is important for the formulation of clinical individual chemotherapy regimen has
Meaning.
At present, the method for UGT1A1 genetic polymorphism detections has：Sequencing, chip method, fluorescence quantitative PCR method.Sequencing
It is the modal method of genetic polymorphism detection, and the goldstandard of gene polymorphism sites detection, although the method has height
Flux, the advantage in many sites, but the method is simultaneously present, and cumbersome, sensitivity is low, sample easily pollutes and instrument cost height etc.
Problem.Chip method needs advanced performing PCR amplification, afterwards by the PCR primer containing purposeful SNP site and mutant probe, wild probe
Hybridization, by comparing the signal intensity judgement sample genotype of two probe hybridization.The method operating process is loaded down with trivial details, it is impossible to realize
Mass and automatization, and interpretation is as a result difficult, false positive probability is high.Fluorescence quantitative PCR method is different according to its principle and can divide
For mutation amplification retarding system (amplification refractory mutation system, ARMS-PCR), Taqman
Sonde method and high-resolution fusion curve (high-resolutionmelt, HRM) method.Fluorescence quantitative PCR method have sensitivity it is high,
The easy interpretation of simple to operate, result, the advantages of the test period is short, and without the need for the subsequent analysis process such as enzyme action, electrophoresis after PCR terminates,
Reduce the risk of cross-contamination.At present, the method for UGT1A1 genetic polymorphism detections is based on sequencing, and high sequencing
It is commonly used in clinical diagnosises that instrument cost limits the method, and quantitative real time PCR Instrument is higher in each hospital's popularity rate, has
UGT1A1 genetic polymorphism detection test kit and method of the necessary exploitation based on quantitative fluorescent PCR, quickly divide for clinical application
Son diagnosis..
The content of the invention
In order to solve for the above-mentioned technical problem of detection UGT1A1 gene pleiomorphisms in prior art, the invention provides
One group be applied to fluorescence quantitative PCR detection said gene polymorphism primer and probe, specificity and sensitivity it is very good, can
The genomic DNA sample of detection as little as 1ng.
UGT1A1 genetic polymorphism detections primer and probe that the present invention is provided, including detection UGT1A1*6SNP sites base
Because of the primer and probe of polymorphism, and the primer and probe of detection UGT1A1*28SNP single nucleotide polymorphisms, nucleotides sequence
Row are as follows：
The primer and probe of detection UGT1A1*6SNP single nucleotide polymorphisms：
Mutant allele forward primer (1M-F)：5'-CCTCGTTGTACATCAGAGCAA-3'(SEQ ID
NO.1),
Wild-type allele forward primer (1W-F)：5'-CCTCGTTGTACATCAGAGCAG-3'(SEQ ID
NO.2),
Allele general reverse primer (1C-R)：5'-AAACATTATGCCCGAGACTA-3'(SEQ ID NO.3),
Specific probe (1P)：FAM 5'-CCTTGAAGACGTACCCTGTGCCA NFQ-MGB(SEQ ID NO.4)；
The primer and probe of detection UGT1A1*28SNP single nucleotide polymorphisms：
Forward primer (2-F)：5'-CCTGCTACCTTTGTGGACTG-3'(SEQ ID NO.5),
Downstream primer (2-R)：5'-TTGCTCCTGCCAGAGGTTCG-3'(SEQ ID NO.6),
Saltant type probe (2P-M)：FAM5'-GCCATATATATATATATATAAGTAGGAGA-3'(SEQ ID NO.7)；
Wild-type probe (2P-W)：FAM5'-TGCCATATATATATATATAAGTAGGAGAG-3'(SEQ ID NO.8)；
Taqman probes two ends are respectively provided with fluorophor and fluorescent quenching group, in normal state, glimmering on probe
Light is quenched, and does not send fluorescence.After proceeding by PCR amplifications, probe is separated with fluorophor by PCR enzyme hydrolysiss, quenching group,
There is fluorescence signal to discharge.The quenching group of MGB probes adopts non-fluorescence quenching group (Non-Fluorescent
Quencher, NFQ), itself does not produce fluorescence, can substantially reduce the intensity of background signal.MGB is also associated with probe simultaneously
The Tm values of probe can be improved 10 DEG C or so by (Minor Groove Binder) modification group.
Present invention also offers a kind of UGT1A1 genetic polymorphism detections test kit, including above-described UGT1A1 genes
Polymorphic detection primer and probe.
Preferably, above-mentioned UGT1A1 genetic polymorphism detections test kit, also including internal control gene GAPDH detection primer and
Probe：
Internal control forward primer (R-F)：5'-CAACAATAGCGAACCTCCATC-3'(SEQ ID NO.9),
Internal control downstream primer (R-R)：5'-CTCAACGCAGTTCAGTTAGGC-3'(SEQ ID NO.10),
Internal control probe (RP)：JOE5'-CGCTCTTTAAAATGCAAGGCTT-3'(SEQ ID NO.11)；
Preferably, above-mentioned UGT1A1 genetic polymorphism detections test kit, also comprising positive control and blank.
Preferably, above-mentioned UGT1A1 genetic polymorphism detections test kit, also including TaqmanMasterMix and ddH2O。
TaqmanMasterMix full name TaqMan Real-Time PCRMasterMixes, purchased from Thermo Fisher
Scientific (Thermo Fischer Scient Inc.).
Present invention also offers the using method of the UGT1A1 genetic polymorphism detection test kits described in any of the above, step
It is as follows：
(1) extraction of sample to be tested genomic DNA；
(2) PCR reactant liquors are prepared：Each PCR reaction systems are prepared, and saltant type detection system are prepared respectively per pleomorphism site
With wild type detection system；The same samples of 2.0 μ L are added in saltant type detection system and wild type detection system PCR pipe respectively
DNA, of short duration centrifugation, mix homogeneously；
(3) fluorescence quantitative PCR detection：The PCR system for preparing is put in fluorescent PCR instrument, quantitative fluorescent PCR is carried out
Augmentation detection；
(4) result interpretation：According to the poor △ Ct of saltant type detection system Ct value and wild type detection system Ct value to sample
Genotype carry out interpretation, if △ Ct<- 3, then the sample gene locis genotype is saltant type；If -3≤△ Ct≤3,
The sample gene locis genotype is heterozygous；If △ is Ct>3, then the sample gene locis genotype is wild type.
Preferably, the using method of above-mentioned UGT1A1 genetic polymorphism detections test kit, fluorescent quantitative PCR program is such as
Under：
First stage：95℃5min；
Second stage：95 DEG C of 20s, 58 DEG C of 30s, circulate 10 times；
Phase III：95 DEG C of 20s, 58 DEG C of 30s, circulate 35 times；
Fluorescence signal is collected during 58 DEG C of extensions of phase III.
The invention has the advantages that：
Primer and probe sequence sensitivity are high, can accurately detect the as little as genomic DNA of 1ng, and gene pleiomorphism is examined
The goldstandard Sanger sequencing of survey requires higher DNA concentration and volume, is unfavorable for that subsequent experimental is carried out；High specificity,
Primer provided by the present invention can only specific amplification target fragment, without non-specific amplification, at the same using Taqman hydrolysis visit
Pin improves the specificity of fluorescent PCR, and detection method provided by the present invention is coincide with sequencing 100%；The inventive method energy
Various genotype, and visual result, easy interpretation is enough accurately distinguished, and sequencing is due to particularity, the design of primers of gene structure
Etc. factor, easily there is sequencing result set peak, affect result interpretation；Detection method provided by the present invention can be complete in 90 minutes
Into detection, required time is far below sequencing, is particularly suitable for clinical application diagnosis；Test kit of the present invention and detection method are applied to
The quantitative real time PCR Instruments such as ABI7500, Roche LC480, Bio-Rad CFX96, the suitability are good.
Description of the drawings
Fig. 1 is UGT1A1*6G/UGT1A1*6G genotype sample amplification curves；
Fig. 2 is UGT1A1*6A/UGT1A1*6A genotype sample amplification curves；
Fig. 3 is UGT1A1*6G/UGT1A1*6A genotype sample amplification curves；
Fig. 4 is 6 genotype sample amplification curves of UGT1A1*28 (TA) 6/ (TA)；
Fig. 5 is 7 genotype sample amplification curves of UGT1A1*28 (TA) 7/ (TA)；
Fig. 6 is 7 genotype sample amplification curves of UGT1A1*28 (TA) 6/ (TA).
Specific embodiment
For may be better understood professional and technical personnel in the field and implementing the present invention, with reference to specific embodiment pair
The present invention is described further, but protection scope of the present invention is not limited to following embodiments.If no special instructions, in embodiment
Reagent used and biomaterial are commercial sources purchase, and the technological means for using are this area routine techniquess means.
1 mankind's UGT1A1 genetic polymorphism detections of embodiment, mainly include：
1st, the design and synthesis of primer and probe
According to NCBI dbSNP data base (https://www.ncbi.nlm.nih.gov/SNP/) UGT1A1*6 that announces
With UGT1A1*28 sequence informations, detection primer and the probe for the two pleomorphism sites is separately designed, obtained through screening
Primer and probe sequence are specific as follows：
(1) it is used for the primer and probe of the detection of UGT1A1*6SNP single nucleotide polymorphisms：
Mutant allele specific PCR forward primer (1M-F)：
5'-CCTCGTTGTACATCAGAGCAA-3',
Wild-type allele specific PCR forward primer (1W-F)：
5'-CCTCGTTGTACATCAGAGCAG-3',
ApoE gene general reverse primer (1C-R)：
5'-AAACATTATGCCCGAGACTA-3',
Specific probe (1P)：FAM 5'-CCTTGAAGACGTACCCTGTGCCANFQ-MGB.(2) it is used for UGT1A1*
The primer and probe of 28SNP single nucleotide polymorphisms detection：
Forward primer (2-F)：5'-CCTGCTACCTTTGTGGACTG-3',
Downstream primer (2-R)：5'-TTGCTCCTGCCAGAGGTTCG-3',
Saltant type probe (2P-M)：FAM5'-GCCATATATATATATATATAAGTAGGAGA-3',
Wild-type probe (2P-W)：FAM5'-TGCCATATATATATATATAAGTAGGAGAG-3'.
For monitoring the amplification efficiency of the effectiveness and DNA profiling of PCR reaction systems, according to mankind house-keeping gene GAPDH's
Sequential design a pair of internal control primers and probe, particular sequence are as follows：
Internal control forward primer (R-F)：5'-CAACAATAGCGAACCTCCATC-3',
Internal control downstream primer (R-R)：5'-CTCAACGCAGTTCAGTTAGGC-3',
Internal control probe (RP)：JOE5'-CGCTCTTTAAAATGCAAGGCTT-3'.
Primer Synesis Company is transferred to synthesize the primer for designing and probe sequence.
2nd, the extraction of clinical sample DNA
With reference to the poba gene group DNA extraction kit explanation of Tiangeng biochemical technology company limited, sample gene group is obtained
DNA.DNA extraction advises carrying out concentration mensuration, sample with 2000 ultramicrospectrophotometers of Nanodrop to sample DNA after finishing
This DNA concentration should be advisable in 20ng/ μ L～200ng/ μ L, and OD260/OD280 is between 1.65～1.80.
3rd, PCR reaction systems are prepared
Each polymorphic detection site includes saltant type detection system and wild type detection system (being shown in Table 1), the above respectively
Detection system is dispensed in 2 PCR reaction tubes after the completion of preparing respectively, is separately added into 2.0 μ L to more than in 2 PCR reaction tubes
Same sample DNA, the of short duration centrifugation several seconds, makes PCR reaction system mix homogeneously.
1 quantitative fluorescent PCR reaction system of table
4th, fluorescent PCR detection
The PCR system for having configured is put in ABI7500 fluorescent PCR instruments, fluorescent PCR augmentation detection is carried out.Reaction bar
Part is：
First stage：95 DEG C of denaturations 5min；
Second stage：95 DEG C of 20s, 58 DEG C of 30s, 10 circulations；
Phase III:95 DEG C of 20s, 58 DEG C of 30s (collecting FAM and JOE signals), 35 circulations.
After PCR EP (end of program), baseline and threshold line are set automatically, the Ct values of sample are obtained.
5th, result interpretation
NTC and positive control analysis：NTC reacting holes otherwise, should illustrate possibility without amplification curve and Ct values under normal circumstances
There is operational pollution, now, it is proposed that behind the source that decontaminates, restart experiment；Positive control reacting hole should have typically " S "
Type amplification curve, and Ct value≤30, otherwise, illustrate that PCR reaction systems are likely to occur exception, have impact on amplification efficiency.
Internal control is analyzed：Usually, internal control JOE signals should have typically " S " type amplification curve, and Ct values 12～30 it
Between.If Ct value≤12, show added sample DNA excessive concentration, DNA applied sample amounts should be reduced as one sees fit；If Ct >=30, show to be loaded
This DNA copy number is low or sample DNA in there may be PCR inhibitor；If internal control JOE signals are without amplification curve, Ke Nengcun
Add in sample DNA leakage, it is proposed that restart experiment.
Sample gene type assay：Under the premise of NTC (negative control), positive control and internal control JOE signals are normal,
According to the difference (△ Ct) of saltant type detection system Ct value and wild type detection system Ct value the genotype of sample is carried out interpretation (see
Table is 2).For any one polymorphic detection site, if △ is Ct<- 3, then the sample gene locis genotype is saltant type；
If -3≤△ Ct >=3, the sample gene locis genotype is heterozygous；If △ is Ct>3, then sample gene locis base
Because type is wild type.
2 UGT1A1 genes of table, two pleomorphism site genotype results judge
Embodiment described above is only for absolutely proving the present invention and the preferred embodiment lifted, the protection model of the present invention
Enclose not limited to this.Equivalent substitute or conversion that those skilled in the art are made on the basis of the present invention, in the present invention
Protection domain within.Protection scope of the present invention is defined by claims.
SEQUENCE LISTING
<110>Wuhan Hygiea Bioscience Co., Ltd.
<120>UGT1A1 genetic polymorphism detection primers, probe and test kit
<130> WH1610020-1
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence (1M-F)
<400> 1
cctcgttgta catcagagca a 21
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence (1W-F)
<400> 2
cctcgttgta catcagagca g 21
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (1C-R)
<400> 3
aaacattatg cccgagacta 20
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence (1P)
<400> 4
ccttgaagac gtaccctgtg cca 23
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (2-F)
<400> 5
cctgctacct ttgtggactg 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence (2-R)
<400> 6
ttgctcctgc cagaggttcg 20
<210> 7
<211> 29
<212> DNA
<213>Artificial sequence (2P-M)
<400> 7
gccatatata tatatatata agtaggaga 29
<210> 8
<211> 29
<212> DNA
<213>Artificial sequence (2P-W)
<400> 8
tgccatatat atatatataa gtaggagag 29
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence (R-F)
<400> 9
caacaatagc gaacctccat c 21
<210> 10
<211> 21
<212> DNA
<213>Artificial sequence (R-R)
<400> 10
ctcaacgcag ttcagttagg c 21
<210> 11
<211> 22
<212> DNA
<213>Artificial sequence (RP)
<400> 11
cgctctttaa aatgcaaggc tt 22
Claims (7)
1.UGT1A1 genetic polymorphism detections primer and probe, including the primer of detection UGT1A1*6SNP single nucleotide polymorphisms
And probe, and the primer and probe of detection UGT1A1*28SNP single nucleotide polymorphisms, it is characterised in that nucleotide sequence is such as
Under：
The primer and probe of detection UGT1A1*6SNP single nucleotide polymorphisms：
Mutant allele forward primer (1M-F)：5'-CCTCGTTGTACATCAGAGCAA-3',
Wild-type allele forward primer (1W-F)：5'-CCTCGTTGTACATCAGAGCAG-3',
Allele general reverse primer (1C-R)：5'-AAACATTATGCCCGAGACTA-3',
Specific probe (1P)：FAM 5'-CCTTGAAGACGTACCCTGTGCCANFQ-MGB；
The primer and probe of detection UGT1A1*28SNP single nucleotide polymorphisms：
Forward primer (2-F)：5'-CCTGCTACCTTTGTGGACTG-3',
Downstream primer (2-R)：5'-TTGCTCCTGCCAGAGGTTCG-3',
Saltant type probe (2P-M)：FAM5'-GCCATATATATATATATATAAGTAGGAGA-3'；
Wild-type probe (2P-W)：FAM5'-TGCCATATATATATATATAAGTAGGAGAG-3'.
2.UGT1A1 genetic polymorphism detection test kits, it is characterised in that including the UGT1A1 gene polymorphics described in claim 1
Property detection primer and probe.
3. UGT1A1 genetic polymorphism detections test kit according to claim 2, it is characterised in that also including internal control gene
The detection primer of GAPDH and probe：
Internal control forward primer (R-F)：5'-CAACAATAGCGAACCTCCATC-3',
Internal control downstream primer (R-R)：5'-CTCAACGCAGTTCAGTTAGGC-3',
Internal control probe (RP)：JOE5'-CGCTCTTTAAAATGCAAGGCTT-3'.
4. UGT1A1 genetic polymorphism detections test kit according to claim 3, it is characterised in that also comprising positive control
And blank.
5. UGT1A1 genetic polymorphism detections test kit according to claim 3, it is characterised in that also including Taqman
Master Mix and ddH2O。
6. the using method of the arbitrary described UGT1A1 genetic polymorphism detection test kits of claim 3-5, it is characterised in that step
It is rapid as follows：
(1) extraction of sample to be tested genomic DNA；
(2) PCR reactant liquors are prepared：Each PCR reaction systems are prepared, and saltant type detection system and open country are prepared respectively per pleomorphism site
Raw type detection system；The same sample DNAs of 2.0 μ L are added in saltant type detection system and wild type detection system PCR pipe respectively,
Of short duration centrifugation, mix homogeneously；
(3) fluorescence quantitative PCR detection：The PCR system for preparing is put in fluorescent PCR instrument, fluorescent quantitative PCR is carried out
Detection；
(4) result interpretation：Base according to the poor △ Ct of saltant type detection system Ct value and wild type detection system Ct value to sample
Because type carries out interpretation, if △ is Ct<- 3, then the sample gene locis genotype is saltant type；If -3≤△ Ct≤3, the sample
Should gene locis genotype be heterozygous；If △ is Ct>3, then the sample gene locis genotype is wild type.
7. the using method of UGT1A1 genetic polymorphism detections test kit according to claim 6, it is characterised in that fluorescence
Quantitative pcr amplification program is as follows：
First stage：95℃5min；
Second stage：95 DEG C of 20s, 58 DEG C of 30s, circulate 10 times；
Phase III：95 DEG C of 20s, 58 DEG C of 30s, circulate 35 times；
Fluorescence signal is collected during 58 DEG C of extensions of phase III.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107299136A (en) * | 2017-07-06 | 2017-10-27 | 广州海思医疗科技有限公司 | It is a kind of at the same detect NUDT15 and UGT1A1 gene multisite mutations kit |
| CN107299137A (en) * | 2017-07-06 | 2017-10-27 | 广州海思医疗科技有限公司 | The NUDT15 primers and probe of one group of high specific |
| CN107868824A (en) * | 2017-11-10 | 2018-04-03 | 广州金域医学检验集团股份有限公司 | Single base extension method detects primer system, the methods and applications of UGT1A1*6 gene pleiomorphisms |
| CN109371127A (en) * | 2018-10-22 | 2019-02-22 | 江苏美因康生物科技有限公司 | The kit and method of a kind of while quick detection UGT1A1*6 type and UGT1A1*28 type gene pleiomorphism |
| CN109504747A (en) * | 2017-09-15 | 2019-03-22 | 益善生物技术股份有限公司 | HCCSP T1A1 genetic polymorphism detection kit based on Taqman-MGB probe |
| CN112592970A (en) * | 2020-11-24 | 2021-04-02 | 首都医科大学附属北京友谊医院 | Gilbert syndrome UGT1A1 gene multi-site variation detection kit |
| WO2023030162A1 (en) * | 2021-09-01 | 2023-03-09 | 上海市儿童医院 | Extended probe-type qpcr detection method for snv |
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| CN107299136A (en) * | 2017-07-06 | 2017-10-27 | 广州海思医疗科技有限公司 | It is a kind of at the same detect NUDT15 and UGT1A1 gene multisite mutations kit |
| CN107299137A (en) * | 2017-07-06 | 2017-10-27 | 广州海思医疗科技有限公司 | The NUDT15 primers and probe of one group of high specific |
| CN109504747A (en) * | 2017-09-15 | 2019-03-22 | 益善生物技术股份有限公司 | HCCSP T1A1 genetic polymorphism detection kit based on Taqman-MGB probe |
| CN107868824A (en) * | 2017-11-10 | 2018-04-03 | 广州金域医学检验集团股份有限公司 | Single base extension method detects primer system, the methods and applications of UGT1A1*6 gene pleiomorphisms |
| CN109371127A (en) * | 2018-10-22 | 2019-02-22 | 江苏美因康生物科技有限公司 | The kit and method of a kind of while quick detection UGT1A1*6 type and UGT1A1*28 type gene pleiomorphism |
| CN112592970A (en) * | 2020-11-24 | 2021-04-02 | 首都医科大学附属北京友谊医院 | Gilbert syndrome UGT1A1 gene multi-site variation detection kit |
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