CN106399565A - Rs12979860 genotyping dual-color fluorescent PCR rapid detection kit - Google Patents

Rs12979860 genotyping dual-color fluorescent PCR rapid detection kit Download PDF

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Publication number
CN106399565A
CN106399565A CN201611039569.4A CN201611039569A CN106399565A CN 106399565 A CN106399565 A CN 106399565A CN 201611039569 A CN201611039569 A CN 201611039569A CN 106399565 A CN106399565 A CN 106399565A
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China
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fluorescence
pcr
probe
genotyping
kit
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CN201611039569.4A
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Chinese (zh)
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赵友云
孙莉军
郑毅
王业富
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Wuhan University WHU
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Wuhan University WHU
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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
    • 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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Abstract

The invention discloses an rs12979860 locus genotyping dual-color fluorescent PCR rapid detection kit. According to the kit, a primer and a TaqMan-MGB probe are redesigned, a reaction system is optimized, bi-component hot-start DNA polymerase forms an enzyme activity automatic regulation system, ROX Reference Dye can eliminate a signal background and correct fluorescence signal errors between holes, therefore, the kit achieves accuracy, high amplification efficiency, high sensitivity, good specificity, good repeatability, easy and convenient operation and shorter detection time, and the technology can be applied and popularized clinically.

Description

A kind of rs12979860 Genotyping two-color fluorescence PCR quick detection kit
Technical field
The present invention relates to biological technical field, it is based on rs12979860 Genotyping Two Colour Fluorescence in particular to a kind of PCR quick detection kit.
Background technology
Hepatitis, are a kind of virus hepatitis being caused by HCV (HCV) infection, mainly through blood transfusion, acupuncture, suction Poison etc. is propagated.Show the rs12979860 loci polymorphism of mankind's IL-28B gene and the spontaneous removing of HCV in existing research, resist The curative effect of viral therapy has correlation.
At present, the discrimination method of rs12979860 single nucleotide polymorphisms have multiple, mainly adopt gene sequencing, gene core The methods such as piece, high-resolution solubility curve (HRM) and digestion, but majority operation is numerous and diverse, it is longer to take, required instrument and equipment is held high Expensive, specificity poor it is difficult to general hospital carry out.
Content of the invention
It is an object of the invention to overcoming the defect of prior art, provide a kind of IL28B gene rs12979860 site base Because of parting two-color fluorescence PCR quick detection kit, this kit has high sensitivity, high specific, easy and simple to handle, time-consuming short The features such as, can efficiently monitor C the and T allele of rs12979860.
For achieving the above object, the rs12979860 Genotyping two-color fluorescence PCR quick detection examination designed by the present invention Agent box, including DNA extract and two-color fluorescence PCR reactant liquor；
Described two-color fluorescence PCR reactant liquor comprises following components：
(1) the primed probe group based on rs12979860 site
Forward primer F, its nucleotide sequence is as shown in SEQ ID NO.1；
Reverse primer R, its nucleotide sequence is as shown in SEQ ID NO.2；
C fluorescence probe S1, its nucleotide sequence is as shown in SEQ ID NO.3；
T fluorescence probe S2, its nucleotide sequence is as shown in SEQ ID NO.4；
(2) PCR amplification system
PCR 10 × buffer, the MgCl of 25mM2, dNTPs contain dUTP, bi-component thermal starting archaeal dna polymerase, 50 × ROX Reference Dye、RNase-Free ddH2O；
Described PCR 10 × buffer includes KCl, 0.8%v/v's of Tris-HCl, 500mM of 100mM pH8.8 Nonidet；Described PCR 10 × buffer does not contain Mg2+；
Described bi-component thermal starting archaeal dna polymerase is HotStar Taq archaeal dna polymerase and the antibody modification of chemical modification Anti Taq archaeal dna polymerase.
Preferably, in mentioned reagent box, each primer and probe concentration in amplification system is as follows：
Preferably, the fluorescent quantitative PCR condition of mentioned reagent box is 95 DEG C of 5min preheating, 95 DEG C of 3s, 60 DEG C of 30s, 25 circulations, 60 DEG C start to collect fluorescence signal.
Preferably, in mentioned reagent box, C fluorescence probe S1 is MGB probe, and its 5' fluorescence is FAM；Described T fluorescence probe S2 For MGB probe, its 5' fluorescence is VIC.
Beneficial effects of the present invention：By having redesigned primer and TaqMan-MGB probe, optimizing reaction system, double groups Divide thermal starting archaeal dna polymerase composition enzyme activity automatic regulating system, ROX Reference Dye can eliminate signal background and correction The fluorescence signal error producing between Kong Yukong, thus realizing accurate, amplification efficiency height, sensitivity is high, specificity is good, repeated Good, the easy and simple to handle and detection used time is shorter, makes this technology be capable of application clinically and popularization.
Brief description
The sequencing result figure in the IL28B gene rs12979860 site that Fig. 1 detects for DNA sequencing method.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Embodiment 1
Present embodiments provide a kind of rs12979860 Genotyping two-color fluorescence PCR quick detection kit
First, kit forms and preparation
1) primer and probe groups
Primed probe group based on rs12979860 site
Forward primer F, its nucleotide sequence is as shown in SEQ ID NO.1；
5'CGGTCGTGCCTGTCGTGT 3'；
Reverse primer R, its nucleotide sequence is as shown in SEQ ID NO.2；
5'AGCGCGGAGTGCAATTCA 3'；
C fluorescence probe S1, its nucleotide sequence is as shown in SEQ ID NO.3；
5'FAM-ACCCTGGTTCGCGCC-NFQ 3'-MGB, FAM probe；
T fluorescence probe S2, its nucleotide sequence is as shown in SEQ ID NO.4；
5'VIC-TGGTTCACGCCTTC-NFQ 3'-MGB, VIC probe；
Shanghai raw work bio-artificial synthesis all entrusted by above-mentioned primer and probe.
2) DNA extract
DNA extract can be using commercially available DNA extraction kit it is also possible to voluntarily prepare.
DNA extract recipe in the present embodiment：(1) 3% gelatin；(2) 200mmol/L NaCl, 100mmol/L are prepared Tris-HCl；(3) 10%SDS 3mg/ml Proteinase K (adding before use)；(4) pH7.8 saturated phenol；(5) absolute ethyl alcohol；(6) 70% ethanol；(7)RNase-Free ddH2O.
3) reaction system of two-color fluorescence PCR is as follows：
Reagent Content in final concentration or unit volume
PCR 10×buffer 5μl
MgCl2 4mmol/L
DNTP (containing dUTP) 0.48mmol/L
Taq archaeal dna polymerase 0.5U
Forward primer F 1.5μl
Reverse primer R 1.5μl
C fluorescence probe S1 1.0μl
T fluorescence probe S2 1.0μl
DNA profiling 5μl
ddH2O Add to 50 μ l
Total reaction volume 50μl
During using mentioned reagent box, the response procedures of two-color fluorescence PCR are：95 DEG C of 5min preheating, 95 DEG C of 3s, 60 DEG C of 30s, 25 circulations, 60 DEG C start to collect fluorescence signal.
Test example 1DNA sequence verification
1st, by the two-color fluorescence PCR kit in embodiment 1, for 50 hepatitis patients whole blood sample DNA carry out double Color fluorescent PCR, the results are shown in Table 1.
Above-mentioned dilution product are utilized the reaction system amplification of two-color fluorescence PCR, amplification condition is 95 DEG C of 5min preheatings, 95 DEG C 3s, 60 DEG C of 30s, 25 circulations, 60 DEG C start to collect fluorescence signal.
Table 1 rs12979860 site SNP type test result compares
2nd, 3 kinds of genotype in the design rs12979860 site to 50 parts of sample DNAs for the sequencing primer are sequenced, and above-mentioned Kit testing result is consistent.Three kinds of bases of C/C, C/T and T/T type in the rs12979860 site that Fig. 1 detects for DNA sequencing method Because of type.
SEQUENCE LISTING
<110>Wuhan University
<120>A kind of rs12979860 Genotyping two-color fluorescence PCR quick detection kit
<130> 2016
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
cggtcgtgcc tgtcgtgt 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
agcgcggagt gcaattca 18
<210> 3
<211> 15
<212> DNA
<213>Artificial sequence
<400> 3
accctggttc gcgcc 15
<210> 4
<211> 14
<212> DNA
<213>Artificial sequence
<400> 4
tggttcacgc cttc 14

Claims (4)

1. a kind of rs12979860 Genotyping two-color fluorescence PCR quick detection kit it is characterised in that：
Described kit includes DNA extract and two-color fluorescence PCR reactant liquor；
Described two-color fluorescence PCR reactant liquor comprises following components：
(1) the primed probe group based on rs12979860 site
Forward primer F, its nucleotide sequence is as shown in SEQ ID NO.1；
Reverse primer R, its nucleotide sequence is as shown in SEQ ID NO.2；
C fluorescence probe S1, its nucleotide sequence is as shown in SEQ ID NO.3；
T fluorescence probe S2, its nucleotide sequence is as shown in SEQ ID NO.4；
(2) PCR reaction system
PCR 10 × buffer, the MgCl of 25mM2, dNTPs contain dUTP, bi-component thermal starting archaeal dna polymerase, 50 × ROX Reference Dye、RNase-Free ddH2O；
Described PCR 10 × buffer includes the Nonidet of KCl, 0.8%v/v of Tris-HCl, 500mM of 100mM pH8.8； Described PCR 10 × buffer does not contain Mg2+；
Described bi-component thermal starting archaeal dna polymerase is HotStar Taq archaeal dna polymerase and the antibody modification of chemical modification Anti Taq archaeal dna polymerase.
2. rs12979860 Genotyping two-color fluorescence PCR quick detection kit according to claim 2, it is special Levy and be：
Each primer and probe concentration in amplification system is as follows：
3. rs12979860 Genotyping two-color fluorescence PCR quick detection kit according to claim 2, it is special Levy and be：The fluorescent quantitative PCR condition of described kit is 95 DEG C of 5min preheatings, 95 DEG C of 3s, 60 DEG C of 30s, 25 circulations, 60 DEG C start to collect fluorescence signal.
4. the rs12979860 Genotyping two-color fluorescence PCR quick detection examination according to any one of claims 1 to 3 Agent box it is characterised in that：Described C fluorescence probe S1 is MGB probe, and its 5' fluorescence is FAM；
Described T fluorescence probe S2 is MGB probe, and its 5' fluorescence is VIC.
CN201611039569.4A 2016-11-21 2016-11-21 Rs12979860 genotyping dual-color fluorescent PCR rapid detection kit Pending CN106399565A (en)

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Application Number Priority Date Filing Date Title
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107502600A (en) * 2017-07-26 2017-12-22 李桂秋 A kind of double thermal starting archaeal dna polymerases and PCR amplification detection methods containing nano antibody
CN109628423A (en) * 2018-12-06 2019-04-16 北京春雷杰创生物科技有限公司 A kind of method of thermal starting Taq archaeal dna polymerase Combinatorial Optimization molecular agents
RU2819836C1 (en) * 2023-12-01 2024-05-27 Федеральное государственное бюджетное образовательное учреждение высшего образования "Курский государственный медицинский университет" Министерства здравоохранения Российской Федерации METHOD FOR GENOTYPING SINGLE-NUCLEOTIDE VARIANT rs13056243 (C>T) OF HUMAN ZNRF3 GENE BY REAL-TIME POLYMERASE CHAIN REACTION

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CN104774918A (en) * 2015-01-07 2015-07-15 上海孚清生物科技有限公司 Real-time fluorescent PCR-based IL28B gene polymorphism detection kit
CN104513864A (en) * 2015-01-21 2015-04-15 山东维真生物科技有限公司 Primers, probes and kit for detecting human EGFR gene mutations
CN105400877A (en) * 2015-12-07 2016-03-16 中国人民解放军第四军医大学 Method for genome SNP locus detection based on immune enzyme-linked reaction
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MARTIN LAGGING等: "Response Prediction in Chronic Hepatitis C by Assessment of IP-10 and IL28B-Related Single Nucleotide Polymorphisms", 《PLOS ONE》 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107502600A (en) * 2017-07-26 2017-12-22 李桂秋 A kind of double thermal starting archaeal dna polymerases and PCR amplification detection methods containing nano antibody
CN109628423A (en) * 2018-12-06 2019-04-16 北京春雷杰创生物科技有限公司 A kind of method of thermal starting Taq archaeal dna polymerase Combinatorial Optimization molecular agents
RU2819836C1 (en) * 2023-12-01 2024-05-27 Федеральное государственное бюджетное образовательное учреждение высшего образования "Курский государственный медицинский университет" Министерства здравоохранения Российской Федерации METHOD FOR GENOTYPING SINGLE-NUCLEOTIDE VARIANT rs13056243 (C>T) OF HUMAN ZNRF3 GENE BY REAL-TIME POLYMERASE CHAIN REACTION
RU2820348C1 (en) * 2023-12-27 2024-06-03 Федеральное государственное бюджетное образовательное учреждение высшего образования "Курский государственный медицинский университет" Министерства здравоохранения Российской Федерации METHOD FOR GENOTYPING POLYMORPHIC LOCUS rs11024032 (CT) OF C11orf58 GENE IN HUMANS BY REAL-TIME PCR USING ALLELE-SPECIFIC FLUORESCENT PROBES

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