CN103789451B - A kind of fluorescence quantitative kit detecting CA 6, A10 type - Google Patents
A kind of fluorescence quantitative kit detecting CA 6, A10 type Download PDFInfo
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Abstract
The invention provides a kind of fluorescence quantitative kit detecting CA 6, A10 type, be made up of quantitative RT-PCR reaction solution, enzyme mixation, primed probe mixed solution, CVA6 and CVA10 standard substance, CVA6 and CVA10 robust positive control product, the weak positive reference substance of CVA6 and CVA10, negative controls.The present invention uses one-step method real-time fluorescent quantitative RT-PCR technology, adopt primer and the fluorescence labeling probe of CVA6 and CVA10 high special, detect the existence whether having CVA6 and CVA10 from stool sample simultaneously, more convenient than substance fluorescence quantifying PCR method rapid, cost-saving.Detect real-time, accurate quantitative analysis, provide early diagnosis for clinical, for the formulation of clinical treatment provides reference frame; Can be applicable to CA 6, laboratory emergency diagnosis that A10 type causes epidemic outbreaks, rapid screening, clinical diagnosis and hand foot mouth disease EPDML research.
Description
Technical field
The invention belongs to biological technical field, relate to fluorescence quantitative RT-PCR detecting kit, be specifically related to the nucleic acid detection method that a kind of single stage method dual real-time fluorescence quantitative RT-PCR detects CA 6 in patient's stool sample, A10 type in same reaction tubes, can be applicable to CA 6, laboratory emergency diagnosis that A10 type causes epidemic outbreaks, rapid screening, clinical diagnosis and hand foot mouth disease EPDML research.
Background technology
Hand foot mouth disease (hand, foot and mouth disease, HFMD) be by Human enterovirus virus (human enterovirus,
HEV) the class children common transmittable caused is sick.Human enterovirus virus has 90 Multi-genotypes at present, be divided into A, B, C, D totally 4 groups, wherein topmost pathogenic agent is enterovirns type 71 (EV71) in HEV-A group and coxsackie virus A 16-type (Coxsackie virus A16, CVAl6).But increasing research shows, some other serotypes in HEV, as CVA6, CVA10, CVA2, CVA5 type etc. also can cause HFMD.
CA 6 type (Coxsackie virus A6, and CA 10 type (Coxsackie virus A10 CVA6), CVAl0) Human enterovirus virus A group is all belonged to, in recent years in Europe and the national hand foot mouth disease epidemic situation broken out of China's peripheral part, CA 6 type and CA 10 type are repeatedly detected by as main causative pathogen.2008, Finland was broken out based on the hand foot mouth disease of CA 6, A10 type.2010, in French brothers' mouth infective pathogen investigation, the ratio of CA 10 and A6 type was considerably beyond EV71 and coxsackie virus A 16-type.And in recent years, the hand-foot-mouth disease epidemic situation based on CA 6 has also been broken out in succession in some Asian countries such as Singapore, Thailand, Japan etc., wherein CA 10 type also account for certain ratio.In China, 2009 to 2011 in Shandong, hand foot mouth disease investigation display that launches of Henan, Chongqing three provinces and cities: CA 10 type and A6 type are only second to EV71 and coxsackie virus A 16-type, occupy third and fourth position.2012, in the hand foot mouth disease investigation of Shenzhen, CA 6 type and A10 type were only second to EV71 and coxsackie virus A 16-type, occupy third and fourth position.And in the former investigation of part month in 2013 hand foot mouth disease of our labs, CA 6 type and A10 type have accounted for high ratio.The hand foot mouth disease etiological diagnosis market of current China, still to detect enterovirus universal or enterovirns type 71 and coxsackie virus A 16-type, cause hand foot mouth disease pathogen detection indefinite or undetected, cause the delay of patient treatment and the waste etc. of diagnostic reagent.Thus a kind of diagnostic reagent for CA 6 type and CA 10 type Pathogen test is developed extremely urgent.
Real-Time Fluorescent Quantitative PCR Technique is with advantages such as its totally-enclosed Single tube amplification, simple and efficient, reproducible, real-time quantitative, pollution are few, overcome the defect of clinical diagnosis in the past, there is specificity and the susceptibility of height, for clinical diagnosis provides laboratory foundation accurately and reliably, can be used as a kind of detection means effectively and rapidly of clinical etiological diagnosis.
Summary of the invention
The object of this invention is to provide a kind of fluorescence quantitative kit detecting CA 6, A10 type, is a kind of single stage method dual fluorescence quantification RT-PCR detection kit detected for CA 6 type and CA 10 type.This test kit comprises quantitative RT-PCR reaction solution, enzyme mixation, primed probe mixed solution, standard substance (CVA6, CVA10), robust positive control product (CVA6, CVA10), weak positive reference substance (CVA6, CVA10), negative controls.
Wherein quantitative RT-PCR reaction solution includes PCR reaction buffer, magnesium chloride and triphosphate deoxyribose nucleotide mixture, enzyme mixation is containing heat-resisting Taq archaeal dna polymerase, RNA enzyme inhibitors and MMLV reversed transcriptive enzyme, primed probe mixed solution comprises CVA6, CVA10 two group-specific primers and corresponding CVA6, CVA10 two kinds of fluorescently-labeled specific probes of difference, negative control is that the DEPC (diethylpyrocarbonate) of autoclave sterilization processes water, CVA6, CVA10 robust positive control product and CVA6, the weak positive reference substance of CVA10 is for comprising CVA6, the positive plasmid sample of the conserved region gene sequence of CVA10.Primed probe mixed solution need be kept at brown pipe.
Multiplex real-time PCR detection upstream primer and downstream primer and corresponding specific probe sequence as follows:
Upstream primer CVA6-F:5 '-AARCCRGATAGYAGGAAATC-3 '
Downstream primer CVA6-R:5 '-GGGGTGGATCACTCAATTTTG-3 '
Specific probe CVA6-P:5 '-FAM-CAATGGCAGACTGCYACYAAYCCGTCG-BHQ1-3 '
Upstream primer CVA10-F:5 '-GAGACTGGACGYGTRCCA-3 '
Downstream primer CVA10-R:5 '-GGGTYTCAATCATGTTYTCATCTG-3 '
Specific probe CVA10-P:5 '-HEX-CAGAGACRGGTGCCACWTCTAAYGCC-BHQ1-3 '
Wherein the specific probe of CVA6, CVA10 adopts FAM, HEX two kinds of different fluorescent marks respectively.
The conserved region gene sequence of above-mentioned CVA6, CVA10 standard substance is as follows:
CVA6 standard substance sequence is:
GTATGTACCACCAGGGGCCCCTAAACCGGATAGTAGGAAATCATACCAATGGCAGACTGCTACTAACCCGTCGGTATTCGCAAAATTGAGTGATCCACCCCCCCAGGTGTCTGTCCCGTTCAT
CVA10 standard substance sequence is:
CCACTCCCAGTTCACACCGATTAGAGACTGGACGCGTGCCAGCGCTACAGGCTGCAGAGACGGGTGCCACTTCTAATGCCACAGATGAGAACATGATTGAAACCCGTTGTGTGGTTAACAGAA
Fluorescence quantitative kit provided by the invention in-20 DEG C of storages, need reduce multigelation as far as possible; Primed probe mixed solution needs lucifuge condition to preserve.
Another object of the present invention is to provide above-mentioned single stage method dual fluorescence quantification RT-PCR detection kit, is applied to the detection of nucleic acids of CA 6 type and CA 10 type.
The using method of test kit of the present invention: positive control and negative control all should be set up in each Samples detection.The Easy Dilution(article No. of standard substance TaKaRa company: 9160) dilution is 1 × 10
2-1 × 10
7copies/ml.
The extraction of fecal sample nucleic acid: get 0.2g fecal sample and be added in EP pipe, add the physiological saline of 1.5ml, concussion mixing 3 times, each 10s, then room temperature leaves standstill 10min, with the centrifugal 5min of 8000r/min.Drawing 200 ml supernatants joins in EP pipe, carries out nucleic acid extraction.Nucleic acid extraction adopts the Viral Nucleic Acid extraction KitII of Geneaid company or the QIAamp Viral RNA Mini Kit of QIAGEN company, extract according to test kit specification sheets, get the testing sample nucleic acid of 5ul extracting as template.
The detection of nucleic acid: get the testing sample nucleic acid of 5ul extracting as template.Reaction cumulative volume is 25
, wherein quantitative RT-PCR reaction solution 12.5
, enzyme mixation 1
, primed probe mixed solution (20 μm of ol/L comprise two group-specific primerses and corresponding two kinds of fluorescent probes) totally 3
, template 5
, add water and complement to 25
.ABI7500 quantitative real time PCR Instrument (or other Two Colour Fluorescences and above PCR instrument) detects, and reaction parameter is: reverse transcription 50 DEG C, 30min; 95 DEG C of 5min warm starts, then 95 DEG C of 15s, 55 DEG C of 45s, carry out double fluorescent detection at 55 DEG C, carry out 40 circulations altogether.
Fluorescent quantitation report the test: the respective C of 1. CA 6 type, the detection of CA 10 type
tthe corresponding corresponding fluorescently-labeled virus of value, detect sample CT value be 40,0 and without numerical value time, be reported as feminine gender.2. sample C is detected
tduring value≤35, be reported as corresponding virus-positive.3. sample C is detected
tvalue>=35 and be less than 40 sample, suggestion recheck, recheck result C
tvalue <, is reported as corresponding virus-positive according to judging criterion 2..According to obtained typical curve, calculate the virus quantity (copies/ml) of sample CA 6 type to be measured or CA 10 type.
This research is for Coxsackie virus high conservative and the VP1 albumen that there is larger difference between type designs the primed probe of CA 6 type and CA 10 type high specific respectively.Adopt single tube single stage method dual real-time fluorescence quantitative RT-PCR, primary first-order equation can detect CA 6 type or/and CA 10 type in single tube simultaneously, not only save reagent consumptive material greatly, shorten detection time, there is high specificity and sensitivity simultaneously.
The present invention uses one-step method real-time fluorescent quantitative RT-PCR technology, adopt primer and the fluorescence labeling probe of CA 6 type and CA 10 type high special, development is used for the dual fluorescence quantification RT-PCR detection kit of CA 6 type and the detection of CA 10 type.This invention is by a PCR reaction, can detect the existence whether having CA 6 type and CA 10 type from stool sample simultaneously, more convenient than substance fluorescence quantifying PCR method rapid, cost-saving.Meanwhile, real-time accurate quantitative analysis is carried out, according to the titre of virus infection, for patient provides early diagnosis clinically, for the formulation of clinical treatment provides reference frame to the virus detected; Can be applicable to CA 6, laboratory emergency diagnosis that A10 type causes epidemic outbreaks, rapid screening, clinical diagnosis and hand foot mouth disease EPDML research.
Accompanying drawing explanation
Fig. 1 is the sensitivity that this test kit detects CA 6 type, and from left to right (1-6) is followed successively by 10
7, 10
6, 10
5, 10
4, 10
3, 10
2copies/ml.
Fig. 2 is this test kit CA 6 type standard substance real-time fluorescence quantitative RT-PCR product gel electrophoresis schematic diagram, and electrophoretic band size is about 77bp, and wherein Lane M is that DL2000 Marker, Lane 1-6 is respectively CA 6 type standard substance (10
7copies/ml) real-time fluorescence quantitative RT-PCR product after ten times of gradient dilutions, Lane 7 is negative control.
Fig. 3 is this test kit CA 6 type standard substance real-time fluorescence quantitative RT-PCR typical curve.
Fig. 4 is the sensitivity that this test kit detects CA 10 type, and from left to right (1-6) is followed successively by 10
7, 10
6, 10
5, 10
4, 10
3, 10
2copies/ml.
Fig. 5 is this test kit CA 10 type standard substance real-time fluorescence quantitative RT-PCR product gel electrophoresis schematic diagram, and electrophoretic band size is about 81bp, and wherein Lane M is that DL2000 Marker, Lane 1-6 is respectively CA 10 type standard substance (10
7copies/ml) real-time fluorescence quantitative RT-PCR product after ten times of gradient dilutions, Lane 7 is negative control.
Fig. 6 is this test kit CA 10 type standard substance real-time fluorescence quantitative RT-PCR typical curve.
Embodiment
Specific embodiment is further elaborated the present invention to the present invention by reference to the accompanying drawings below, but these embodiments are only limitted to the present invention is described and does not limit the scope of the invention.
embodiment 1
Detect a dual real-time fluorescence quantitative RT-PCR detecting kit for CA 6, A10 type, comprise: quantitative RT-PCR reaction solution, enzyme mixation, primed probe mixed solution, standard substance (CVA6, CVA10), robust positive control product (CVA6, CVA10), weak positive reference substance (CVA6, CVA10), negative controls.
Wherein quantitative RT-PCR reaction solution includes PCR reaction buffer, magnesium chloride and triphosphate deoxyribose nucleotide mixture, enzyme mixation is containing heat-resisting Taq archaeal dna polymerase, RNA enzyme inhibitors and MMLV reversed transcriptive enzyme, primed probe mixed solution comprises CVA6, CVA10 two group-specific primers and corresponding CVA6, CVA10 two kinds of fluorescently-labeled specific probes of difference, negative control is that the DEPC (diethylpyrocarbonate) of autoclave sterilization processes water, CVA6, CVA10 robust positive control product and CVA6, the weak positive reference substance of CVA10 is for comprising CVA6, the positive plasmid sample of the conserved region gene sequence of CVA10.Robust positive control product plasmid concentration is 10
6copies/ml, weak positive reference substance plasmid concentration is 10
3copies/ml.Primed probe mixed solution pipe need be kept at brown pipe.
Multiplex real-time PCR detection upstream primer and downstream primer and corresponding specific probe sequence as follows:
Upstream primer CVA6-F:5 '-AARCCRGATAGYAGGAAATC-3 ',
Downstream primer CVA6-R:5 '-GGGGTGGATCACTCAATTTTG-3 ',
Specific probe CVA6-P:5 '-FAM-CAATGGCAGACTGCYACYAAYCCGTCG-BHQ1-3 ',
Upstream primer CVA10-F:5 '-GAGACTGGACGYGTRCCA-3 ',
Downstream primer CVA10-R:5 '-GGGTYTCAATCATGTTYTCATCTG-3 ',
Specific probe CVA10-P:5 '-HEX-CAGAGACRGGTGCCACWTCTAAYGCC-BHQ1-3 '.
Wherein the specific probe of CVA6, CVA10 adopts FAM, HEX two kinds of different fluorescent marks respectively.
Above-mentioned standard substance comprise CVA6, CVA10 standard substance, and its conserved region gene sequence is as follows:
CVA6 standard substance sequence is:
GTATGTACCACCAGGGGCCCCTAAACCGGATAGTAGGAAATCATACCAATGGCAGACTGCTACTAACCCGTCGGTATTCGCAAAATTGAGTGATCCACCCCCCCAGGTGTCTGTCCCGTTCAT。
CVA10 standard substance sequence is:
CCACTCCCAGTTCACACCGATTAGAGACTGGACGCGTGCCAGCGCTACAGGCTGCAGAGACGGGTGCCACTTCTAATGCCACAGATGAGAACATGATTGAAACCCGTTGTGTGGTTAACAGAA。
Fluorescence quantitative kit provided by the invention in-20 DEG C of storages, need reduce multigelation as far as possible; Primed probe mixed solution needs lucifuge condition to preserve.
embodiment 2
1 materials and methods
1.1 clinical samples and viral nucleic acid:
The clinical sample of CA 6, A10 type derives from the stool sample of other Ji Jia hospitals hand foot mouth disease patient diagnosed and suspected patient in Zhejiang University Medical College The First Affiliated Hospital and Zhejiang Province, is transported to laboratory after sample collection.In addition, other enteroviruses as enterovirns type 71, coxsackie virus A 16-type, CA 2 type, CA 5 type, Coxsackie B virus 1 type, Coxsackie virus type B3, dust gram virus 30 type, and influenza A virus, respiratory syncytial virus, bocavirus positive nucleic acid provided by transmissible disease diagnosis and treatment National Key Laboratory.
1.2 primers and probe
Many the gene orders containing domestic and international CA 6, A10 type have been downloaded from NCBI gene pool.Utilize DNAman software to carry out tetraploid rice to it, determine above virus genomic conserved regions.Use Primer Express 3.0 software at the primer of its conserved regions design high degree of specificity and Taqman probe, primer and probe sequence are all verified by Blast, have better specificity.Primer and probe sequence as follows:
Upstream primer CVA6-F:5 '-AARCCRGATAGYAGGAAATC-3 ',
Downstream primer CVA6-R:5 '-GGGGTGGATCACTCAATTTTG-3 ',
Specific probe CVA6-P:5 '-FAM-CAATGGCAGACTGCYACYAAYCCGTCG-BHQ1-3 ',
Upstream primer CVA10-F:5 '-GAGACTGGACGYGTRCCA-3 ',
Downstream primer CVA10-R:5 '-GGGTYTCAATCATGTTYTCATCTG-3 ',
Specific probe CVA10-P:5 '-HEX-CAGAGACRGGTGCCACWTCTAAYGCC-BHQ1-3 ',
Above primer and probe entrust Sangon Biotech (Shanghai) Co., Ltd. to synthesize.
The extraction of 1.3 viral nucleic acids and standard substance quantitative criterion:
Get 0.2g fecal sample to be added in EP pipe, add the physiological saline of 1.5ml, concussion mixing 3 times, each 10s, then room temperature leaves standstill 10min, with the centrifugal 5min of 8000r/min.Draw 200
supernatant joins in EP pipe, adopt the Viral Nucleic Acid extraction KitII of Geneaid company or the Rneasy Mini Kit of QIAGEN company, extract according to test kit specification sheets, get the testing sample nucleic acid of 5ul extracting as template.
Synthesize each gene standard substance fragment, be connected to plasmid vector pMDTM19-T Simple Vector(TaKaRa company).Cultivate after transforming.Extract plasmid DNA after qualification, utilize NanoDrop ND-2000 Spectrophotometer to measure the concentration of plasmid DNA, determine that the copy number of DNA is as the quantitative mother liquor of standard substance.Experimentally need, quantitative for standard substance mother liquor is diluted to required maximum concentration, and do ten times and be diluted to minimum concentration, cryopreservation is for subsequent use.
The optimization of 1.4 dual real-time fluorescence quantitative RT-PCR reaction systems and condition:
Reaction cumulative volume is 25
, wherein quantitative RT-PCR reaction solution 12.5
, enzyme mixation 1
, primed probe mixed solution (20 μm of ol/l comprise the Auele Specific Primer of two-strain and corresponding two kinds of fluorescent probes) totally 3.0
, template 5
, DEPC water complements to 25
.ABI7500 quantitative real time PCR Instrument detects, and reaction parameter is: reverse transcription 50 DEG C, 30min; 95 DEG C of 5min warm starts, then 95 DEG C of 15s, 55 DEG C of 45s, carry out quadruple fluoroscopic examination at 55 DEG C, carry out 40 circulations altogether.Result judges: selection fluoroscopic examination model F AM, HEX fluorescence baseline adjustment get the fluorescent signal mean value of 3-15 circulation, and threshold setting is with the vertex of threshold line just above negative controls, and sample is typical amplification curve, is judged as the positive.Without typical amplification curve, be judged as feminine gender.The optimization Test of system, in the reaction system being template with the positive nucleic acid of same concentrations, primer concentration is from 1 ~ 20 μM, concentration and probe concentration is from 1 ~ 20 μM, adopt the optimum concn of the preferred primer of matrix method and probe, select best primer and concentration and probe concentration according to minimum Ct value and most high fluorescent increased value (Δ Rn).
1.5 dual real-time fluorescence quantitative RT-PCR specificitys, susceptibility and replica test
Select the positive nucleic acid (all being identified by gene sequencing) of CA 6 type and CA 10 type and other enteroviruses as enterovirns type 71, coxsackie virus A 16-type, CA 2 type, CA 5 type, Coxsackie B virus 1 type, Coxsackie virus type B3, dust gram virus 30 type respectively, and influenza A virus, respiratory syncytial virus, bocavirus positive nucleic acid, verify the specificity of present method with dual real-time fluorescence quantitative RT-PCR; To the CA 6 type synthesis fragment (10 of demarcating copy number (copies/ml)
7copies/ml) and COxsackie sick
poisona10 type synthesis fragment (10
7copies/ml), respectively after dilution, parallelly carry out Fluorescence PCR, compare its sensitivity.In addition, make 3 duplicate detection to the positive nucleic acid diluent of each prescribed concentration, the Ct value obtained calculates its standard deviation and the variation coefficient, the repeatability of checking the method.
The foundation of 1.6 dual real-time fluorescence quantitative RT-PCR typical curves
To demarcate the CA 6 type standard substance (10 of copy number (copies/ml)
7copies/ml), CA 10 type standard substance (10
7copies/ml) difference ten times of gradient dilutions to 10
2copies/ml.After carrying out real-time fluorescence quantitative PCR amplification as template with this test kit respectively, with the logarithmic value of standard concentration for X-axis, cycle number is Y-axis, drawing standard curve.
2 results
2.1 dual real-time fluorescence quantitative RT-PCR reaction system and conditions
The reaction cumulative volume of the method is 25
, wherein quantitative RT-PCR reaction solution 12.5
, enzyme mixation 1
, primed probe mixed solution (20 μm of ol/l comprise two group-specific primerses and corresponding two kinds of fluorescent probes) totally 3
, template 5
, DEPC water complements to 25
.ABI7500 quantitative real time PCR Instrument detects, and reaction parameter is: reverse transcription 50 DEG C, 30min; 95 DEG C of 5min warm starts, then 95 DEG C of 15s, 55 DEG C of 45s, carry out triple fluorescent detection at 55 DEG C, carry out 40 circulations altogether.Minimum Ct value and most high fluorescent can be obtained.
2.2 specific test
The single stage method Multiplex real-time PCR method that the present invention sets up has fabulous specificity to CA 6 type and CA 10 type, can detect the positive clinical sample gathered in the recent period completely.Dual primed probe in the present invention and other enteroviruses as enterovirns type 71, coxsackie virus A 16-type, CA 2 type, CA 5 type, Coxsackie B virus 1 type, Coxsackie virus type B3, dust gram virus 30 type, and influenza A virus, respiratory syncytial virus, bocavirus the equal no cross reaction of positive nucleic acid.
2.3 sensitivity test
To CA 6 type and the test of CA 10 type sensitivity Detection, will demarcate the CA 6 type synthesis fragment (10 of copy number (copies/ml)
7copies/ml), CA 10 type synthesis fragment (10
7copies/ml), after difference ten times of gradient dilutions, detect with this test kit, result the method detection sensitivity reaches 10 respectively
2, 10
2copies/ml.Result is see Fig. 1, Fig. 4.Get real-time fluorescence quantitative RT-PCR product 3
, 120V constant voltage electrophoresis 20min, gel imaging system is taken pictures.Result is see Fig. 2, Fig. 5.Wherein Lane M is that DL2000 Marker, Lane 1-6 is respectively fragment (10
8copies/ml) real-time fluorescence quantitative RT-PCR product after ten times of gradient dilutions, Lane 7 is negative control., electrophoretic band is single, and brightness step is clearly demarcated, and illustrate that this reagent specificity is good, quantitative result is relatively accurate.
2.4 replica test
(final concentration is 10 to get CA 6 type synthesis fragment respectively
6copies/ml), CA 10 type synthesis fragment (10
6copies/ml) 3 different concentration are become by 10 times of gradient dilutions, 3 duplicate detection are done to the sample of each concentration, result different IPs acid concentration detection Ct value standard deviation is separately between 0.122 ~ 0.375, and the variation coefficient, all lower than 1.38%, has good repeatability (the results are shown in Table 1).
The foundation of 2.5 typical curves
After real-time fluorescence quantitative PCR amplification, with the logarithmic value of standard concentration for X-axis, cycle number is Y-axis, drawing standard curve.As shown in Figure 3, wherein slope is 3.240 to CA 6 type standard substance real-time fluorescence quantitative RT-PCR typical curve, and intercept is 40.136, and relation conefficient is 0.996.As shown in Figure 6, wherein slope is 3.128 to CA 10 type standard substance real-time fluorescence quantitative RT-PCR typical curve, and intercept is 39.224, and relation conefficient is 0.998.As can be seen here, the logarithmic value of standard concentration and cycle number have good linear relationship.
embodiment 3
The detection of this test kit to clinical sample is adopted mainly to rely on " 12 " key special subjects-infectious disease pathogens detection technique platform project (2012ZX10004-210).The clinical sample gathered to be mainly derived between year June in March, 2013 to 2013 other Ji Jia hospitals hand foot mouth disease patient and suspected patient stool sample 419 parts altogether in Zhejiang University Medical College The First Affiliated Hospital and Zhejiang Province.Adopt the dual real-time fluorescence quantitative RT-PCR in present method to verify the sample collected, detected result is as follows: positive 171 parts of CA 6 type, positive rate 40.8%; Positive 27 parts of CA 10 type, positive rate 6.4%.Detection positive findings and its degree of conformity of having reported the result reach 100%.
<110> Zhejiang University
<120> mono-kind detects the fluorescence quantitative kit of CA 6, A10 type
<160> 8
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> detects upstream primer sequence according to the PCR of CA 6 type VP1 gene design
<400> 1
AARCCRGATAGYAGGAAATC 20
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> detects downstream primer sequence according to the PCR according to strange viral A6 type VP1 gene design
<400> 2
GGGGTGGATCACTCAATTTTG 21
<210> 3
<211> 27
<212> DNA
<213> artificial sequence
<220>
<223> is according to the TaqMan fluorescent quantitation detection probes sequence of CA 6 type VP1 gene design
<400> 3
CAATGGCAGACTGCYACYAAYCCGTCG 27
<210> 4
<211> 123
<212> DNA
<213> artificial sequence
<220>
<223> is according to the fluorescent quantitation examination criteria product sequence of CA 6 type VP1 gene design
<400> 4
GTATGTACCACCAGGGGCCCCTAAACCGGATAGTAGGAAATCATACCAATGGCAGACTGCTACTAACCCGTCGGTATTCGCAAAATTGAGTGATCCACCCCCCCAGGTGTCTGTCCCGTTCAT 123
<210> 5
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> detects upstream primer sequence according to the PCR of CA 10 type VP1 gene design
<400> 5
GAGACTGGACGYGTRCCA 18
<210> 6
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> detects downstream primer sequence according to the PCR of CA 10 type VP1 gene design
<400> 6
GGGTYTCAATCATGTTYTCATCTG 24
<210> 7
<211> 26
<212> DNA
<213> artificial sequence
<220>
<223> is according to the TaqMan fluorescent quantitation detection probes sequence of CA 10 type VP1 gene design
<400> 7
CAGAGACRGGTGCCACWTCTAAYGCC 26
<210> 8
<211> 123
<212> DNA
<213> artificial sequence
<220>
The fluorescent quantitation examination criteria product sequence that <223> designs according to CA 10 type VP1 gene
<400> 8
CCACTCCCAGTTCACACCGATTAGAGACTGGACGCGTGCCAGCGCTACAGGCTGCAGAGACGGGTGCCACTTCTAATGCCACAGATGAGAACATGATTGAAACCCGTTGTGTGGTTAACAGAA 123
Claims (5)
1. one kind is detected the fluorescence quantitative kit of CA 6, A10 type, it is characterized in that, be made up of quantitative RT-PCR reaction solution, enzyme mixation, primed probe mixed solution, CVA6 standard substance, CVA10 standard substance, CVA6 robust positive control product, CVA10 robust positive control product, the weak positive reference substance of CVA6, the weak positive reference substance of CVA10, negative controls, wherein quantitative RT-PCR reaction solution has PCR reaction buffer, magnesium chloride and triphosphate deoxyribose nucleotide mixture, enzyme mixation is containing heat-resisting Taq archaeal dna polymerase, RNA enzyme inhibitors and MMLV reversed transcriptive enzyme, primed probe mixed solution comprises CVA6, CVA10 two group-specific primers and corresponding CVA6, CVA10 two kinds of fluorescently-labeled specific probes, negative control is the diethylpyrocarbonate process water of autoclave sterilization, CVA6, CVA10 robust positive control product and CVA6, the weak positive reference substance of CVA10 is for comprising CVA6, the positive plasmid sample of the gene order of CVA10, robust positive control product plasmid concentration is 10
6copies/ml, weak positive reference substance plasmid concentration is 10
3copies/ml,
Auele Specific Primer and specific probe sequence as follows:
Upstream primer CVA6-F:5 '-AARCCRGATAGYAGGAAATC-3 ',
Downstream primer CVA6-R:5 '-GGGGTGGATCACTCAATTTTG-3 ',
Specific probe CVA6-P:5 '-FAM-CAATGGCAGACTGCYACYAAYCCGTCG-BHQ1-3 ',
Upstream primer CVA10-F:5 '-GAGACTGGACGYGTRCCA-3 ',
Downstream primer CVA10-R:5 '-GGGTYTCAATCATGTTYTCATCTG-3 ',
Specific probe CVA10-P:5 '-HEX-CAGAGACRGGTGCCACWTCTAAYGCC-BHQ1-3 '.
2. a kind of fluorescence quantitative kit detecting CA 6, A10 type according to claim 1, is characterized in that, the specific probe of CVA6, CVA10 adopts FAM, HEX two kinds of fluorescent marks respectively.
3. a kind of fluorescence quantitative kit detecting CA 6, A10 type according to claim 1, it is characterized in that, the gene order of CVA6, CVA10 standard substance is as follows:
CVA6 standard substance sequence is:
GTATGTACCACCAGGGGCCCCTAAACCGGATAGTAGGAAATCATACCAATGGCAGACTGCTACTAACCCGTCGGTATTCGCAAAATTGAGTGATCCACCCCCCCAGGTGTCTGTCCCGTTCAT，
CVA10 standard substance sequence is:
CCACTCCCAGTTCACACCGATTAGAGACTGGACGCGTGCCAGCGCTACAGGCTGCAGAGACGGGTGCCACTTCTAATGCCACAGATGAGAACATGATTGAAACCCGTTGTGTGGTTAACAGAA。
4. a kind of fluorescence quantitative kit detecting CA 6, A10 type according to claim 1, it is characterized in that, primed probe mixed solution is kept at brown pipe.
5. a kind of fluorescence quantitative kit detecting CA 6, A10 type according to claim 1, is characterized in that, described test kit in-20 DEG C of storages, need reduce multigelation as far as possible.
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| CN104195264A (en) * | 2014-08-13 | 2014-12-10 | 江苏硕世生物科技有限公司 | Nucleic acid test kit for rapidly detecting Coxsackie virus A6 type/A10 type and application thereof |
| CN104846122A (en) * | 2015-05-13 | 2015-08-19 | 宝瑞源生物技术(北京)有限公司 | Nucleic acid detection kit of enterovirus type 71 (EV71) and coxsackievirus type A16 (CA16) (fluorescent polymerase chain reaction (PCR) method) |
| CN105331748A (en) * | 2015-12-11 | 2016-02-17 | 湖南圣湘生物科技有限公司 | Reagent kit for Coxsackie virus CA10 type fluorescent quantitative PCR detection |
| CN105349706A (en) * | 2015-12-11 | 2016-02-24 | 湖南圣湘生物科技有限公司 | Coxsackievirus CA6 type fluorescent quantitation PCR (Polymerase Chain Reaction) detection kit |
| CN107513584B (en) * | 2017-09-25 | 2018-12-04 | 浙江大学 | A kind of five heavy fluorescence quantitative kits detecting enterovirus |
| CN107746832B (en) * | 2017-10-12 | 2020-06-09 | 山东第一医科大学(山东省医学科学院) | High-titer Coxsackie virus A10 domesticated strain and application thereof |
| CN107744530B (en) * | 2017-10-12 | 2020-03-27 | 泰山医学院 | Establishment and evaluation of animal model infected with coxsackie virus A10 domesticated strain TA151R-1 |
| CN107881257A (en) * | 2017-11-01 | 2018-04-06 | 杨兴林 | The type of Coxsackie virus A 6 and A10 types combined detection kit and its application |
| CN109554508A (en) * | 2019-01-31 | 2019-04-02 | 福建省农业科学院畜牧兽医研究所 | For identifying the detection primer and probe of anomaly PEDV and TGEV |
| CN109856408A (en) * | 2019-04-09 | 2019-06-07 | 潍坊市康华生物技术有限公司 | A kind of 6 type of Coxsackie virus A and A10 type IgM antibody combined detection kit and preparation method thereof |
| CN109897919B (en) * | 2019-04-23 | 2022-08-02 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | PCR method and kit for simultaneously and accurately quantifying coxsackie A6 type and A10 type |
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