CN107557493B - A kind of enterovirus parting detecting reagent - Google Patents
A kind of enterovirus parting detecting reagent Download PDFInfo
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Abstract
The present invention provides a kind of enterovirus parting detecting reagent, and the enterovirus parting includes enterovirus and its multiple hypotypes：EV71,CVAl6,CVA6,CVA10,CVA2,CVA5,CVA4,CVA9,CVA12,Echo6,Echo9,Echo30,CVB2,CVB3,CVB4.The present invention uses one-step method multiplex real-time reverse transcriptase PCR, using EV and its primer and fluorescence labeling probe of multiple subtype virus high specials, pass through an eight connecting leg simultaneous reactions of PCR, it is detected whether simultaneously with the presence of enterovirus in sample, and is carried out at the same time the Classification Identification of primary bowel Virus type.This method is more convenient rapid, more cost-effective than substance fluorescence PCR method.Viral diagnosis parting is accurate, can be used for enterovirus and causes the emergent rapid screening parting in the laboratory of epidemic outbreaks, for clinical diagnosis and the research of the epidemiology of hand-foot-and-mouth disease.
Description
Technical field
The invention belongs to biotechnologies, are related to real-time fluorescent RT-PCR detection reagent box, and in particular to a kind of one-step method
Multiplex real-time reverse transcriptase PCR detects enterovirus and multiple enterovirus hypotypes in patient's sample in eight connecting leg of PCR
Nucleic acid detection method, can be applied to enterovirus and cause the laboratory emergency diagnosis of epidemic outbreaks, enterovirus rapid screening
The research of the epidemiology of parting, clinical diagnosis and hand-foot-and-mouth disease.
Background technology
Enterovirus (Enterovirus, EV) belongs to Picornaviridae enterovirus genus.World virus taxis committee at present
Member's meeting (ICTV) is classified as EV-A, EV-B, EV-C, EV-D, EV-E, EV-F, EV-G, EV-H, EV-J, RV-A, RV-B, RV-C
12 totally kinds of www.picornaviridae.com (ICTV Master Species List 2015), wherein people can be infected
Enterovirus mainly have EV-A (21 type), EV-B (59 type), EV-C (23 type), EV-D (4 type), type is up to over one hundred kind.
Human enterovirus virus (human enterovirus, HEV) can lead to a variety of diseases, such as hand-foot-and-mouth disease, encephalitis, nothing
Bacterium property meningitis, AFP Cases, respiratory disease, enterogastritis etc..Hand-foot-and-mouth disease (hand, foot and mouth
Disease, HFMD) 5 years old or less children are mostly occurred in, there is fash symptom as main clinic symptoms using hand, foot, mouth, buttocks
Common transmittable disease, mainly based on mild, but severe and death also when have been reported that.Report about brothers mouthful epidemic situation in the past
In, enterovirns type 71 (EV-71) and coxsackie virus A 16-type (Coxsackie virus A16, CV-A16) they are to cause brothers
The most important pathogen of stomatosis virus infection.But the frequency that other types of enterovirus break out and are reported in recent years
It is higher and higher.2010, the main pathogens of Finland's hand-foot-and-mouth disease epidemic situation were CV-A6 and CV-A10；2010 in Japan and platform
Gulf area, it is CV-A6 to cause the main pathogens of hand-foot-and-mouth disease epidemic situation；2014-2015, French hand-foot-and-mouth disease epidemic situation it is main
Pathogen is CV-A6, CV-A10, CV-A16 and EV-71.Domestic investigation shows, 2013, Nanjing, Shanghai, Shenzhen etc.
The main pathogen that hand-foot-and-mouth disease epidemic situation is played in area is CV-A6 and EV-71；2014, the main pathogen of Beijing area be CV-A16,
EV-71, CV-A6 and CV-A10；We investigate the main pathogens of the In Hangzhou Region of Zhe Jiang Province 2013-2014 hand foot and mouth disease early period
It was found that CV-A6 substitutions EV-71 becomes the highest enterovirus of In Hangzhou Region of Zhe Jiang Province detection positive rate, EV-71 comes second.CV-
A10 recall rates are listed in the third position of main pathogen also above CV-A16.In addition, other enterovirus types such as CV-A2, CV-
A4 etc. has outburst and detection in the multiple provinces in China.2012, in brothers' mouthful epidemic situation of Yunnan Province's outburst, CV-B2 and severe
Hand-foot-and-mouth disease is related；2010-2012, Shijiazhuang District brothers mouthful epidemic situation have the prevalence of CV-B3.Angstrom 6 type of gram virus
The enteroviruses type such as (Echoviruses 6, E-6), E-9, E-30 and CV-A5 also has relevant report.It can be seen that causing hand
Human enterovirus virus's type of sufficient stomatosis is more, and cause of disease spectrum is easy to happen the transition in time and space.
The enterovirus molecule in China diagnoses market at present, still to detect enterovirus universal or enterovirns type 71
Based on coxsackie virus A 16-type, cause hand-foot-and-mouth disease pathogen detection indefinite or missing inspection etc., the delay for causing patient to treat
And the waste etc. of diagnostic reagent.Hence it is imperative that exploitation one kind can detect enterovirus and be carried out to main infection type
The detection reagent of parting.
Current enterovirus detection method includes mainly three classes：Virus purification culture, immunological method, molecular biology side
Method.Virus purification culture is the goldstandard of viral disease diagnosis, but its is cumbersome, and detection time is long, and positive rate is relatively low.
Immunological method is the main method of traditional parting, but sero-fast source is very limited, cannot be satisfied wanting for clinical etiological diagnosis
It asks.And the molecular biology method of the relevant technologies such as based on PCR is sensitive with its, the advantages such as quick obtain tremendous expansion.Multiple reality
When fluorescent quantitative PCR technique with the advantages such as its totally-enclosed amplification, simple and efficient, reproducible, real-time detection, pollution be few, overcome
The defect of previous clinical diagnosis, specificity and sensibility with height, provides for clinical diagnosis and accurately and reliably tests
Room foundation can be used as a kind of effectively and rapidly detection means of clinical etiological diagnosis.
Invention content
The object of the present invention is to provide a kind of enterovirus parting detecting reagent, the enterovirus parting includes enteron aisle
Virus and its multiple hypotype：EV71,CVAl6,CVA6,CVA10,CVA2,CVA5,CVA4,CVA9,CVA12,Echo6,
Echo9, Echo30, CVB2, CVB3, CVB4 are a kind of one-step method multiplex real-time reverse transcriptase PCR detection kits.
This kit includes enterovirus (EV) parting detection mixed liquor, enzyme mixation, EV positive reference substances, negative control
Product, inner mark solution.Wherein EV partings detection mixed liquor, which is loaded into, is encoded in 1.-eight connecting legs 8., wherein each of eight connecting legs
Contain PCR reaction buffers (magnesium chloride containing and triphosphate deoxyribose nucleotide mixture etc.) and primed probe mixed liquor in pipe
(probe of each combination primer and corresponding different fluorescent markers).Eight connecting legs 1. -8. in different primed probe mixed liquors be：①
EV+Rnase P、②EV71+CVA16+CVA6、③CVA10+CVA2+CVA5、④CVA4+CVA9+CVA12、⑤Echo6+
Echo9+Echo30,6. CVB2+CVB3+CVB4,7. EV+Rnase P, 8. EV+Rnase P, wherein 7. negative right for detecting
According to product, 8. it is used for detecting EV positive reference substances；Enzyme mixation is reversed containing heat-resisting Taq archaeal dna polymerases, RNase inhibitor and MMLV
Record enzyme；Negative controls are that the DEPC (pyrocarbonic acid diethyl ester) of autoclave sterilization handles water；EV positive reference substances are to include intestines
The virus-like particle solution of road virus 5 ' end non-transcribed code area RNA sequence；Inner mark solution is to include ribonuclease P (Rnase
P) the virus-like particle solution of sequence.
Each group sense primer and downstream primer and the probe sequence of corresponding different fluorescent markers in primed probe mixed liquor
Row are as follows：
Sense primer EV-F:5 '-CCCTGAATGCGGCTAATCC-3 ',
Downstream primer EV-R:5 '-ATTGTCACCATAAGCAGCCA-3 ',
Specific probe EV-P:5 '-ROX-AACCGACTACTTTGGGTGTCCGTGTTTC-BHQ2-3 ',
Sense primer EV71-F:5 '-GAGYATGATYGARACACGCTG-3 ',
Downstream primer EV71-R:5 '-CGCTCTRCTRAAGAARCTATC-3 ',
Specific probe EV71-P:5 '-FAM-AACTCGCACAGTACRGCTGARACCAC-BHQ1-3 ',
Sense primer CVA16-F:5 '-CCCAATGGYGAGYTAGTCCCC-3 ',
Downstream primer CVA16-R:5 '-GTTGGTRGCWGTYTGCCAAGC-3 ',
Specific probe CVA16-P:5 '-HEX-AGTAYATGTATGTCCCRCCAGGGGCT-BHQ1-3 ',
Sense primer CVA6-F:5 '-AARCCRGATAGYAGGAAATC-3 ',
Downstream primer CVA6-R:5 '-GGGGTGGATCACTCAATTTTG-3 ',
Specific probe CVA6-P:5 '-ROX-CAATGGCAGACTGCYACYAAYCCGTCG-BHQ2-3 ',
Sense primer CVA10-F:5 '-GAGACTGGACGYGTRCCA-3 ',
Downstream primer CVA10-R:5 '-GGGTYTCAATCATGTTYTCATCTG-3 ',
Specific probe CVA10-P:5 '-FAM-CAGAGACRGGTGCCACWTCTAAYGCC-BHQ1-3 ',
Sense primer CVA2-F:5'-AGTCGRCCAGTGTCTCAYTCA-3'
Downstream primer CVA2-R：5'-TACACGCCCRGTCTCAATGG-3'
Specific probe CVA2-P:5'-HEX-AACAGCTGCWAACACCCAGGTRAGCC-BHQ1-3'
Sense primer CVA5-F:5'-AAGCGGCGGAGACAGGAG-3'
Downstream primer CVA5-R:5'-CCATGCCTGTTGACCACACA-3'
Specific probe CVA5-P:5'-ROX-CAAAYGCCACCGAYGARAGCATGA-BHQ2-3'
Sense primer CVA4-F:5'-GGRTTYTCAAACTGGGATATTG-3'
Downstream primer CVA4-R:5'-CGCATRTATGTRAATGCYTCAAG-3'
Specific probe CVA4-P:5'-FAM-CATTATGGCRTTTGTGCAAYTGCG-BHQ1-3'
Sense primer CVA9-F:5'-CCYAGYATYTTCTGGACRGA-3'
Downstream primer CVA9-R：5'-GCTRTATGCATTYCCTATACTRATGA-3'
Specific probe CVA9-P:5'-HEX-AACGCACCAGCRCGCATGTCAATC-BHQ1-3'
Sense primer CVA12-F:5'-CGCCGYAAGCTAGAGATCTTCA-3'
Downstream primer CVA12-R:5'-GACTGGTDTTYCCRTTRCGCTC-3'
Specific probe CVA12-P:5'-ROX-CATGCGCTTTGAYGCAGAGTTCACTT-BHQ2-3'
Sense primer Echo6-F:5'-TYAGRCAYGTGAATGACAAAACAA-3'
Downstream primer Echo6-R:5'-AYGCTTTCACGTGYTTTGGC-3'
Specific probe Echo6-P:5'-FAM-AGYCCHATWACRAGCAAAGTKCGCA-BHQ1-3'
Sense primer Echo9-F:5'-CGYTTTGATGCRTGGGAGAT-3'
Downstream primer Echo9-R:5'-AAGCGCAAGTATGTRAACATCTC-3'
Specific probe Echo9-P:5'-HEX-ACATGGTCCAAYTGCGCCGCA-BHQ1-3'
Sense primer Echo30-F:5'-CAGTGGCGTATTGAGCGATGTA-3'
Downstream primer Echo30-R:5'-ATCAACTACCACACCAGATCAGAGTC-3'
Specific probe Echo30-P:5'-ROX-CACGCCGCTCTACCCATAAAGTTTTCTATTGA-BHQ2-3'
Sense primer CVB2-F:5'-GATCAGCATGTGTGTTTTACACGA-3'
Downstream primer CVB2-R:5'-GCCTGTCTAACACTCACCTTCCA-3'
Specific probe CVB2-P:5'-FAM-TACACCAACAGCAAAAATGCAGCCAA-BHQ1-3'
Sense primer CVB3-F:5'-CCAGTAMCAGATAAAGTGGATTCRTA-3'
Downstream primer CVB3-R：5'-AGGAARGGTATRGACATGCGTG-3'
Specific probe CVB3-P:5'-HEX-CCAAGTGTGTTCTGGACAGAGGGTAATGC-BHQ1-3'
Sense primer CVB4-F:5'-CCGAACAAATCCCAGCCTTA-3'
Downstream primer CVB4-R:5'-CTGCATCGTGTCACTTGGGT-3'
Specific probe CVB4-P:5'-ROX-CAGCCGTTGAAACTGGGCATACCTCTC-BHQ2-3'
Sense primer RNase P-F:5'-AGATTTGGACCTGCGAGCG-3'
Downstream primer RNase P-R：5'-GAGCGGCTGTCTCCACAAGT-3'
Specific probe RNase P-P:5'-HEX-TTCTGACCTGAAGGCTCTGCGCG-BHQ1-3'
The end of specific probe sequence 5 ' of wherein EV, EV71, CVA10, CVA4, Echo6, CVB2 use FAM fluorophors
BHQ1 quenching groups label corresponding with fluorophor is respectively adopted in label, 3 ' ends；CVA16,CVA2,CVA9,Echo9,CVB3
The end of specific probe sequence 5 ' marked using HEX fluorophors, 3 ' ends are respectively adopted BHQ1 corresponding with fluorophor and are quenched
Group marks；The end of specific probe sequence 5 ' of CVA6, CVA5, CVA12, Echo30, CVB4 are marked using ROX fluorophors,
BHQ2 quenching groups label corresponding with fluorophor is respectively adopted in 3 ' ends.
Enterovirus parting detecting reagent provided by the invention need to store under the conditions of -20 DEG C, reduce to the greatest extent and freeze repeatedly
Melt；EV partings detection mixed liquor need to be protected from light condition preservation.
It is a further object to provide above-mentioned one-step method multiplex real-time reverse transcriptase PCR detection kits in intestines
It is applied in the detection of nucleic acids of road virus and its multiple hypotype.
The application method of kit of the present invention：Positive control and negative control, and sun should be set up in each Samples detection
Property reference substance and negative controls need to participate in nucleic acid extraction process.Inner mark solution need to participate in nucleic acid extraction process, in every 200 μ l sun
Nucleic acid extraction is carried out after 4 μ l inner mark solutions are added in property reference substance, negative controls and sample.
The extraction of fecal sample nucleic acid：It takes about 0.2g fecal samples to be added in EP pipes, the physiological saline of 1.5ml is added, shake
Swing mixing 3 times, then each 10s is stored at room temperature 10min, 5min is centrifuged with 8000r/min.It draws 200ml supernatants and is added to EP
Guan Zhong carries out nucleic acid extraction.Nucleic acid extraction uses the Viral Nucleic Acid extraction of Geneaid companies
The QIAamp Viral RNA Mini Kit of KitII or QIAGEN companies, are carried out according to kit specification extraction, have taken 5ul
The sample to be tested nucleic acid of extracting is as template.
The detection of nucleic acid：Sample to be tested, negative controls and the positive reference substance nucleic acid extracted is as template.Reaction is total
Volume be 25 μ l, wherein eight connecting legs often pipe include pre- packing EV partings detection 19 μ l of mixed liquor, need to set on centrifuge before use
Centrifugation.The 1 μ l of enzyme mixed liquor into each pipe, 5 μ l of template.It is detected on ABI7500 fluorescence quantitative PCR instruments, probe in detecting
Pattern is arranged according to above-mentioned fluorescence marker groups and corresponding fluorescent quenching group；Response parameter is：50 DEG C of reverse transcription, 15min；
95 DEG C of 5min thermal startings, then 95 DEG C of 15s, 55 DEG C of 45s, fluoroscopic examination is carried out at 55 DEG C, carries out 40 cycles altogether.
Fluorescent quantitation result is reported：1) pipe 1. EV, pipe 2.-the respective Ct values that 6. detect correspond to the disease of corresponding fluorescent marker
Poison, detection sample Ct values are 40,0 and when without numerical value, are reported as feminine gender.2) when detection sample Ct values≤37, it is reported as corresponding disease
It is malicious positive, middle pipe 2. -6. virus-positive, pipe 1. EV virus-positives need to be met simultaneously；Only EV virus-positives, then be reported as other
Enterovirus is positive.3) detection sample Ct values > 37 and the sample less than 40, it is proposed that reinspection, value≤37 reinspection result Ct, according to
It is reported as corresponding virus-positive according to criterion 2).
This research is for there are the designs of the VP1 gene orders of larger difference between the main type of enterovirus is highly conserved and type
The primed probe of high specific.Using one-step method multiplex real-time reverse transcriptase PCR, can simultaneously be detected in eight connecting leg of PCR
Go out enterovirus and its multiple hypotype.Convenient experimental operation, greatly shortens the detection time of enterovirus parting, and has
There are higher specificity and sensitivity.
The present invention uses one-step method multiplex real-time reverse transcriptase PCR, using drawing for EV and its multiple subtype virus high specials
Object and fluorescence labeling probe, develop for enterovirus and its multiple hypotype (EV71, CVAl6, CVA6, CVA10, CVA2,
CVA5, CVA4, CVA9, CVA12, Echo6, Echo9, Echo30, CVB2, CVB3, CVB4) detection multiple real time fluorescence RT-
PCR detection kit.The invention be by an eight connecting leg simultaneous reactions of PCR, can from stool sample simultaneously detect be
No have the presence of enterovirus, and is carried out at the same time the Classification Identification of primary bowel Virus type.This method is than substance fluorescent PCR side
Method is more convenient rapid, cost-effective.Meanwhile accurate parting is carried out to the virus of detection, according to the type of virus infection, for clinic
The formulation of therapeutic scheme provides reference frame；It can be applied to enterovirus and cause the emergent rapid screening point in the laboratory of epidemic outbreaks
Type, for clinical diagnosis and the research of the epidemiology of hand-foot-and-mouth disease.
Description of the drawings
Fig. 1 is this reagent cartridge configuration schematic diagram.
Fig. 2 is the sensitivity that this kit detects enterovirus, and from left to right (1-6) is followed successively by 108、107、106、105、
104、103copies/ml。
Fig. 3 is this kit enterovirus sensitivity experiment real-time fluorescence quantitative RT-PCR product gel electrophoresis schematic diagram,
Electrophoretic band size about 146bp, wherein Lane M are DL2000Marker, and Lane 1-6 are respectively EV positive plasmids
(108Copies/ml) real-time fluorescence quantitative RT-PCR product after ten times of gradient dilutions, Lane 7 are negative control.
Fig. 4 is that this kit is used to detect the example for making a definite diagnosis enterovirus infection patient's stool sample EV71 virus-positives,
1. EV is positive for eight connecting legs, and internal standard is positive.
Fig. 5 is that this kit is used to detect the example for making a definite diagnosis enterovirus infection patient's stool sample EV71 virus-positives,
2. EV71 is positive for eight connecting legs.
Fig. 6 is that this kit is used to detect the example for making a definite diagnosis enterovirus infection patient's stool sample EV71 virus-positives,
7. eight connecting legs are negative control, EV is negative, and internal standard is positive
Fig. 7 is that this kit is used to detect the example for making a definite diagnosis enterovirus infection patient's stool sample EV71 virus-positives,
8. eight connecting legs are positive control, EV is positive, and internal standard is positive.
Specific implementation mode
The present invention is further elaborated to the present invention in the attached drawing of specific embodiment combination below, but these embodiments are only limitted to
Illustrate the present invention and does not limit the scope of the invention.
Embodiment 1
A kind of enterovirus parting detecting reagent referring to Fig. 1, including：EV partings detect mixed liquor, enzyme mixation, EV sun
Property reference substance, negative controls, inner mark solution.EV partings detection mixed liquor, which is loaded into, to be encoded in 1.-eight connecting legs 8., wherein
Contain PCR reaction buffers (magnesium chloride containing and triphosphate deoxyribose nucleotide mixture etc.) in each of eight connecting legs pipe and draws
Physical prospecting needle mixed liquor (probe of each combination primer and corresponding different fluorescent markers), eight connecting legs 1. -8. in different primer visit
Needle mixed liquor is：①EV+Rnase P,②EV71+CVA16+CVA6,③CVA10+CVA2+CVA5,④CVA4+CVA9+
CVA12,⑤Echo6+Echo9+Echo30,⑥CVB2+CVB3+CVB4,⑦EV+Rnase P,⑧EV+Rnase P；Enzyme mixes
Liquid contains heat-resisting Taq archaeal dna polymerases, RNase inhibitor and MMLV reverse transcriptases；Negative controls are autoclave sterilization
DEPC (pyrocarbonic acid diethyl ester) handles water；EV positive reference substances are to include enterovirus 5 ' to hold non-transcribed code area RNA sequence
Virus-like particle solution；Inner mark solution is the virus-like particle solution for including ribonuclease P (Rnase P) sequence.
Each group sense primer and downstream primer and the probe sequence of corresponding different fluorescent markers in primed probe mixed liquor
Row are as follows：
Sense primer EV-F:5 '-CCCTGAATGCGGCTAATCC-3 ',
Downstream primer EV-R:5 '-ATTGTCACCATAAGCAGCCA-3 ',
Specific probe EV-P:5 '-ROX-AACCGACTACTTTGGGTGTCCGTGTTTC-BHQ2-3 ',
Sense primer EV71-F:5 '-GAGYATGATYGARACACGCTG-3 ',
Downstream primer EV71-R:5 '-CGCTCTRCTRAAGAARCTATC-3 ',
Specific probe EV71-P:5 '-FAM-AACTCGCACAGTACRGCTGARACCAC-BHQ1-3 ',
Sense primer CVA16-F:5 '-CCCAATGGYGAGYTAGTCCCC-3 ',
Downstream primer CVA16-R:5 '-GTTGGTRGCWGTYTGCCAAGC-3 ',
Specific probe CVA16-P:5 '-HEX-AGTAYATGTATGTCCCRCCAGGGGCT-BHQ1-3 ',
Sense primer CVA6-F:5 '-AARCCRGATAGYAGGAAATC-3 ',
Downstream primer CVA6-R:5 '-GGGGTGGATCACTCAATTTTG-3 ',
Specific probe CVA6-P:5 '-ROX-CAATGGCAGACTGCYACYAAYCCGTCG-BHQ2-3 ',
Sense primer CVA10-F:5 '-GAGACTGGACGYGTRCCA-3 ',
Downstream primer CVA10-R:5 '-GGGTYTCAATCATGTTYTCATCTG-3 ',
Specific probe CVA10-P:5 '-FAM-CAGAGACRGGTGCCACWTCTAAYGCC-BHQ1-3 ',
Sense primer CVA2-F:5'-AGTCGRCCAGTGTCTCAYTCA-3'
Downstream primer CVA2-R：5'-TACACGCCCRGTCTCAATGG-3'
Specific probe CVA2-P:5'-HEX-AACAGCTGCWAACACCCAGGTRAGCC-BHQ1-3'
Sense primer CVA5-F:5'-AAGCGGCGGAGACAGGAG-3'
Downstream primer CVA5-R:5'-CCATGCCTGTTGACCACACA-3'
Specific probe CVA5-P:5'-ROX-CAAAYGCCACCGAYGARAGCATGA-BHQ2-3'
Sense primer CVA4-F:5'-GGRTTYTCAAACTGGGATATTG-3'
Downstream primer CVA4-R:5'-CGCATRTATGTRAATGCYTCAAG-3'
Specific probe CVA4-P:5'-FAM-CATTATGGCRTTTGTGCAAYTGCG-BHQ1-3'
Sense primer CVA9-F:5'-CCYAGYATYTTCTGGACRGA-3'
Downstream primer CVA9-R：5'-GCTRTATGCATTYCCTATACTRATGA-3'
Specific probe CVA9-P:5'-HEX-AACGCACCAGCRCGCATGTCAATC-BHQ1-3'
Sense primer CVA12-F:5'-CGCCGYAAGCTAGAGATCTTCA-3'
Downstream primer CVA12-R:5'-GACTGGTDTTYCCRTTRCGCTC-3'
Specific probe CVA12-P:5'-ROX-CATGCGCTTTGAYGCAGAGTTCACTT-BHQ2-3'
Sense primer Echo6-F:5'-TYAGRCAYGTGAATGACAAAACAA-3'
Downstream primer Echo6-R:5'-AYGCTTTCACGTGYTTTGGC-3'
Specific probe Echo6-P:5'-FAM-AGYCCHATWACRAGCAAAGTKCGCA-BHQ1-3'
Sense primer Echo9-F:5'-CGYTTTGATGCRTGGGAGAT-3'
Downstream primer Echo9-R:5'-AAGCGCAAGTATGTRAACATCTC-3'
Specific probe Echo9-P:5'-HEX-ACATGGTCCAAYTGCGCCGCA-BHQ1-3'
Sense primer Echo30-F:5'-CAGTGGCGTATTGAGCGATGTA-3'
Downstream primer Echo30-R:5'-ATCAACTACCACACCAGATCAGAGTC-3'
Specific probe Echo30-P:5'-ROX-CACGCCGCTCTACCCATAAAGTTTTCTATTGA-BHQ2-3'
Sense primer CVB2-F:5'-GATCAGCATGTGTGTTTTACACGA-3'
Downstream primer CVB2-R:5'-GCCTGTCTAACACTCACCTTCCA-3'
Specific probe CVB2-P:5'-FAM-TACACCAACAGCAAAAATGCAGCCAA-BHQ1-3'
Sense primer CVB3-F:5'-CCAGTAMCAGATAAAGTGGATTCRTA-3'
Downstream primer CVB3-R：5'-AGGAARGGTATRGACATGCGTG-3'
Specific probe CVB3-P:5'-HEX-CCAAGTGTGTTCTGGACAGAGGGTAATGC-BHQ1-3'
Sense primer CVB4-F:5'-CCGAACAAATCCCAGCCTTA-3'
Downstream primer CVB4-R:5'-CTGCATCGTGTCACTTGGGT-3'
Specific probe CVB4-P:5'-ROX-CAGCCGTTGAAACTGGGCATACCTCTC-BHQ2-3'
Sense primer RNase P-F:5'-AGATTTGGACCTGCGAGCG-3'
Downstream primer RNase P-R：5'-GAGCGGCTGTCTCCACAAGT-3'
Specific probe RNase P-P:5'-HEX-TTCTGACCTGAAGGCTCTGCGCG-BHQ1-3'
The end of specific probe sequence 5 ' of wherein EV, EV71, CVA10, CVA4, Echo6, CVB2 use FAM fluorophors
BHQ1 quenching groups label corresponding with fluorophor is respectively adopted in label, 3 ' ends；CVA16,CVA2,CVA9,Echo9,CVB3
The end of specific probe sequence 5 ' marked using HEX fluorophors, 3 ' ends are respectively adopted BHQ1 corresponding with fluorophor and are quenched
Group marks；The end of specific probe sequence 5 ' of CVA6, CVA5, CVA12, Echo30, CVB4 are marked using ROX fluorophors,
BHQ2 quenching groups label corresponding with fluorophor is respectively adopted in 3 ' ends.
Enterovirus parting detecting reagent provided by the invention need to store under the conditions of -20 DEG C, reduce to the greatest extent and freeze repeatedly
Melt；EV partings detection mixed liquor need to be protected from light condition preservation.
Embodiment 2
1 materials and methods
1.1 clinical samples and viral nucleic acid：
Clinical sample other Ji Jia hospitals brothers mouthful in Zhejiang University Medical College The First Affiliated Hospital and Zhejiang Province
The stool sample of sick patient diagnosed and suspected patient is transported to laboratory after sample collection.EV71, CVA16, CVA6, CVA10 and
Other enteroviruses for example 5 type of Coxsackie virus A, 2 type of Coxsackie virus A, Coxsackie virus B 1-B4 types, angstrom 9 type of gram virus, angstrom
Gram 30 type of virus and influenza A virus, influenza B virus, Respiratory Syncytial Virus(RSV), bocavirus, mycoplasma, golden yellow
The positive nucleic acid of staphylococcus etc. is provided by infectious disease diagnosis and treatment National Key Laboratory.
1.2 primers and probe
Downloaded from NCBI gene pools cover domestic and international various other enterovirus and EV71, CVA16, CVA6, CVA10,
The a plurality of gene sequence of CVA2, CVA5, CVA4, CVA9, CVA12, Echo6, Echo9, Echo30, CVB2, CVB3, CVB4 virus
Row.Tetraploid rice is carried out to it using BioEditor softwares, determines the above virus genomic conserved region.Use Primer
For 3.0 softwares of Express in primer and the Taqman probes of its conserved regions design high degree of specificity, primer and probe sequence is logical
Blast verifications are crossed, there is preferably specificity.The primer probe sequence finally screened after design is as described above, primer and probe is entrusted
Hold in the palm Sangon Biotech's synthesis.
The extraction of 1.3 viral nucleic acids：
It takes about 0.2g fecal samples to be added in EP pipes, the physiological saline of 1.5ml is added, shake mixing 3 times, each 10s, so
After be stored at room temperature 10min, with 8000r/min centrifuge 5min.It draws 200 μ l supernatants to be added in EP pipes, using Geneaid companies
Viral Nucleic Acid extraction KitII or QIAGEN companies Rneasy Mini Kit, according to kit
Specification extracts, and takes the sample to be tested nucleic acid that 5ul has been extracted as template.
The optimization of 1.4 reaction systems and condition：
Reaction total volume be 25 μ l, wherein eight connecting legs often pipe include pre- packing EV partings detection 19 μ l of mixed liquor, use
Before need to set on centrifuge and centrifuge, 1 μ l of enzyme mixation, 5 μ l of template.It is detected, reacts on ABI7500 fluorescence quantitative PCR instruments
Parameter is：50 DEG C of reverse transcription, 15min；95 DEG C of 5min thermal startings, then 95 DEG C of 15s, 55 DEG C of 45s, fluorescence inspection is carried out at 55 DEG C
It surveys, carries out 40 cycles altogether.
As a result judge：Fluoroscopic examination model F AM, VIC, ROX fluorescence, baseline adjustment is selected to take the fluorescence letter of 3-15 cycle
Number average value, threshold value setting are just above the peak of negative controls with threshold line, and sample is in typical amplification curve, is judged
For the positive.Without typical amplification curve, it is judged as feminine gender.The Optimum Experiment of system, using same concentrations positive nucleic acid as template
Reaction system in, primer and probe adjust within the scope of 0.1~1 μM of final concentration, using the preferred primer and probe of matrix method
Optimum proportioning selects best primer and probe concentration according to minimum Ct values and highest fluorescence intensity value added (Δ Rn).
1.5 specificity, sensibility and repetitive test
Detect EV71, CVA16, CVA6, CVA10 virus and other enteroviruses such as Coxsack disease respectively using this kit
Malicious A5 types, 2 type of Coxsackie virus A, Coxsackie virus B 1-B4 types, angstrom 9 type of gram virus, angstrom 30 type of gram virus, to verify this reagent
Covering detectability of the box to different subtype enterovirus.The hylon of EV71, CVA16, CVA6, CVA10 virus is selected respectively
Sour (being identified by gene sequencing) and other enteroviruses such as 5 type of Coxsackie virus A, 2 type of Coxsackie virus A, Coxsack disease
Malicious B1-B4 types, angstrom 9 type of gram virus, angstrom 30 type of gram virus and influenza A virus, influenza B virus, respiratory syncystial disease
The positive nucleic acid of poison, bocavirus, mycoplasma, staphylococcus aureus etc. verifies its specificity with this kit；To having demarcated
After the EV positive pseudovirion samples dilution of copy number (copies/ml), it is sensitive to compare its for parallel carry out Fluorescence PCR
Degree.It is detected in addition, making 3 repetitions to the EV positive pseudovirion samples of each prescribed concentration, obtained Ct values calculate it
Standard deviation and the coefficient of variation verify the repeatability of this method.
2 results
2.1 reaction systems and reaction condition
The reaction total volume of this method is 25 μ l, and wherein fluorescent PCR detects 19 μ l (wherein PCR reaction buffers of mixed liquor
12.5 μ l, primed probe mixed liquor and water are supplemented to 19 μ l；EV,EV71,CVA6,CVA2,CVA5,CVA4,CVA9,Echo6,
Echo9, Echo30, CVB2, CVB3, RNase P primers and correspondent probe concentration ratio are 2:1, CVAl6, CVA10, CVB4 primer
It is 3 with correspondent probe concentration ratio:2, CVA12 primers and correspondent probe concentration ratio are 1:1), 1 μ l of enzyme mixation, 5 μ l of template, add
Water complements to 25 μ l.It is detected on ABI7500 fluorescence quantitative PCR instruments, response parameter is：50 DEG C of reverse transcription, 15min；95
DEG C 5min thermal startings, then 95 DEG C of 15s, 55 DEG C of 45s, fluoroscopic examination is carried out at 55 DEG C, carries out 40 cycles altogether.It can get minimum
Ct values and highest fluorescence intensity.
2.2 specific test
Enterovirus parting that the present invention establishes detection box to enterovirus and EV71, CVAl6, CVA6, CVA10, CVA2,
CVA5, CVA4, CVA9, CVA12, Echo6, Echo9, Echo30, CVB2, CVB3, CVB4 virus all have preferable specificity.
This kit can detect the enterovirus of the above different subtype, and mutual no cross reaction.
Primed probe in the present invention and other enteroviruses such as enterovirns type 71, coxsackie virus A 16-type, Coxsack
Viral A6 types, 10 type of Coxsackie virus A, 1 type of Coxsackie virus B, Coxsackie virus type B3, angstrom 30 type of gram virus and A type stream
The equal no cross reaction of positive nucleic acid of Influenza Virus, Respiratory Syncytial Virus(RSV), bocavirus.With other enteroviruses such as Coxsack disease
Malicious A5 types, 2 type of Coxsackie virus A, Coxsackie virus B 1-B4 types, angstrom 9 type of gram virus, angstrom 30 type of gram virus and Flu-A disease
The equal no cross reaction such as poison, influenza B virus, Respiratory Syncytial Virus(RSV), bocavirus, mycoplasma, staphylococcus aureus.
2.3 sensitivity tests
After having demarcated EV positive pseudovirions ten times of gradient dilutions of sample of copy number (copies/ml), tried with this
Agent box is detected, and as a result shows that this method detection sensitivity reaches 103copies/ml.As a result referring to Fig. 2.Take real-time fluorescence
3 μ l, 120V constant pressure electrophoresis 20min of quantitative RT-PCR product, gel imaging system are taken pictures.As a result referring to Fig. 3.Wherein Lane M are
DL2000Marker, Lane 1-6 are respectively segment (108Copies/ml) real-time fluorescence quantitative RT-PCR after ten times of gradient dilutions
Product, Lane 7 are negative control.Electrophoretic band is single, and brightness step is clearly demarcated, illustrates that this kit specificity is good, sensibility
It is high.
2.4 repetitive test
Take the EV positive pseudovirions sample (final concentration of 10 for having demarcated copy number (copies/ml)6copies/ml、
105copies/ml、104Copies/ml), make 3 repetitions to the sample of each concentration to detect, as a result different nucleic acid concentrations are each
From detection Ct value standard deviations between 0.30~0.33, the coefficient of variation is below 1.24%, has preferable repeated (result
It is shown in Table 1).
1 multiplex real-time reverse transcriptase PCR of table detects the repetitive test of EV positive pseudovirions
Embodiment 3
" 13 " key special subjects-infectious disease pathogens are mainly relied on to detect skill the detection of clinical sample using this kit
Art platform project and Zhejiang Province's public good technology application study project.The clinical sample of acquisition is mainly derived from March, 2013 extremely
It other Ji Jia hospitals hand-foot-and-mouth disease patient and is doubted in Zhejiang University Medical College The First Affiliated Hospital and Zhejiang Province between in April, 2014
Like patient's stool sample.It randomly selects 200 enterovirus positive samples and uses the enterovirus parting kit in the present invention
It is verified, testing result is as follows：Enterovirus is 200 positive, positive coincidence rate 100%；Wherein EV71 virus-positives 36,
Accounting 18.0%；CVA16 virus-positives 9, accounting 4.5%；CVA6 virus-positives 102, accounting 51.0%；CVA10 viruses
It is 18 positive, accounting 9.0%；CVA2 virus-positives 9, accounting 4.5%；CVA5 virus-positives 6, accounting 3.0%；Echo9
Virus-positive 3, accounting 1.5%；CVA9 virus-positives 2, accounting 1.0%；CVA12, Echo30, CVB3, CVB4 virus sun
1 each, accounting 0.5% of property；The positive is not detected in CVA4, Echo6, CVB2 virus.Total 189 enterovirus positive samples are realized
Parting, parting success rate are up to 94.5%.This kit makes a definite diagnosis enterovirus infection patient's stool sample EV71 diseases for detecting
Malicious positive example as shown in figs. 4-7, wherein：1. EV is positive for eight connecting legs, and internal standard is positive, sees Fig. 4；2. EV71 is positive, Fig. 5 is seen；
6. 3. being feminine gender；7. being negative control, EV is negative, and internal standard is positive, sees Fig. 6；8. being positive control, EV is positive, internal standard sun
Property, see Fig. 7.
Sequence table
<110>Zhejiang University
<120>A kind of enterovirus parting detecting reagent
<160> 51
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence (Unknown)
<400> 1
ccctgaatgc ggctaatcc 19
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Unknown)
<400> 2
attgtcacca taagcagcca 20
<210> 3
<211> 28
<212> DNA
<213>Artificial sequence (Unknown)
<400> 3
aaccgactac tttgggtgtc cgtgtttc 28
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence (Unknown)
<400> 4
gagyatgaty garacacgct g 21
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence (Unknown)
<400> 5
cgctctrctr aagaarctat c 21
<210> 6
<211> 26
<212> DNA
<213>Artificial sequence (Unknown)
<400> 6
aactcgcaca gtacrgctga raccac 26
<210> 7
<211> 21
<212> DNA
<213>Artificial sequence (Unknown)
<400> 7
cccaatggyg agytagtccc c 21
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence (Unknown)
<400> 8
gttggtrgcw gtytgccaag c 21
<210> 9
<211> 26
<212> DNA
<213>Artificial sequence (Unknown)
<400> 9
agtayatgta tgtcccrcca ggggct 26
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence (Unknown)
<400> 10
aarccrgata gyaggaaatc 20
<210> 11
<211> 21
<212> DNA
<213>Artificial sequence (Unknown)
<400> 11
ggggtggatc actcaatttt g 21
<210> 12
<211> 27
<212> DNA
<213>Artificial sequence (Unknown)
<400> 12
caatggcaga ctgcyacyaa yccgtcg 27
<210> 13
<211> 18
<212> DNA
<213>Artificial sequence (Unknown)
<400> 13
gagactggac gygtrcca 18
<210> 14
<211> 24
<212> DNA
<213>Artificial sequence (Unknown)
<400> 14
gggtytcaat catgttytca tctg 24
<210> 15
<211> 26
<212> DNA
<213>Artificial sequence (Unknown)
<400> 15
cagagacrgg tgccacwtct aaygcc 26
<210> 16
<211> 21
<212> DNA
<213>Artificial sequence (Unknown)
<400> 16
agtcgrccag tgtctcaytc a 21
<210> 17
<211> 20
<212> DNA
<213>Artificial sequence (Unknown)
<400> 17
tacacgcccr gtctcaatgg 20
<210> 18
<211> 26
<212> DNA
<213>Artificial sequence (Unknown)
<400> 18
aacagctgcw aacacccagg tragcc 26
<210> 19
<211> 18
<212> DNA
<213>Artificial sequence (Unknown)
<400> 19
aagcggcgga gacaggag 18
<210> 20
<211> 20
<212> DNA
<213>Artificial sequence (Unknown)
<400> 20
ccatgcctgt tgaccacaca 20
<210> 21
<211> 24
<212> DNA
<213>Artificial sequence (Unknown)
<400> 21
caaaygccac cgaygaragc atga 24
<210> 22
<211> 22
<212> DNA
<213>Artificial sequence (Unknown)
<400> 22
ggrttytcaa actgggatat tg 22
<210> 23
<211> 23
<212> DNA
<213>Artificial sequence (Unknown)
<400> 23
cgcatrtatg traatgcytc aag 23
<210> 24
<211> 24
<212> DNA
<213>Artificial sequence (Unknown)
<400> 24
cattatggcr tttgtgcaay tgcg 24
<210> 25
<211> 20
<212> DNA
<213>Artificial sequence (Unknown)
<400> 25
ccyagyatyt tctggacrga 20
<210> 26
<211> 26
<212> DNA
<213>Artificial sequence (Unknown)
<400> 26
gctrtatgca ttycctatac tratga 26
<210> 27
<211> 24
<212> DNA
<213>Artificial sequence (Unknown)
<400> 27
aacgcaccag crcgcatgtc aatc 24
<210> 28
<211> 22
<212> DNA
<213>Artificial sequence (Unknown)
<400> 28
cgccgyaagc tagagatctt ca 22
<210> 29
<211> 22
<212> DNA
<213>Artificial sequence (Unknown)
<400> 29
gactggtdtt yccrttrcgc tc 22
<210> 30
<211> 26
<212> DNA
<213>Artificial sequence (Unknown)
<400> 30
catgcgcttt gaygcagagt tcactt 26
<210> 31
<211> 24
<212> DNA
<213>Artificial sequence (Unknown)
<400> 31
tyagrcaygt gaatgacaaa acaa 24
<210> 32
<211> 20
<212> DNA
<213>Artificial sequence (Unknown)
<400> 32
aygctttcac gtgytttggc 20
<210> 33
<211> 25
<212> DNA
<213>Artificial sequence (Unknown)
<400> 33
agycchatwa cragcaaagt kcgca 25
<210> 34
<211> 20
<212> DNA
<213>Artificial sequence (Unknown)
<400> 34
cgytttgatg crtgggagat 20
<210> 35
<211> 23
<212> DNA
<213>Artificial sequence (Unknown)
<400> 35
aagcgcaagt atgtraacat ctc 23
<210> 36
<211> 21
<212> DNA
<213>Artificial sequence (Unknown)
<400> 36
acatggtcca aytgcgccgc a 21
<210> 37
<211> 22
<212> DNA
<213>Artificial sequence (Unknown)
<400> 37
cagtggcgta ttgagcgatg ta 22
<210> 38
<211> 26
<212> DNA
<213>Artificial sequence (Unknown)
<400> 38
atcaactacc acaccagatc agagtc 26
<210> 39
<211> 32
<212> DNA
<213>Artificial sequence (Unknown)
<400> 39
cacgccgctc tacccataaa gttttctatt ga 32
<210> 40
<211> 24
<212> DNA
<213>Artificial sequence (Unknown)
<400> 40
gatcagcatg tgtgttttac acga 24
<210> 41
<211> 23
<212> DNA
<213>Artificial sequence (Unknown)
<400> 41
gcctgtctaa cactcacctt cca 23
<210> 42
<211> 27
<212> DNA
<213>Artificial sequence (Unknown)
<400> 42
caatggcaga ctgcyacyaa yccgtcg 27
<210> 43
<211> 26
<212> DNA
<213>Artificial sequence (Unknown)
<400> 43
ccagtamcag ataaagtgga ttcrta 26
<210> 44
<211> 22
<212> DNA
<213>Artificial sequence (Unknown)
<400> 44
aggaarggta trgacatgcg tg 22
<210> 45
<211> 29
<212> DNA
<213>Artificial sequence (Unknown)
<400> 45
ccaagtgtgt tctggacaga gggtaatgc 29
<210> 46
<211> 20
<212> DNA
<213>Artificial sequence (Unknown)
<400> 46
ccgaacaaat cccagcctta 20
<210> 47
<211> 20
<212> DNA
<213>Artificial sequence (Unknown)
<400> 47
ctgcatcgtg tcacttgggt 20
<210> 48
<211> 27
<212> DNA
<213>Artificial sequence (Unknown)
<400> 48
cagccgttga aactgggcat acctctc 27
<210> 49
<211> 19
<212> DNA
<213>Artificial sequence (Unknown)
<400> 49
agatttggac ctgcgagcg 19
<210> 50
<211> 20
<212> DNA
<213>Artificial sequence (Unknown)
<400> 50
gagcggctgt ctccacaagt 20
<210> 51
<211> 23
<212> DNA
<213>Artificial sequence (Unknown)
<400> 51
ttctgacctg aaggctctgc gcg 23
Claims (3)
1. a kind of enterovirus parting detecting reagent, the enterovirus parting includes enterovirus and its multiple hypotypes：
EV71、CVAl6、CVA6、CVA10、CVA2、CVA5、CVA4、CVA9、CVA12、Echo6、Echo9、Echo30、CVB2、CVB3、
CVB4, the kit include enterovirus parting detection mixed liquor, enzyme mixation, EV positive reference substances, negative controls, internal standard
Solution, which is characterized in that wherein enterovirus parting detection mixed liquor, which is loaded into, is encoded in 1.-eight connecting legs 8., wherein eight
Contain PCR reaction buffers and primed probe mixed liquor in each of connecting leg pipe, number 1.-eight connecting legs 8. in have different draw
Physical prospecting needle mixed liquor, respectively：①EV+Rnase P,②EV71+CVA16+CVA6,③CVA10+CVA2+CVA5,④CVA4+
CVA9+CVA12,5. Echo6+Echo9+Echo30,6. CVB2+CVB3+CVB4,7. EV+Rnase P, 8. EV+Rnase P,
In 7. be used for detect negative controls, 8. be used for detect EV positive reference substances；Enzyme mixation is containing heat-resisting Taq archaeal dna polymerases, RNA
Enzyme inhibitor and MMLV reverse transcriptases；
The probe sequence of each group sense primer and downstream primer and corresponding different fluorescent markers is such as in primed probe mixed liquor
Under：
Sense primer EV-F：5 '-CCCTGAATGCGGCTAATCC-3 ',
Downstream primer EV-R：5 '-ATTGTCACCATAAGCAGCCA-3 ',
Specific probe EV-P：5 '-ROX- AACCGACTACTTTGGGTGTCCGTGTTTC-BHQ2-3 ',
Sense primer EV71-F：5 '-GAGYATGATYGARACACGCTG-3 ',
Downstream primer EV71-R：5 '-CGCTCTRCTRAAGAARCTATC-3 ',
Specific probe EV71-P：5 '-FAM- AACTCGCACAGTACRGCTGARACCAC-BHQ1-3 ',
Sense primer CVA16-F：5 '-CCCAATGGYGAGYTAGTCCCC-3 ',
Downstream primer CVA16-R：5 '-GTTGGTRGCWGTYTGCCAAGC-3 ',
Specific probe CVA16-P：5 '-HEX- AGTAYATGTATGTCCCRCCAGGGGCT-BHQ1-3 ',
Sense primer CVA6-F：5 '-AARCCRGATAGYAGGAAATC -3 ',
Downstream primer CVA6-R：5 '-GGGGTGGATCACTCAATTTTG -3 ',
Specific probe CVA6-P：5 '-ROX- CAATGGCAGACTGCYACYAAYCCGTCG-BHQ2-3 ',
Sense primer CVA10-F：5 '-GAGACTGGACGYGTRCCA-3 ',
Downstream primer CVA10-R：5 '-GGGTYTCAATCATGTTYTCATCTG-3 ',
Specific probe CVA10-P：5 '-FAM-CAGAGACRGGTGCCACWTCTAAYGCC-BHQ1-3 ',
Sense primer CVA2-F： 5'- AGTCGRCCAGTGTCTCAYTCA-3'
Downstream primer CVA2-R：5'-TACACGCCCRGTCTCAATGG-3'
Specific probe CVA2-P： 5'- HEX- AACAGCTGCWAACACCCAGGTRAGCC-BHQ1-3'
Sense primer CVA5-F： 5'- AAGCGGCGGAGACAGGAG-3'
Downstream primer CVA5-R： 5'- CCATGCCTGTTGACCACACA-3'
Specific probe CVA5-P： 5'-ROX- CAAAYGCCACCGAYGARAGCATGA-BHQ2-3'
Sense primer CVA4-F： 5'- GGRTTYTCAAACTGGGATATTG-3'
Downstream primer CVA4-R： 5'- CGCATRTATGTRAATGCYTCAAG-3'
Specific probe CVA4-P： 5'-FAM- CATTATGGCRTTTGTGCAAYTGCG-BHQ1-3'
Sense primer CVA9-F： 5'- CCYAGYATYTTCTGGACRGA-3'
Downstream primer CVA9-R：5'- GCTRTATGCATTYCCTATACTRATGA-3'
Specific probe CVA9-P： 5'- HEX- AACGCACCAGCRCGCATGTCAATC-BHQ1-3'
Sense primer CVA12-F： 5'- CGCCGYAAGCTAGAGATCTTCA-3'
Downstream primer CVA12-R： 5'- GACTGGTDTTYCCRTTRCGCTC-3'
Specific probe CVA12-P： 5'-ROX- CATGCGCTTTGAYGCAGAGTTCACTT-BHQ2-3'
Sense primer Echo6-F： 5'- TYAGRCAYGTGAATGACAAAACAA-3'
Downstream primer Echo6-R： 5'- AYGCTTTCACGTGYTTTGGC-3'
Specific probe Echo6-P： 5'-FAM- AGYCCHATWACRAGCAAAGTKCGCA-BHQ1-3'
Sense primer Echo9-F： 5'- CGYTTTGATGCRTGGGAGAT-3'
Downstream primer Echo9-R： 5'- AAGCGCAAGTATGTRAACATCTC-3'
Specific probe Echo9-P： 5'-HEX- ACATGGTCCAAYTGCGCCGCA-BHQ1-3'
Sense primer Echo30-F： 5'- CAGTGGCGTATTGAGCGATGTA-3'
Downstream primer Echo30-R： 5'- ATCAACTACCACACCAGATCAGAGTC-3'
Specific probe Echo30-P： 5'-ROX- CACGCCGCTCTACCCATAAAGTTTTCTATTGA-BHQ2-3'
Sense primer CVB2-F： 5'- GATCAGCATGTGTGTTTTACACGA-3'
Downstream primer CVB2-R： 5'- GCCTGTCTAACACTCACCTTCCA-3'
Specific probe CVB2-P： 5'-FAM- TACACCAACAGCAAAAATGCAGCCAA-BHQ1-3'
Sense primer CVB3-F： 5'- CCAGTAMCAGATAAAGTGGATTCRTA-3'
Downstream primer CVB3-R：5'- AGGAARGGTATRGACATGCGTG-3'
Specific probe CVB3-P： 5'-HEX- CCAAGTGTGTTCTGGACAGAGGGTAATGC-BHQ1-3'
Sense primer CVB4-F： 5'- CCGAACAAATCCCAGCCTTA-3'
Downstream primer CVB4-R： 5'- CTGCATCGTGTCACTTGGGT-3'
Specific probe CVB4-P： 5'-ROX- CAGCCGTTGAAACTGGGCATACCTCTC-BHQ2-3'
Sense primer RNase P-F： 5'- AGATTTGGACCTGCGAGCG-3'
Downstream primer RNase P-R：5'- GAGCGGCTGTCTCCACAAGT-3'
Specific probe RNase P-P： 5'- HEX - TTCTGACCTGAAGGCTCTGCGCG-BHQ1-3'；
PCR reaction buffers magnesium chloride containing and triphosphate deoxyribose nucleotide mixture.
2. a kind of enterovirus parting detecting reagent according to right 1, which is characterized in that negative controls are that high temperature is high
The pyrocarbonic acid diethyl ester of pressure sterilizing handles water；EV positive reference substances are to include enterovirus 5 ' to hold non-transcribed code area RNA sequence
Virus-like particle solution；Inner mark solution is the virus-like particle solution for including ribonuclease P sequence.
3. a kind of enterovirus parting detecting reagent according to right 1, which is characterized in that wherein EV partings detection mixing
Liquid need to be kept in dark place；The kit need to store under the conditions of -20 DEG C.
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| CN108531661A (en) * | 2018-06-08 | 2018-09-14 | 东莞市第八人民医院 | A kind of fluorescence quantification PCR primer, probe, kit and the method for detection CVB3 |
| CN109609689B (en) * | 2018-12-18 | 2019-10-08 | 北京卓诚惠生生物科技股份有限公司 | For detecting the nucleic acid reagent, kit and system of enterovirus |
| CN109680100B (en) * | 2018-12-29 | 2022-10-14 | 深圳市刚竹医疗科技有限公司 | Nucleic acid composition for detecting enterovirus, kit and use method of kit |
| CN110760618A (en) * | 2019-11-29 | 2020-02-07 | 广东龙帆生物科技有限公司 | Paraenterovirus type 3 real-time fluorescent quantitative PCR detection primer, probe, detection kit, detection method and application thereof |
| CN111153990B (en) * | 2020-01-15 | 2020-10-27 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | Monoclonal antibody aiming at Echo30, preparation method and application thereof |
| CN112593011A (en) * | 2020-12-25 | 2021-04-02 | 中山大学 | Primer and probe for detecting coxsackie virus B group |
| CN116286803B (en) * | 2023-02-07 | 2023-11-24 | 广州凯普医药科技有限公司 | Detection primer probe combination and gene chip for synchronously detecting 22 hand-foot-mouth viruses |
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| CN106148570A (en) * | 2016-08-31 | 2016-11-23 | 中华人民共和国大榭出入境检验检疫局 | The primer of a kind of enterovirus genus virus screenings and parting and detection method |
| CN106435026A (en) * | 2016-10-13 | 2017-02-22 | 山东省疾病预防控制中心 | Primer set, probe and test kit for detection of enteroviruses |
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| CN106148570A (en) * | 2016-08-31 | 2016-11-23 | 中华人民共和国大榭出入境检验检疫局 | The primer of a kind of enterovirus genus virus screenings and parting and detection method |
| CN106435026A (en) * | 2016-10-13 | 2017-02-22 | 山东省疾病预防控制中心 | Primer set, probe and test kit for detection of enteroviruses |
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