CN107513584A - A kind of five heavy fluorescence quantitative kits for detecting enterovirus - Google Patents

A kind of five heavy fluorescence quantitative kits for detecting enterovirus Download PDF

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CN107513584A
CN107513584A CN201710875664.6A CN201710875664A CN107513584A CN 107513584 A CN107513584 A CN 107513584A CN 201710875664 A CN201710875664 A CN 201710875664A CN 107513584 A CN107513584 A CN 107513584A
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enterovirus
cva6
cva10
sense primer
virus
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陈瑜
谢国良
崔大伟
楼滨
郑书发
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Zhejiang University ZJU
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Abstract

A kind of five heavy fluorescence quantitative kits for detecting enterovirus of this offer, four hypotypes of enterovirus are EV71, CVAl6, CVA6, CVA10.The present invention uses one-step method real-time fluorescent quantitative RT-PCR technology, using EV and the primer and fluorescence labeling probe of EV71, CVAl6, CVA6, CVA10 virus high special, detect the presence of enterovirus simultaneously from stool sample by a PCR reaction, and Viral typing identification is carried out simultaneously, it is more convenient rapid, more cost-effective than substance fluorescence quantifying PCR method.The present invention carries out real-time accurate quantitative analysis to the virus of detection, according to the titre of virus infection, provides instrument for clinic early diagnosis, reference frame is provided for the formulation of clinical treatment.It can be applied to the research that enterovirus causes the epidemiology of the laboratory emergency diagnosis of epidemic outbreaks, enterovirus rapid screening parting, clinical diagnosis and hand-foot-and-mouth disease.

Description

A kind of five heavy fluorescence quantitative kits for detecting enterovirus
Technical field
The invention belongs to biological technical field, is related to fluorescence quantitative RT-PCR detecting kit, and in particular to one kind detection intestines Five heavy fluorescence quantitative kits of road virus, it is that the weight real-time fluorescence quantitative RT-PCR of one-step method five detects in same reaction tube The detection of nucleic acids of enterovirus and its four hypotypes (EV71, CVAl6, CVA6, CVA10) in patient's excrement or oropharyngeal swab specimen Method, it can be applied to enterovirus and cause the laboratory emergency diagnosis of epidemic outbreaks, enterovirus rapid screening parting, clinic to be examined The research of disconnected and hand-foot-and-mouth disease epidemiology.
Background technology
Enterovirus belongs to Picornaviridae enterovirus genus, is traditionally divided into poliovirus (Poliovirus, PV), ECHO virus (Echovirus, Echo), Coxsackie virus A, B groups (CoxsackievirusA, B, CVA, B) and newtype enteroviru.In newest enterovirus classification, International Commission on Virus Classification (ICTV) is according to biology Learn and hereditary capacity is classified as 4 groups, respectively enterovirus A, B, C, D groups.So far the enterovirus serotype reported More than hundred kinds.
Hand-foot-and-mouth disease (hand, foot and mouth disease, HFMD) be as caused by infecting Human enterovirus virus, Using hand, foot, mouth, buttocks occur fash symptom as main clinic symptoms common transmittable disease, mainly based on mild, but severe and Death also when have been reported that.According to the morbidity of CDC whole nation notifiable infectious diseases, necrology Latest data shows (http://www.chinacdc.cn/tjsj/fdcrbbg/), it is big from 2008 in July, 2016, China 15,432,764 hand-foot-and-mouth disease examples of Lu Gongyou, wherein 3,486 death.Infection situation of the hand-foot-mouth disease in China is still tight It is high.
In conventional most report, the pathogen for causing hand-foot-and-mouth disease is enterovirns type 71 (EV71) and Ke's Sa Strange virus A 16-type (Coxsackie virus A16, CVA16).And in the range of world in recent years, in addition to EV71 and CVA16 Hand-foot-and-mouth disease case caused by other enteroviruses and epidemic situation event are more and more.Singapore CVA6 in 2008 and CVA10 infects Cause 35.3% hand-foot-and-mouth disease case；Break out in Finland within 2008 and mainly caused by CVA6 infection characterized by loss of finger-nail Hand-foot-and-mouth disease epidemic situation.2010, in France and Finland, the main pathogens of hand-foot-and-mouth disease case epidemic situation were CVA6 and CVA10； 2010 are CVA6 in Japan and Taiwan, the main pathogens for causing hand-foot-and-mouth disease epidemic situation；2012, in India's brothers' mouth The pathogen of sick epidemic situation is mainly CVA16 and CVA6, and EV71 and CVA10 are then seldom.The other types prevalence of enterovirus Frequency more and more higher, all had been reported that in the area such as the Shandong in China, Henan, Chongqing and Shenzhen.Shenzhen area in 2012 by EV71, Hand-foot-and-mouth disease caused by tetra- kinds of cause of diseases of CVA16, CVA6 and CVA10 amounts to and has accounted for the 89.8% of enterovirus.2013-2014 Great change occurs for the main pathogens of In Hangzhou Region of Zhe Jiang Province hand foot and mouth disease, and CVA6 substitutions EV-71 turns into In Hangzhou Region of Zhe Jiang Province detection sun The enterovirus of property rate highest, In Hangzhou Region of Zhe Jiang Province hand-foot-and-mouth disease as caused by tetra- kinds of cause of diseases of EV71, CVA16, CVA6 and CVA10 are total Meter has accounted for the 85.4% of enterovirus.Above-mentioned document report shows that in recent years, domestic and international hand-foot-and-mouth disease cause of disease composes composition Large change, CVA6 and CVA10 are increasingly becoming the main and important pathogen of hand-foot-and-mouth disease.
The hand-foot-and-mouth disease etiological diagnosis market in China 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 waste of diagnostic reagent etc..It can detect enterovirus hence it is imperative that exploitation is a kind of and main infection type is carried out The detection reagent of parting.
Enterovirus detection method mainly includes three classes at present：Virus purification culture, immunological method, molecular biology side Method.Virus purification culture is the goldstandard of viral disease diagnosis, but its is cumbersome, detection time length, and positive rate is relatively low. Immunological method is the main method of traditional parting, but sero-fast source is very limited, can not meet wanting for clinical etiological diagnosis Ask.And the molecular biology method of the correlation technique such as PCR-based obtains tremendous expansion with its advantage such as sensitive, quick.Multiple reality When fluorescent quantitative PCR technique with the advantage such as its totally-enclosed Single tube amplification, simple and efficient, reproducible, real-time quantitative, pollution be few, The defects of overcoming conventional clinical diagnosis, there is the specificity and sensitiveness of height, provided accurately and reliably for clinical diagnosis Laboratory foundation, can be as a kind of effectively and rapidly detection means of clinical etiological diagnosis.
The content of the invention
It is an object of the invention to provide a kind of five heavy fluorescence quantitative kits for detecting enterovirus, are related to a kind of enteron aisle disease The fluorescence quantitative kit of poison and its four hypotypes (EV71, CVAl6, CVA6, CVA10), it is that a kind of weight of one-step method five is real-time Fluorescence quantitative RT-PCR detecting kit.
It is right that this kit includes fluorescent PCR detection mixed liquor, enzyme mixation, enterovirus (EV) positive reference substance, feminine gender According to product, EV plasmid standards for quantitation No. 1-5 number.Wherein contain PCR reaction buffers (magnesium chloride containing and three in fluorescent PCR detection mixed liquor Phosphoric acid deoxyribonucleotide mixture etc.) and primed probe mixed liquor (draw containing EV and five groups of EV71, CVAl6, CVA6, CVA10 The probe of thing and corresponding five kinds of different fluorescence labelings)；Enzyme mixation containing heat-resisting Taq archaeal dna polymerases, RNase inhibitor and MMLV reverse transcriptases；Negative controls handle water for the DEPC (pyrocarbonic acid diethyl ester) of autoclave sterilization；EV positive reference substances To include the virus-like particle that enterovirus 5 ' holds non-transcribed code area RNA sequence；EV plasmid standards for quantitation 1-5 be concentration 1 × 103copies/ml-1×107The copies/ml positive plasmid sample for including enterovirus 5 ' and holding non-transcribed code area DNA sequence dna Product.
The spy of five groups of sense primers and anti-sense primer and corresponding five kinds of different fluorescence labelings in primed probe mixed liquor Pin sequence is as follows：
Sense primer EV-F:5 '-CCCTGAATGCGGCTAATCC-3 ',
Anti-sense primer EV-R:5 '-ATTGTCACCATAAGCAGCCA-3 ',
Specific probe EV-P:5 '-ROX-AACCGACTACTTTGGGTGTCCGTGTTTC-BHQ2-3 ',
Sense primer EV71-F:5 '-GAGYATGATYGARACACGCTG-3 ',
Anti-sense primer EV71-R:5 '-CGCTCTRCTRAAGAARCTATC-3 ',
Specific probe EV71-P:5 '-FAM-AACTCGCACAGTACRGCTGARACCAC-BHQ1-3 ',
Sense primer CVA16-F:5 '-CCCAATGGYGAGYTAGTCCCC-3 ',
Anti-sense primer CVA16-R:5 '-GTTGGTRGCWGTYTGCCAAGC-3 ',
Specific probe CVA16-P:5 '-VIC-AGTAYATGTATGTCCCRCCAGGGGCT-BHQ1-3 ',
Sense primer CVA6-F:5 '-AARCCRGATAGYAGGAAATC-3 ',
Anti-sense primer CVA6-R:5 '-GGGGTGGATCACTCAATTTTG-3 ',
Specific probe CVA6-P:5 '-CY5-CAATGGCAGACTGCYACYAAYCCGTCG-BHQ3-3 ',
Sense primer CVA10-F:5 '-GAGACTGGACGYGTRCCA-3 ',
Anti-sense primer CVA10-R:5 '-GGGTYTCAATCATGTTYTCATCTG-3 ',
Specific probe CVA10-P:5 '-TAMRA-CAGAGACRGGTGCCACWTCTAAYGCC-BHQ2-3 ',
Wherein EV, EV71, CVA16, CVA6, CVA10 specific probe sequence 5 ' end be respectively adopted ROX, FAM, VIC, The different fluorophor marks of 5 kinds of CY5, TAMARA etc., 3 ' ends are respectively adopted BHQ1, BHQ2 or BHQ3 corresponding with fluorophor and quenched The group that goes out marks.
Enterovirus fluorescence quantitative kit provided by the invention need to store under the conditions of -20 DEG C, reduce and freeze repeatedly as far as possible Melt；Fluorescent PCR detection mixed liquor needs lucifuge condition to preserve.
It is a further object to provide above-mentioned detection kit in enterovirus and the nucleic acid of its four hypotypes Applied in detection.Four hypotypes of the enterovirus are respectively EV71, CVA16, CVA6, CVA10.
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.EV plasmid standards for quantitation 1-5 is concentration 1 × 103copies/ml-1 ×107Copies/ml positive plasmid sample, is not required to extract.As when only doing qualitative experiment, EV plasmid standards for quantitation can be not involved in examining Survey.
The extraction of excrement or throat swab sample nucleic acid：Take about 0.2g fecal samples to be added in EP pipes, add 1.5ml physiology Salt solution, concussion are mixed 3 times, each 10s, are then stored at room temperature 10min, and 5min is centrifuged with 8000r/min.Draw 200ml supernatants It is added in EP pipes, carries out nucleic acid extraction.The processing of oropharyngeal swab specimen need to only be directly added into 1.5ml physiological saline, and concussion is mixed After even, the same fecal sample of remaining processing procedure.Nucleic acid extraction uses the Viral Nucleic Acid of Geneaid companies The QIAamp Viral RNAMini Kit of extraction KitII or QIAGEN companies, are carried according to kit specification Take, take the testing sample nucleic acid that 5ul has been extracted as template.
The detection of nucleic acid：The testing sample nucleic acid that 5ul has been extracted is taken as template.Reaction cumulative volume is 25 μ l, wherein glimmering Light PCR detects the μ l of mixed liquor 15, the μ l of enzyme mixation 1, the μ l of template 5, adds water to complement to 25 μ l.In ABI7500 quantitative real time PCR Instruments On detected, 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, 55 DEG C of progress fluoroscopic examinations, carry out 40 circulations altogether.
Fluorescent quantitation result is reported：1. the respective Ct values of EV, EV71, CVAl6, CVA6, CVA10 detection are corresponding corresponding glimmering The virus of signal, detection sample CTIt is worth for 40,0 and during without numerical value, is reported as feminine gender.2. detect sample Ct values≤37, report Accuse as corresponding virus-positive, wherein EV71, CVAl6, CVA6, CVA10 virus-positive, EV virus-positives need to be met simultaneously；Only EV Virus-positive, then it is reported as other enteroviruses positive.3. detect sample Ct values ＞ 37 and the sample less than 40, it is proposed that recheck, Result Ct value≤37 are rechecked, corresponding virus-positive is reported as according to criterion 2..According to the standard curve obtained, meter Calculate the virus quantity (copies/ml) of the corresponding virus of sample to be measured.
This research for highly conserved and various VP1 gene that larger difference be present of enterovirus separately design EV71, CVAl6, CVA6, CVA10 primed probe., can be one in single tube using five heavy real-time fluorescence quantitative RT-PCR of single tube one-step method Secondary response detects enterovirus and its four hypotypes (EV71, CVAl6, CVA6, CVA10) simultaneously, not only greatly saves Reagent consumptive material, shortens detection time, and still have high specificity and sensitivity.
The present invention uses one-step method real-time fluorescent quantitative RT-PCR technology, using EV and EV71, CVAl6, CVA6, CVA10 The primer and fluorescence labeling probe of viral high special, develop for enterovirus and its four hypotype (EV71, CVAl6, CVA6, CVA10) detection five heavy real-time fluorescence quantitative RT-PCR detection reagent kits.The invention is reacted by a PCR, can To detect whether the presence of enterovirus simultaneously from stool sample, and the parting of primary bowel Virus type is carried out simultaneously Identification, it is more convenient rapid, more cost-effective than substance fluorescence quantifying PCR method.Meanwhile the virus of detection in real time accurately determine Amount, according to virus infection titre, provide early diagnosis for clinically patient, for clinical treatment formulation provide reference according to According to；It can be applied to enterovirus and cause the laboratory emergency diagnosis of epidemic outbreaks, enterovirus rapid screening parting, clinical diagnosis And the research of the epidemiology of hand-foot-and-mouth disease.
Brief description of the drawings
Fig. 1 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. 2 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. 3 is this kit EV standard items real-time fluorescence quantitative RT-PCR standard curves.
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.
Fig. 5 is that this kit is used to detect the example for making a definite diagnosis enterovirus infection patient's stool sample CVA6 virus-positives.
Fig. 6 is that this kit is used to detect the reality for making a definite diagnosis the other enteroviruses positives of enterovirus infection patient's stool sample Example.
Embodiment
The accompanying drawing of specific embodiment combination below the present invention is further elaborated the present invention, but these embodiments are only limitted to Illustrate the present invention and do not limit the scope of the invention.
Embodiment 1
A kind of five heavy fluorescence quantitative kits for detecting enterovirus, comprising：Fluorescent PCR detection mixed liquor, enzyme mixation, Primed probe mixed liquor, mark EV positive reference substances, negative controls, EV plasmid standards for quantitation 1-5.Wherein fluorescent PCR detection is mixed Close in liquid and mixed containing PCR reaction buffers (magnesium chloride containing and triphosphate deoxyribose nucleotide mixture etc.) and primed probe Liquid (probe containing five groups of primers of EV and EV71, CVAl6, CVA6, CVA10 and corresponding five kinds of different fluorescence labelings)；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；EV plasmid standards for quantitation 1-5 is positive plasmid sample, and the wherein plasmid concentration of EV plasmid standards for quantitation 1 is 103The plasmid concentration of copies/ml, EV plasmid standards for quantitation 2 is 104The plasmid concentration of copies/ml, EV plasmid standards for quantitation 3 is 105The plasmid concentration of copies/ml, EV plasmid standards for quantitation 4 is 106The plasmid concentration of copies/ml, EV plasmid standards for quantitation 5 is 107copies/ml.Fluorescent PCR detection mixed liquor need to deposit in brown pipe.
The spy of five groups of sense primers and anti-sense primer and corresponding five kinds of different fluorescence labelings in primed probe mixed liquor Pin sequence is as follows：
Sense primer EV-F:5 '-CCCTGAATGCGGCTAATCC-3 ',
Anti-sense primer EV-R:5 '-ATTGTCACCATAAGCAGCCA-3 ',
Specific probe EV-P:5 '-ROX-AACCGACTACTTTGGGTGTCCGTGTTTC-BHQ2-3 ',
Sense primer EV71-F:5 '-GAGYATGATYGARACACGCTG-3 ',
Anti-sense primer EV71-R:5 '-CGCTCTRCTRAAGAARCTATC-3 ',
Specific probe EV71-P:5 '-FAM-AACTCGCACAGTACRGCTGARACCAC-BHQ1-3 ',
Sense primer CVA16-F:5 '-CCCAATGGYGAGYTAGTCCCC-3 ',
Anti-sense primer CVA16-R:5 '-GTTGGTRGCWGTYTGCCAAGC-3 ',
Specific probe CVA16-P:5 '-VIC-AGTAYATGTATGTCCCRCCAGGGGCT-BHQ1-3 ',
Sense primer CVA6-F:5 '-AARCCRGATAGYAGGAAATC-3 ',
Anti-sense primer CVA6-R:5 '-GGGGTGGATCACTCAATTTTG-3 ',
Specific probe CVA6-P:5 '-CY5-CAATGGCAGACTGCYACYAAYCCGTCG-BHQ3-3 ',
Sense primer CVA10-F:5 '-GAGACTGGACGYGTRCCA-3 ',
Anti-sense primer CVA10-R:5 '-GGGTYTCAATCATGTTYTCATCTG-3 ',
Specific probe CVA10-P:5 '-TAMRA-CAGAGACRGGTGCCACWTCTAAYGCC-BHQ2-3 ',
Wherein EV, EV71, CVA16, CVA6, CVA10 specific probe sequence 5 ' end be respectively adopted ROX, FAM, VIC, The different fluorophor marks of 5 kinds of CY5, TAMARA etc., 3 ' ends are respectively adopted BHQ1, BHQ2 or BHQ3 corresponding with fluorophor and quenched The group that goes out marks.
Fluorescence quantitative kit provided by the invention need to reduce multigelation as far as possible in -20 DEG C of storages；Fluorescent PCR detects Mixed liquor needs lucifuge condition to preserve.
Embodiment 2
1 materials and methods
1.1 clinical samples and viral nucleic acid：
Clinical sample other Ji Jia hospitals brothers' mouths in Zhejiang University Medical College The First Affiliated Hospital and Zhejiang Province Sick patient diagnosed and the stool sample of suspected patient, laboratory is transported to after sample collection.EV71, CVA16, CVA6, CVA10 and Other enteroviruses for example the type of Coxsackie virus A 5, the type of Coxsackie virus A 2, Coxsackie virus B 1-B4 types, angstrom type of gram virus 9, angstrom Gram type of virus 30, 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 and covered domestic and international various other enterovirus and EV71, CVA16, CVA6, CVA10 disease The a plurality of gene order of poison.Tetraploid rice is carried out to it using BioEditor softwares, it is determined that virus genomic conservative above Area.Using the softwares of Primer Express 3.0 in the primer and Taqman probes of its conserved regions design high degree of specificity, primer Verify that there is preferably specificity by Blast with probe sequence.The primer probe sequence finally screened after design as above institute State, primer and probe entrusts Sangon Biotech (Shanghai) Co., Ltd. to synthesize.
The extraction of 1.3 viral nucleic acids and standard items quantitative criterion：
Take about 0.2g fecal samples to be added in EP pipes, add 1.5ml physiological saline, shake mixing 3 times, each 10s, so After be stored at room temperature 10min, with 8000r/min centrifuge 5min.The processing of oropharyngeal swab specimen need to only be directly added into 1.5ml physiology salt Water, after concussion mixes, the same fecal sample of remaining processing procedure.200 μ l supernatants are drawn to be added in EP pipes, it is public using Geneaid The Rneasy Mini Kit of the Viral Nucleic Acid extraction KitII or QIAGEN companies of department, according to reagent Box specification is extracted, and takes the testing sample nucleic acid that 5ul has been extracted as template.
EV plasmid standards for quantitation positive nucleic acid fragments are synthesized, are connected to plasmid vector pMDTM19-T Simple Vector (TaKaRa companies), cultivates after conversion.DNA is extracted after identification, utilizes NanoDrop ND-2000 Spectrophotometer measures the concentration of DNA, determines DNA copy number as the quantitative mother liquor of standard items.According to experiment Need, standard items are quantified into mother liquor is diluted to required maximum concentration, and does ten times and be diluted to least concentration, and Cord blood is standby. The optimization of 1.4 5 heavy real-time fluorescence quantitative RT-PCR reaction systems and condition：
Reaction cumulative volume is 25 μ l, wherein the fluorescent PCR detection μ l of mixed liquor 15, the μ l of enzyme mixation 1, the μ l of template 5, adds water to mend Enough to 25 μ l.Detected on ABI7500 quantitative real time PCR Instruments, response parameter is：50 DEG C of reverse transcription, 15min；95℃ 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 circulations altogether.
As a result judge：The baseline adjustment of selection fluoroscopic examination model F AM, VIC, ROX, CY5, TAMARA fluorescence takes 3-15 to follow The fluorescence signal average value of ring, threshold value setting are just above the peak of negative controls with threshold line, and sample expands in typical Increase curve, be judged as the positive.Without typical amplification curve, it is judged as feminine gender.The Optimum Experiment of system, positive with same concentrations For in the reaction system of template, primer and probe adjust nucleic acid in the range of 0.1~1 μM of final concentration, are preferably drawn using matrix method The optimum proportioning of thing and probe, according to minimum CTValue and highest fluorescence intensity value added (Δ Rn) select optimal primer and probe dense Degree.
Specificity, sensitiveness and the replica test of 1.5 5 heavy real-time fluorescence quantitative RT-PCR kits
Detect EV71, CVA16, CVA6, CVA10 virus and other enteroviruses such as COxsackie disease respectively using this kit Malicious A5 types, the type of Coxsackie virus A 2, the type of Coxsackie virus A 4, Coxsackie virus B 1-B4 types, angstrom type of gram virus 9, angstrom gram virus 30 Type, to verify covering detectability of this kit to different subtype enterovirus.Respectively select EV71, CVA16, CVA6, The positive nucleic acid (being identified by gene sequencing) and other enteroviruses such as type of Coxsackie virus A 5, COxsackie of CVA10 viruses Viral A2 types, the type of Coxsackie virus A 4, Coxsackie virus B 1-B4 types, angstrom type of gram virus 9, angstrom type of gram virus 30, and Flu-A The positive nucleic acid of virus, influenza B virus, Respiratory Syncytial Virus(RSV), bocavirus, mycoplasma, staphylococcus aureus etc., Its specificity is verified with this kit；After being diluted to the EV positive pseudovirions sample for having demarcated copy number (copies/ml), Parallel carry out Fluorescence PCR, compares its sensitivity.In addition, the EV positive pseudovirion samples to each prescribed concentration Make 3 repetitions to detect, obtained Ct values calculate its standard deviation and the coefficient of variation, verify the repeatability of this method.
The foundation of 1.6 5 heavy real-time fluorescence quantitative RT-PCR standard curves
Use the EV plasmid standards for quantitation 1 (10 for having demarcated copy number (copies/ml)3Copies/ml), EV plasmid standards for quantitation 2(104Copies/ml), EV plasmid standards for quantitation 3 (105Copies/ml), EV plasmid standards for quantitation 4 (106Copies/ml), EV is quantitative Standard items 5 (107Copies/ml after) carrying out real-time fluorescence quantitative PCR amplification with this kit for template, with standard concentration Logarithm value is X-axis, and period is Y-axis, draws standard curve.
2 results
2.1 5 heavy real-time fluorescence quantitative RT-PCR reaction systems and condition
The reaction cumulative volume of this method is 25 μ l, wherein fluorescent PCR detection μ l (the wherein PCR reaction buffers of mixed liquor 15 The 12.5 μ l and μ l of primed probe mixed liquor 2.5；EV, EV71, CVA6 primer and correspondent probe concentration ratio are 2:1, CVAl6, CVA10 Primer and correspondent probe concentration ratio are 3:2), the μ l of enzyme mixation 1, the μ l of template 5, add water to complement to 25 μ l.Determine in ABI7500 fluorescence Detected in amount PCR instrument, 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 45s, fluoroscopic examination is carried out at 55 DEG C, carries out 40 circulations altogether.Minimum Ct values and highest fluorescence intensity can be obtained.
2.2 specific test
One-step method five that the present invention establishes weight fluorescent quantitative RT-PCR method to enterovirus and EV71, CVA16, CVA6, CVA10 viruses have fabulous specificity, can detect positive clinical sample completely.This kit can detect the intestines of variant hypotype Road virus, and no cross reaction between EV71, CVA16, CVA6, CVA10.
EV71, CVA16, CVA6, CVA10 primed probe and other enteroviruses such as Coxsackie virus A 5 in the present invention Type, the type of Coxsackie virus A 2, the type of Coxsackie virus A 4, Coxsackie virus B 1-B4 types, angstrom type of gram virus 9, angstrom type of gram virus 30, And the equal no cross reaction such as influenza A 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 102copies/ml.As a result referring to Fig. 1.Take real-time fluorescence μ l, 120V the constant pressure electrophoresis 20min of quantitative RT-PCR product 3, gel imaging system are taken pictures.As a result referring to Fig. 2.Wherein Lane M are DL2000 Marker, Lane 1-6 are respectively fragment (108Copies/ml) real time fluorescent quantitative RT- after ten times of gradient dilutions PCR primer, Lane 7 are negative control.Electrophoretic band is single, and brightness step is clearly demarcated, illustrates that this kit specificity is good, quantitative As a result it is relatively accurate.
2.4 replica test
Take the EV positive pseudovirions sample (final concentration of 10 for having demarcated copy number (copies/ml)6copies/ml、 105copies/ml、104Copies/ml), 3 repetition detections are made to the sample of each concentration, as a result various concentrations are respective Detect CTIt is worth standard deviation between 0.40~0.45, the coefficient of variation is below 1.96%, and there is preferably repeatability (the results are shown in Table 1)。
The replica test of the weight real-time fluorescence quantitative RT-PCR detection EV positive pseudovirions of table 1 five
The foundation of 2.5 standard curves
After real-time fluorescence quantitative PCR amplification, using the logarithm value of standard concentration as X-axis, period is Y-axis, draws standard Curve.EV standard items real-time fluorescence quantitative RT-PCR standard curves as shown in figure 3, wherein slope be 3.552, intercept 43.98, Coefficient correlation is 0.995.As can be seen here, the logarithm value of standard concentration has preferable linear relationship with period.
Embodiment 3
" 13 " key special subjects-infectious disease pathogens detection skill is mainly relied on using detection of this kit to clinical sample Art platform project (2017ZX10103008) and Zhejiang Province's public good technology application study project (2017C33044).The clinic of collection Sample is mainly derived from March, 2013 to Zhejiang University Medical College The First Affiliated Hospital between in April, 2014 and Zhejiang Province it 1340 parts altogether of his Ji Jia hospitals hand-foot-and-mouth disease patient and suspected patient stool sample.Using five heavy real-time fluorescences in this method Quantitative RT-PCR verifies that testing result is as follows to the sample being collected into：Enterovirus is positive 1090 parts, positive rate 81.34%.194 parts of EV71 virus-positives, account for the 17.80% of enterovirus；51 parts of CVA16 virus-positives, account for enterovirus 4.68%；550 parts of CVA6 virus-positives, account for the 50.46% of enterovirus；114 parts of CVA10 virus-positives, account for enterovirus 10.46%.The sum of EV71, CVA16, CVA6, CVA10 four kinds of enterovirus types of virus accounts for detection enterovirus 83.39%.Wherein, this kit is used to detect the example for making a definite diagnosis enterovirus infection patient's stool sample EV71 virus-positives such as Shown in Fig. 4；The example that enterovirus infection patient's stool sample CVA6 virus-positives are made a definite diagnosis in detection is as shown in Figure 5；Detection is made a definite diagnosis The positive example of other enteroviruses of enterovirus infection patient stool sample is as shown in Figure 6.Detection positive findings has been reported with it Result is accused completely to be consistent.
Sequence table
<110>Zhejiang University
<120>A kind of five heavy fluorescence quantitative kits for detecting enterovirus
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<170> SIPOSequenceListing 1.0
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<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
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<213>Artificial sequence (Unknown)
<400> 6
aactcgcaca gtacrgctga raccac 26
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<213>Artificial sequence (Unknown)
<400> 7
cccaatggyg agytagtccc c 21
<210> 8
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<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
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<211> 20
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<213>Artificial sequence (Unknown)
<400> 10
aarccrgata gyaggaaatc 20
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<212> DNA
<213>Artificial sequence (Unknown)
<400> 11
ggggtggatc actcaatttt g 21
<210> 12
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<212> DNA
<213>Artificial sequence (Unknown)
<400> 12
caatggcaga ctgcyacyaa yccgtcg 27
<210> 13
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<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
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<211> 26
<212> DNA
<213>Artificial sequence (Unknown)
<400> 15
cagagacrgg tgccacwtct aaygcc 26

Claims (5)

1. a kind of five heavy fluorescence quantitative kits for detecting enterovirus, it is characterised in that mixed liquor, enzyme are detected by fluorescent PCR Mixed liquor, enterovirus positive reference substance, negative controls, wherein No. 1-5 composition of enterovirus plasmid standards for quantitation, fluorescent PCR Contain PCR reaction buffers and primed probe mixed liquor in detection mixed liquor, primed probe mixed liquor contain EV, EV71, CVAl6, The probe of five groups of primers of CVA6, CVA10 and corresponding five kinds of different fluorescence labelings, enzyme mixation containing heat-resisting Taq archaeal dna polymerases, RNase inhibitor and MMLV reverse transcriptases, negative controls handle water, enteron aisle disease for the pyrocarbonic acid diethyl ester of autoclave sterilization Malicious positive reference substance is to include the virus-like particle that enterovirus 5 ' holds non-transcribed code area RNA sequence, and enterovirus is quantitatively marked Quasi- product 1-5 are concentration：103copies/ml-1×107The copies/ml enterovirus 5 ' that includes holds non-transcribed code area DNA The positive plasmid sample of sequence；
The probe sequence of five groups of sense primers and anti-sense primer and corresponding five kinds of different fluorescence labelings in primed probe mixed liquor Row are as follows：
Sense primer EV-F:5 '-CCCTGAATGCGGCTAATCC-3 ',
Anti-sense primer EV-R:5 '-ATTGTCACCATAAGCAGCCA-3 ',
Specific probe EV-P:5 '-ROX-AACCGACTACTTTGGGTGTCCGTGTTTC-BHQ2-3 ',
Sense primer EV71-F:5 '-GAGYATGATYGARACACGCTG-3 ',
Anti-sense primer EV71-R:5 '-CGCTCTRCTRAAGAARCTATC-3 ',
Specific probe EV71-P:5 '-FAM-AACTCGCACAGTACRGCTGARACCAC-BHQ1-3 ',
Sense primer CVA16-F:5 '-CCCAATGGYGAGYTAGTCCCC-3 ',
Anti-sense primer CVA16-R:5 '-GTTGGTRGCWGTYTGCCAAGC-3 ',
Specific probe CVA16-P:5 '-VIC-AGTAYATGTATGTCCCRCCAGGGGCT-BHQ1-3 ',
Sense primer CVA6-F:5 '-AARCCRGATAGYAGGAAATC-3 ',
Anti-sense primer CVA6-R:5 '-GGGGTGGATCACTCAATTTTG-3 ',
Specific probe CVA6-P:5 '-CY5-CAATGGCAGACTGCYACYAAYCCGTCG-BHQ3-3 ',
Sense primer CVA10-F:5 '-GAGACTGGACGYGTRCCA-3 ',
Anti-sense primer CVA10-R:5 '-GGGTYTCAATCATGTTYTCATCTG-3 ',
Specific probe CVA10-P:5’-TAMRA-CAGAGACRGGTGCCACWTCTAAYGCC-BHQ2-3’.
2. a kind of five heavy fluorescence quantitative kits for detecting enterovirus according to claim 1, it is characterised in that wherein ROX, FAM, VIC, CY5, TAMARA 5 is respectively adopted in EV, EV71, CVA16, CVA6, CVA10 end of specific probe sequence 5 ' BHQ1, BHQ2 or BHQ3 quenching group mark corresponding with fluorophor is respectively adopted in the different fluorophors marks of kind, 3 ' ends.
A kind of 3. five heavy fluorescence quantitative kits for detecting enterovirus according to claim 1, it is characterised in that PCR Reaction buffer magnesium chloride containing and triphosphate deoxyribose nucleotide mixture.
A kind of 4. five heavy fluorescence quantitative kits for detecting enterovirus according to claim 1, it is characterised in that reagent Box stores under the conditions of -20 DEG C, reduces multigelation, and fluorescent PCR detection mixed liquor needs lucifuge condition to preserve.
5. a kind of five weight fluorescence quantitative kits for detecting enterovirus according to claim 1 are in enterovirus and its four Applied in the detection of nucleic acids of individual hypotype, four hypotypes of the enterovirus are respectively EV71, CVA16, CVA6, CVA10.
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CN109609689A (en) * 2018-12-18 2019-04-12 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
CN109680100A (en) * 2018-12-29 2019-04-26 深圳市刚竹医疗科技有限公司 Detect the nucleic acid compositions of enterovirus, the application method of kit and kit
CN110863065A (en) * 2019-10-21 2020-03-06 珞可为科技(武汉)有限公司 Kit for detecting four enteroviruses and detection method thereof
CN111321206A (en) * 2020-03-06 2020-06-23 杭州博日科技有限公司 Detection system of quintuple fluorescent PCR (polymerase chain reaction), application and product thereof
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CN109182608A (en) * 2018-11-09 2019-01-11 辽宁佰昊生物科技有限公司 For detecting and/or assisting detection to cause primer sets, reagent, kit and the detection method of hand-foot-and-mouth disease poison CVA16
CN109609689A (en) * 2018-12-18 2019-04-12 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
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
CN109680100A (en) * 2018-12-29 2019-04-26 深圳市刚竹医疗科技有限公司 Detect the nucleic acid compositions of enterovirus, the application method of kit and kit
CN110863065A (en) * 2019-10-21 2020-03-06 珞可为科技(武汉)有限公司 Kit for detecting four enteroviruses and detection method thereof
CN111321206B (en) * 2020-03-06 2023-09-22 杭州博日科技股份有限公司 Detection system of quintuple fluorescent PCR (polymerase chain reaction), application and product thereof
CN111321206A (en) * 2020-03-06 2020-06-23 杭州博日科技有限公司 Detection system of quintuple fluorescent PCR (polymerase chain reaction), application and product thereof
CN111748650A (en) * 2020-06-23 2020-10-09 深圳市艾伟迪生物科技有限公司 Nucleic acid composition for simultaneously detecting four B-group coxsackie virus subtypes and kit and detection method thereof
CN112322796A (en) * 2020-12-10 2021-02-05 广东省妇幼保健院 Visual kit for detecting coxsackievirus A group 6 type nucleic acid and application
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