CN107151711A - A kind of double fluorescent quantitative RT PCR kits for detecting dengue virus and zika virus - Google Patents

A kind of double fluorescent quantitative RT PCR kits for detecting dengue virus and zika virus Download PDF

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CN107151711A
CN107151711A CN201710497287.7A CN201710497287A CN107151711A CN 107151711 A CN107151711 A CN 107151711A CN 201710497287 A CN201710497287 A CN 201710497287A CN 107151711 A CN107151711 A CN 107151711A
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zikv
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kit
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CN107151711B (en
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王翔
温晓红
崔大伟
郭慧慧
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First Peoples Hospital of Huzhou
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Abstract

The present invention provides a kind of Multiplex real-time PCR kit for detecting dengue virus and zika virus.This kit includes quantitative RT-PCR reaction solution, enzyme mixation, primed probe mixed liquor, standard items (DENV and ZIKV), robust positive control product (DENV and ZIKV), weakly positive reference substance (DENV and ZIKV), negative controls.The present invention respectively according to DENV and ZIKV be highly conserved and type between there is smaller difference polyprotein protein regions design the primed probes of DENV and ZIKV high specifics.Using single tube one-step method dual real-time fluorescence quantitative RT-PCR, primary first-order equation dengue fever virus and zika virus can be detected simultaneously in single tube.The kit has higher specificity and sensitivity, can be applied to dengue virus and zika virus cause epidemic outbreaks laboratory emergency diagnosis, rapid screening, clinical diagnosis and the exanthema virus that generates heat to epidemiology research.

Description

A kind of Multiplex real-time PCR kit for detecting dengue virus and zika virus
Technical field
The invention belongs to biological technical field, it is related to a kind of fluorescence quantitative RT-PCR detecting kit, and in particular to Yi Zhongyi Footwork dual real-time fluorescence quantitative RT-PCR detects dengue virus and zika virus in people or mosquito sample in same reaction tube Nucleic acid detection method, can be applied to dengue virus and zika virus cause the laboratory emergency diagnosis of epidemic outbreaks, quick sieve Look into, the research of the epidemiology of clinical diagnosis and dengue virus and zika virus.
Background technology
Dengue virus (dengue virus, DENV) and zika virus (Zika virus, ZIKV) belong to flaviviridae, Flavivirus, genome is single-stranded positive RNA.DENV is divided into 4 kinds of serotypes according to antigenic difference：DENV-1、DENV-2、 DENV-3 and DENV-4, antigenicity between antigenic difference, and each type, which may be present, in the different strains of same type is present necessarily Intersect.Zika virus is divided into according to genotype：Two genotype of Asian type and African type.The natural host bag of dengue virus infection People and mosquito etc. are included, mainly passes through the insect-borne transmissions such as Aedes aegypti and aedes albopictus.Zika virus is dynamic in non-human primates Circulated between thing and mosquito matchmaker, Genus Homo is and universal susceptible in occasional host, and the virus is mainly propagated by yellow-fever mosquito, and Aedes aegypti is Primary vehicle, and the yellow-fever mosquito such as aedes albopictus and aedes africanus can also propagate, and also there is vertical transmission, spread through sex intercourse and blood The modes such as liquid propagation.China is in subtropical area, and southern some areas (such as Guangdong and Guangxi) are Aedes aegypti and lineae ablicantes The Prevalent district of the mosquito matchmaker such as yellow-fever mosquito, particularly yellow-fever mosquito summer and autumn occurred frequently, faces dengue virus and zika virus outbreak of epidemic High-risk areas.
Dengue virus and zika virus are widely current in Perenniporia martius countries and regions, more than 125 countries and There is dengue virus infection in area, and 70% infection population occurs in the Asian-Pacific area, just gradually to spreading all over the world.Zika virus There is the sick outbreak of epidemic in Africa, Asia, America and the pacific island state area, epidemic situation is in quick spreading trend.China in The virus is found in Foshan first within 1978, be found on Hainan, Guangxi, Fujian, Zhejiang, Yunnan and other places later, it is special It is not that peak in the late two decades is presented in Dengue epidemic in Guangdong Province prevalence in 2014, number of the infected is more than 40,000, and number people is dead；2015 Year Taiwan dengue prevalence causes to infect more than 30,000 people, dead people more than 140.Zika virus is in nineteen forty-seven first in Uganda It is found in rhesus monkeys, nineteen fifty-two is separated in the human body of Uganda and Tanzania.Before 2007, the whole world is only reported The sick Sporadic cases of 14 zika viruses, finds zika virus in 2007 on the Ya Pu islands of the pacific island state Micronesia first Epidemic outbreaks.There is the outbreak of epidemic of zika virus in Brazil within 2015, and there is epidemic situation report more than 46 countries and regions thereafter, I State occurred many cases Introduced cases zika virus cases of infection in 2016, and epidemic situation has global spread trend.
Dengue virus infection majority is asymptomatic subclinical infection, and hair patient main clinical characteristics are dengue fever (dengue Fever, DF), dengue hemorrhagic fever (dengue hemorrhagic fever, DHF) and dengue shock syndrome (dengue Shock syndrome, DSS), the death rate of both clinical symptoms is higher after appearance.Due to dengue virus be prone to variation and There are 4 Serotypes, the serum antibody of primary infection person's convalescence only plays certain protective effect to the serotype, to other Serotype infection is capable of the infection of enhanced virus, causes even more serious clinic on the contrary not only without cross-protection Symptom.Zika virus infection asymptomatic person is common, and symptom occurs in only about 20% the infected, and main clinical manifestation is low-heat, joint Pain and skin maculopapule etc., are not easy to the viral disease difference of the heating eruption such as dengue fever.In addition zika virus has extremely strong Neural invasion, its infect with actue infectious polyradiculoneuritis, neonate's microcephalus disease presence necessarily associates, this sick case fatality rate is low, Lifetime immunity can be obtained after infection.
At present, there is no safely and effectively vaccine go through application.Control communication media, prevent bite by mosquitos from being to prevent and treat Dengue The important measures that virus and zika virus infect, early diagnosis, early treatment are to reduce the effective means of the death rate.Dengue virus and stockaded village The detection for blocking virus is main by methods such as Virus culture, Serologic detection, viral nucleic acid detections.Real-time fluorescence quantitative PCR skill Art is overcome conventional clinic and examined with the advantage such as its totally-enclosed Single tube amplification, simple and efficient, reproducible, real-time quantitative, pollution be few Disconnected defect, specificity and sensitiveness with height, accurately and reliably laboratory foundation is provided for clinical diagnosis, can conduct A kind of effectively and rapidly detection means of clinical etiological diagnosis.
Due to dengue virus and zika virus infected patient clinical symptoms and its it is similar be not easy to difference, and in laboratory The mostly monochromatic real-time fluorescence quantitative RT-PCR reagent of detection dengue virus or zika virus, therefore this research is directed to dengue virus Exist between (DENV-1, DENV-2, DENV-3 and DENV-4) and zika virus (Asian type and African type) highly conserved and type compared with The non-structural protein code area of small difference separately designs the primed probe of dengue virus and the universal high specific of zika virus.Adopt With single tube one-step method dual real-time fluorescence quantitative RT-PCR, primary first-order equation dengue virus and stockaded village's card can be detected simultaneously in single tube Virus, is not only greatly saved reagent consumptive material, shortens detection time, while having high specificity and sensitivity.
The content of the invention
It is an object of the invention to provide a kind of dual glimmering for dengue virus and the one-step method of the universal detection of zika virus Light quantitative RT-PCR detecting kit.This kit comprising quantitative RT-PCR reaction solution, enzyme mixation, primed probe mixed liquor, Standard items (DENV and ZIKV), (DENV and ZIKV, plasmid copy number are 2 × 10 to robust positive control product6Copies/mL it is), weak (DENV and ZIKV, plasmid copy number are 2 × 10 to positive reference substance4Copies/mL), negative controls.Container is provided with box Hole, respectively place quantitative RT-PCR reaction liquid pipe, enzyme mixation pipe, primed probe mixing liquid pipe, standard QC (DENV and ZIKV), robust positive control QC (DENV and ZIKV), weakly positive control QC (DENV and ZIKV), negative control QC.
By force, weakly positive control is the positive plasmid sample of the high and low concentration of the conserved region gene sequence comprising DENV and ZIKV Product.The copy number of wherein DENV and ZIKV high concentrations plasmid (i.e. robust positive control product) is 2 × 106Copies/mL, and it is low dense The copy number for spending (i.e. weakly positive reference substance) plasmid is 2 × 104copies/mL。
Wherein quantitative RT-PCR reaction liquid pipe is included：PCR reaction buffers pipe (magnesium chloride containing and triphosphoric acid deoxyribose core Thuja acid mixture etc.), enzyme mixation pipe (containing heat-resisting Taq archaeal dna polymerases, RNase inhibitor and MMLV reverse transcriptases etc.), draw The physical prospecting pin mixing liquid pipe probe of corresponding two kinds of different fluorescence labelings (two groups of primers with), and primed probe mixes liquid pipe and need to be Brown pipe.Negative control is DEPC (pyrocarbonic acid diethyl ester) the processing water of autoclave sterilization, and positive control is DENV and ZIKV Positive plasmid sample.
Multiplex real-time PCR detection sense primer and anti-sense primer and corresponding specific probe sequence are such as Under：
Sense primer DENV-F:5’-GACTAGYGGTTAGAGGAGACCCCTC-3’
Anti-sense primer DENV-R：5’-GCGYTCTGTGCCTGGAWTGAT-3’
Specific probe DENV-P:5’-FAM-AGACCAGAGATCCTGCTGT-MGB-3’
Sense primer ZIKV-F:5’-GACTGGGTACCAACTGGGAGAAC-3’
Anti-sense primer ZIKV-R:5’-GGTCGTTCTCCTCAATCCACAC-3’
Specific probe ZIKV-P:5’-HEX-GAAAGGGAGAATGGATGAC-MGB-3’
5 ' ends of wherein DENV specific probes use FAM fluorescence labelings, and 5 ' ends of ZIKV specific probes are glimmering using HEX Signal；The fluorescent quenching label at 3 ' ends of DENV and ZIKV specific probes uses MGB fluorochrome labels.
Above-mentioned standard product include DENV, ZIKV standard items, and its conserved region gene sequence is as follows：
DENV standard items sequences are：
GACTAGCGGTTAGAGGAGACCCCTCCCATCACTGACAAAACGCAGCAAAAAAGGGGGCCCGAAGCCAGG AGGAAGCTGTACTCCTGGTGGAAGGACTAGAGGTTAGAGGAGACCCCCCCAACACAAAAACAGCATATTGACGCTGG GAAAGACCAGAGATCCTGCTGTCTCTGCAACATCAATCCAGGCACAGAGCGC
ZIKV standard items sequences are：
GACTGGGTACCAACTGGGAGAACCACCTGGTCAATCCATGGAAAGGGAGAATGGATGACCACTGAGGAC ATGCTCATGGTGTGGAATAGAGTGTGGATTGAGGAGAACGACC
The fluorescence quantitative kit that the present invention is provided need to reduce multigelation as far as possible in -20 DEG C of storages；Primed probe is mixed Liquid needs lucifuge condition to preserve.
It is a further object to provide above-mentioned dual fluorescence quantification RT-PCR detection kit in detection DENV With the application in ZIKV nucleic acid.
The application method of kit of the present invention：Positive control and negative control all should be set up in each Samples detection.Mark Easy Dilution (the article No.s of quasi- product TaKaRa companies：9160) it is diluted to 1 × 102-1×107copies/ml。
The extraction of sample nucleic acid：Take 200 μ l serum, blood plasma or urine specimen to be added in EP pipes, carry out nucleic acid extraction.Nucleic acid QIAamp Viral RNA Mini Kit or Qiagen the RNeasy Mini Kit using QIAGEN companies are extracted, according to examination Agent box specification is extracted, and takes the testing sample nucleic acid that 5 μ l have 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 fixed Measure RT-PCR reaction solutions 12.5 μ l, the μ l of enzyme mixation 1, primed probe mixed liquor (20 μm of ol/L, comprising two group-specific primerses and Corresponding two kinds of fluorescence probes) totally 3 μ l, the μ l of template 5, add water and complement to 25 μ l.ABI7500 quantitative real time PCR Instruments (or its The PCR instrument of his Two Colour Fluorescence and the above) on detected, response parameter is：50 DEG C of reverse transcription, 20min；95 DEG C of 5min heat are opened Dynamic, then 95 DEG C of 15s, 60 DEG C of 45s, double fluorescent detection is carried out at 60 DEG C, and 40 circulations are carried out altogether.
Fluorescent quantitation result is reported：1. the respective C that DENV, ZIKV are detectedTThe virus of the corresponding fluorescence labeling of value correspondence, inspection Test sample this CT value is 40,0 and during without numerical value, is reported as feminine gender.2. sample C is detectedTValue<When 35, corresponding virus-positive is reported as. 3. sample C is detectedTValue >=35 and the sample less than 40, it is proposed that recheck, recheck result CTValue ＜, and fluorescence curve is preferably, according to Corresponding virus-positive is reported as according to criterion 2..According to the standard curve obtained, calculate sample DENV to be measured or ZIKV virus quantity (copies/ml).
Usefulness of the present invention is：It is high using DENV and ZIKV with one-step method real-time fluorescent quantitative RT-PCR technology Degree special primer and fluorescence labeling probe, develop the Multiplex real-time PCR detection reagent for DENV and ZIKV detections Box.The invention is reacted by a RT-PCR, can simultaneously have been detected whether from serum, blood plasma and/or urine specimen DENV and ZIKV presence is more convenient rapider, cost-effective than substance fluorescence quantifying PCR method.Meanwhile, the virus to detection is entered The real-time accurate quantitative analysis of row, according to the titre of virus infection, provides early diagnosis for clinically patient, is the system of clinical treatment Reference frame is provided surely；Can be applied to DENV and ZIKV causes the laboratory emergency diagnosis, rapid screening, clinic of epidemic outbreaks to be examined The research of disconnected and epidemiology.
Brief description of the drawings
Fig. 1 is this reagent cartridge configuration schematic diagram, wherein 1 is that quantitative RT-PCR reacts liquid pipe, 2 be enzyme mixation pipe, and 3 be to draw Physical prospecting pin mixes liquid pipe, and 4 be negative control QC, and 5 be standard QC (DENV, ZIKV 2 are managed totally), and 6 be robust positive control QC (DENV, ZIKV 2 are managed totally), 7 be weakly positive control QC (DENV, ZIKV 2 are managed totally).
Fig. 2 is the sensitivity that this kit detects DENV, and from left to right (1-6) is followed successively by 107、106、105、104、103、 102copies/ml。
Fig. 3 is this kit DENV standard items real-time fluorescence quantitative RT-PCR product gel electrophoresis schematic diagrames, electrophoretic band Size about 77bp, wherein Lane M are DL2000Marker, and Lane 1-6 are respectively DENV standard items (107Copies/ml) ten Real-time fluorescence quantitative RT-PCR product after times gradient dilution, Lane 7 is negative control.
Fig. 4 is the sensitivity that this kit detects ZIKV, and from left to right (1-6) is followed successively by 107、106、105、104、103、 102copies/ml。
Fig. 5 is this kit ZIKV standard items real-time fluorescence quantitative RT-PCR product gel electrophoresis schematic diagrames, electrophoretic band Size about 81bp, wherein Lane M are DL2000Marker, and Lane 1-6 are respectively ZIKV standard items (107Copies/ml) ten Real-time fluorescence quantitative RT-PCR product after times gradient dilution, Lane 7 is negative control.
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 detection DENV and ZIKV dual real-time fluorescence quantitative RT-PCR detecting kit, comprising：Quantitative RT-PCR Reaction solution, enzyme mixation, primed probe mixed liquor, standard items (DENV, ZIKV), robust positive control product (DENV, ZIKV), weak sun Property reference substance (DENV, ZIKV), negative controls.This kit is provided with container hole, corresponding with kit each component.Box body Provided with container hole, it is respectively used to placement quantitative RT-PCR reaction liquid pipe (label 1), enzyme mixation pipe (label 2), primed probe and mixes Close liquid pipe (label 3), negative control QC (label 4), standard QC (including DENV, ZIKV standard items, totally 2 pipe, label 5), Robust positive control QC (including DENV, ZIKV positive reference substance, totally 2 pipe, label 6), robust positive control QC (including DENV, ZIKV positive reference substances, 2 are managed, label 7 totally), as shown in Figure 1.
By force, weakly positive control is the positive plasmid sample of the high and low concentration of the conserved region gene sequence comprising DENV and ZIKV Product.The copy number of wherein DENV and ZIKV high concentrations plasmid (i.e. robust positive control product) is 2 × 106Copies/mL, and it is low dense The copy number for spending (i.e. weakly positive reference substance) plasmid is 2 × 104copies/mL。
Wherein quantitative RT-PCR reaction liquid pipe is included：PCR reaction buffers pipe (magnesium chloride containing and triphosphoric acid deoxyribose core Thuja acid mixture etc.), enzyme mixation pipe (containing heat-resisting Taq archaeal dna polymerases, RNase inhibitor and MMLV reverse transcriptases etc.), draw The physical prospecting pin mixing liquid pipe probe of corresponding two kinds of different fluorescence labelings (two groups of primers with), and primed probe mixes liquid pipe and need to be Brown pipe.Negative control is DEPC (pyrocarbonic acid diethyl ester) the processing water of autoclave sterilization, and positive control is DENV and ZIKV Positive plasmid sample.
Multiplex real-time PCR detection sense primer and anti-sense primer and corresponding specific probe sequence are such as Under：
Sense primer DENV-F:5’-GACTAGYGGTTAGAGGAGACCCCTC-3’
Anti-sense primer DENV-R：5’-GCGYTCTGTGCCTGGAWTGAT-3’
Specific probe DENV-P:5’-FAM-AGACCAGAGATCCTGCTGT-MGB-3’
Sense primer ZIKV-F:5’-GACTGGGTACCAACTGGGAGAAC-3’
Anti-sense primer ZIKV-R:5’-GGTCGTTCTCCTCAATCCACAC-3’
Specific probe ZIKV-P:5’-HEX-GAAAGGGAGAATGGATGAC-MGB-3’
5 ' ends of wherein DENV specific probes use FAM fluorescence labelings, and 5 ' ends of ZIKV specific probes are glimmering using HEX Signal；The fluorescent quenching label at 3 ' ends of DENV and ZIKV specific probes uses MGB fluorochrome labels.
Above-mentioned standard product include DENV, ZIKV standard items, and its conserved region gene sequence is as follows：
DENV standard items sequences are：
GACTAGCGGTTAGAGGAGACCCCTCCCATCACTGACAAAACGCAGCAAAAAAGGGGGCCCGAAGCCAGG AGGAAGCTGTACTCCTGGTGGAAGGACTAGAGGTTAGAGGAGACCCCCCCAACACAAAAACAGCATATTGACGCTGG GAAAGACCAGAGATCCTGCTGTCTCTGCAACATCAATCCAGGCACAGAGCGC
ZIKV standard items sequences are：
GACTGGGTACCAACTGGGAGAACCACCTGGTCAATCCATGGAAAGGGAGAATGGATGACCACTGAGGAC ATGCTCATGGTGTGGAATAGAGTGTGGATTGAGGAGAACGACC
The fluorescence quantitative kit that the present invention is provided need to reduce multigelation as far as possible in -20 DEG C of storages；Primed probe is mixed Liquid needs lucifuge condition to preserve.
Embodiment 2
1 materials and methods
1.1 clinical samples and viral nucleic acid：
DENV and ZIKV positive clinical sample is pre- by Huzhou City of Zhejiang Province disease prevention and control center and Zhejiang Province's disease Anti- control centre provides, doubtful samples sources in Huzhou City of Zhejiang Province First People's Hospital, Huzhou City Central Hospital, Zhejiang Prov. and Blood plasma, serum and/or the urine specimen of other Ji Jia hospitals suspected patients, laboratory is transported to after sample collection in Zhejiang Province.Separately Outside, other viruses related to heating eruption such as measles virus, rubella virus, viral 71 types in road, coxsackie virus A 16-type and Ke Sa Qi virus A6 types, and the virus such as A type, influenza B virus positive nucleic acid in the prevention and control of diseases of Huzhou City of Zhejiang Province The heart and Huzhou City of Zhejiang Province First People's Hospital are provided.
1.2 primers and probe
The a plurality of gene order for covering domestic and international DENV hypotypes and ZIKV genotype has been downloaded from NCBI gene pools.Utilize MEGA6.06 softwares carry out tetraploid rice to it, it is determined that virus genomic conserved region above.Use Primer Express 3.0 softwares are in primer and the TaqMan-MGB probes of its conserved regions design high degree of specificity, and primer and probe sequence passes through Blast is verified, with preferable specificity.Primer and probe sequence is as follows,：
Sense primer DENV-F:5’-GACTAGYGGTTAGAGGAGACCCCTC-3’
Anti-sense primer DENV-R：5’-GCGYTCTGTGCCTGGAWTGAT-3’
Specific probe DENV-P:5’-FAM-AGACCAGAGATCCTGCTGT-MGB-3’
Sense primer ZIKV-F:5’-GACTGGGTACCAACTGGGAGAAC-3’
Anti-sense primer ZIKV-R:5’-GGTCGTTCTCCTCAATCCACAC-3’
Specific probe ZIKV-P:5’-HEX-GAAAGGGAGAATGGATGAC-MGB-3’
Above primer and probe commission Shanghai Hui Rui bio tech ltd synthesis.
The extraction of 1.3 viral nucleic acids and standard items quantitative criterion：
Draw 200 μ l serum, blood plasma or urine supernatant to be added in EP pipes, using the QIAamp Viral of QIAGEN companies RNA Mini Kit or Qiagen RNeasy Mini Kit, are extracted according to kit specification, take what 5ul extracted to treat Survey sample nucleic and be used as template.
Each gene standard items fragment is synthesized, plasmid vector pMDTM19-T Simple Vector (TaKaRa are connected to Company).Cultivated after conversion.DNA is extracted after identification, is measured using NanoDrop ND-2000Spectrophotometer The concentration of DNA, determines DNA copy number as the quantitative mother liquor of standard items.According to experiment needs, standard items are quantified into mother liquor Required maximum concentration is diluted to, and does ten times and is diluted to least concentration, Cord blood is standby.
The optimization of 1.4 dual real-time fluorescence quantitative RT-PCR reaction systems and condition：
Reaction cumulative volume is 25 μ l, the wherein μ l of quantitative RT-PCR reaction solution 12.5, the μ l of enzyme mixation 1, primed probe mixing Liquid (20 μm of ol/l include two kinds of viral specific primers and corresponding two kinds of fluorescence probes) totally 3.0 μ l, template 5 μ l, DEPC Water complements to 25 μ l.Detected on ABI7500 quantitative real time PCR Instruments, response parameter is：50 DEG C of reverse transcription, 20min；95 DEG C 5min thermal startings, then 95 DEG C of 15s, 60 DEG C of 45s, double fluorescent detections are carried out at 60 DEG C, and 40 circulations are carried out altogether.As a result sentence It is disconnected：Selection fluoroscopic examination model F AM, HEX fluorescence baseline adjustment takes the fluorescence signal average value of 3-15 circulation, threshold value set with Threshold line is just above the peak of negative controls, and sample is in typical amplification curve, is judged as the positive.Without typical amplification Curve, is judged as feminine gender.The Optimum Experiment of system, in using the positive nucleic acid of same concentrations as the reaction system of template, primer is dense Degree is from 1~20 μM, and concentration and probe concentration is from 1~20 μM, using the optium concentration of the preferred primer and probe of matrix method, according to minimum Ct values Optimal primer and probe concentration is selected with highest fluorescence intensity value added (Δ Rn).
1.5 dual real-time fluorescence quantitative RT-PCRs specificity, sensitiveness and replica test
Respectively selection DENV and ZIKV positive nucleic acid (being identified by gene sequencing) and other to generate heat eruption it is related Virus such as measles virus, rubella virus, viral 71 types in road, coxsackie virus A 16-type and the type of Coxsackie virus A 6, and A type and The positive nucleic acid of the virus such as influenza B virus, the specificity of this method is verified with dual real-time fluorescence quantitative RT-PCR；To Demarcate the DENV synthesis fragments (10 of copy number (copies/ml)7Copies/ml) and ZIKV synthesis fragment (107copies/ml) After diluting respectively, parallel carry out Fluorescence PCR compares its sensitivity.In addition, dilute to the positive nucleic acid of each prescribed concentration Release liquid and make 3 repetition detections, obtained Ct values calculate its standard deviation and the coefficient of variation, verify the repeatability of this method.
2 results
2.1 dual real-time fluorescence quantitative RT-PCR reaction systems and condition
The reaction cumulative volume of this method is 25 μ l, the wherein μ l of quantitative RT-PCR reaction solution 12.5, the μ l of enzyme mixation 1, primer Probe mixed liquor (20 μm of ol/l include two group-specific primerses and corresponding two kinds of fluorescence probes) totally 3 μ l, template 5 μ l, DEPC Water complements to 25 μ l.Detected on ABI7500 quantitative real time PCR Instruments, response parameter is：50 DEG C of reverse transcription, 20min；95 DEG C 5min thermal startings, then 95 DEG C of 15s, 60 DEG C of 45s, double fluorescent detections are carried out at 60 DEG C, and 40 circulations are carried out altogether.It can obtain Minimum Ct values and highest fluorescence intensity.
2.2 specific test
The one-step method Multiplex real-time PCR method that the present invention is set up has fabulous specificity to DENV and ZIKV, The positive clinical sample gathered in the recent period can be detected completely.Dual primed probe in the present invention disease related to other heating eruptions Poison：Viral 71 types in measles virus, rubella virus, road, coxsackie virus A 16-type and the type of Coxsackie virus A 6, and A type and B-mode The equal no cross reaction of positive nucleic acid of the virus such as influenza virus.
2.3 sensitivity tests
DENV and ZIKV sensitivity Detections are tested, the DENV for having demarcated copy number (copies/ml) is synthesized into fragment (107Copies/ml), ZIKV synthesizes fragment (107Copies/ml) examined respectively after ten times of gradient dilutions with this kit Survey, as a result this method detection sensitivity respectively reaches 102、102copies/ml.As a result referring to Fig. 2 (ZIKV), Fig. 4 (DENV).Take μ l, 120V the constant pressure electrophoresis 20min of real-time fluorescence quantitative RT-PCR product 3, gel imaging system is taken pictures.As a result referring to Fig. 3 (ZIKV), Fig. 5 (DENV).Wherein Lane M are DL2000Marker, and Lane 1-6 are respectively fragment (108Copies/ml) ten Real-time fluorescence quantitative RT-PCR product after times gradient dilution, Lane 7 is negative control., electrophoretic band is single, brightness step point It is bright, illustrate that this reagent specificity is good, quantitative result is relatively accurate.
2.4 replica test
DENV is taken to synthesize fragment (final concentration of 10 respectively6Copies/ml), ZIKV synthesizes fragment (106Copies/ml) press 10 times of gradient dilutions make 3 repetition detections, as a result different nucleic acid concentrations into 3 different concentration to the sample of each concentration Respective detection Ct value standard deviations are between 0.091~0.266, and the coefficient of variation is below 1.0%, with preferable repeatability (the results are shown in Table 1).
The dual real-time fluorescence quantitative RT-PCR of table 1 detects the replica test of nucleic acid
Embodiment 3
Detection using this kit to clinical sample：DENV (4 parts) and ZIKV (2 parts) positive clinical sample are by Zhejiang Huzhou Center For Disease Control ＆ Prevention of province and Zhejiang Center For Disease Control and Prevention provide, and doubtful samples sources are in Zhejiang Province Huzhou Blood plasma, the serum of other Ji Jia hospitals suspected patients in First People's Hospital of city, Huzhou City Central Hospital, Zhejiang Prov. and Zhejiang Province And/or 325 parts altogether of urine specimen.The sample being collected into is carried out using the dual real-time fluorescence quantitative RT-PCR in this method Checking, testing result is as follows：DENV is positive 4 parts, positive rate 1.2%；ZIKV is positive 2 parts, positive rate 0.6%.The positive knot of detection Fruit has reported result degree of conformity up to 100% with it.
<110>The First People's Hospital of Huzhou
<120>A kind of Multiplex real-time PCR kit for detecting dengue virus and zika virus
<160> 8
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>The PCR designed according to DENV genes conserved region sequence detects upstream primer sequence
<400> 1
GACTAGYGGTTAGAGGAGACCCCTC 25
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>The PCR designed according to DENV genes conserved region sequence detects downstream primer sequence
<400> 2
GCGYTCTGTGCCTGGAWTGAT 21
<210> 3
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>The TaqMan-MGB fluorogenic quantitative detection probe sequences designed according to DENV genes conserved region sequence
<400> 3
AGACCAGAGATCCTGCTGT 19
<210> 4
<211> 198
<212> DNA
<213>Artificial sequence
<220>
<223>The fluorogenic quantitative detection standard items sequence designed according to DENV genes
<400> 4
GACTAGCGGTTAGAGGAGACCCCTCCCATCACTGACAAAACGCAGCAAAAAAGGGGGCCCGAAGCCAGGAGGA AGCTGTACTCCTGGTGGAAGGACTAGAGGTTAGAGGAGACCCCCCCAACACAAAAACAGCATATTGACGCTGGGAAA GACCAGAGATCCTGCTGTCTCTGCAACATCAATCCAGGCACAGAGCGC 198
<210> 5
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>The PCR designed according to ZIKV genes conserved region sequence detects upstream primer sequence
<400> 5
GACTGGGTACCAACTGGGAGAAC 23
<210> 6
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>The PCR designed according to ZIKV genes conserved region sequence detects downstream primer sequence
<400> 6
GGTCGTTCTCCTCAATCCACAC 22
<210> 7
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>The TaqMan-MGB fluorogenic quantitative detection probe sequences designed according to ZIKV genes conserved region sequence
<400> 7
GAAAGGGAGAATGGATGAC 19
<210> 8
<211> 113
<212> DNA
<213>Artificial sequence
<220>
<223>The fluorogenic quantitative detection standard items sequence designed according to ZIKV genes conserved region sequence
<400> 8
GACTGGGTACCAACTGGGAGAACCACCTGGTCAATCCATGGAAAGGGAGAATGGATGACCACTGAGGACATGC TCATGGTGTGGAATAGAGTGTGGATTGAGGAGAACGACC 113

Claims (7)

1. a kind of Multiplex real-time PCR kit for detecting dengue virus and zika virus, it is characterised in that the reagent Box includes following components：Quantitative RT-PCR reaction solution, enzyme mixation, primed probe mixed liquor, DENV standard items, ZIKV standards Product, DENV robust positive controls product, ZIKV robust positive controls product, DENV weakly positives reference substance, ZIKV weakly positives reference substance, feminine gender are right According to product, wherein quantitative RT-PCR reaction liquid pipe includes PCR reaction buffers pipe, enzymatic mixture pipe, primed probe mixing liquid pipe TaqMan-MGB probes comprising two groups of primers with corresponding two kinds of different fluorescence labelings；The PCR reaction buffers pipe is chloride Change magnesium and triphosphate deoxyribose nucleotide mixture, the enzymatic mixture pipe suppresses containing heat-resisting Taq archaeal dna polymerases, RNase Agent and MMLV reverse transcriptases,
Multiplex real-time PCR detection sense primer and anti-sense primer and corresponding specific probe sequence are as follows：
Sense primer DENV-F:5’-GACTAGYGGTTAGAGGAGACCCCTC-3’
Anti-sense primer DENV-R：5’-GCGYTCTGTGCCTGGAWTGAT-3’
Specific probe DENV-P:5’-FAM-AGACCAGAGATCCTGCTGT-MGB-3’
Sense primer ZIKV-F:5’-GACTGGGTACCAACTGGGAGAAC-3’
Anti-sense primer ZIKV-R:5’-GGTCGTTCTCCTCAATCCACAC-3’
Specific probe ZIKV-P:5’-HEX-GAAAGGGAGAATGGATGAC-MGB-3’.
2. the Multiplex real-time PCR reagent of a kind of detection dengue virus and zika virus according to claims 1 Box, it is characterised in that 5 ' ends of DENV specific probes use FAM fluorescence labelings, 5 ' ends of ZIKV specific probes use HEX Fluorescence labeling；The fluorescent quenching label at 3 ' ends of DENV and ZIKV specific probes uses MGB fluorochrome labels.
3. the Multiplex real-time PCR reagent of a kind of detection dengue virus and zika virus according to claims 1 Box, it is characterised in that the conserved region gene sequence of the DENV standard items and ZIKV standard items is as follows：
DENV standard items sequences are：
GACTAGCGGTTAGAGGAGACCCCTCCCATCACTGACAAAACGCAGCAAAAAAGGGGGCCCGAAGCCAGGAGGA AGCTGTACTCCTGGTGGAAGGACTAGAGGTTAGAGGAGACCCCCCCAACACAAAAACAGCATATTGACGCTGGGAAA GACCAGAGATCCTGCTGTCTCTGCAACATCAATCCAGGCACAGAGCGC
ZIKV standard items sequences are：
GACTGGGTACCAACTGGGAGAACCACCTGGTCAATCCATGGAAAGGGAGAATGGATGACCACTGAGGACATGC TCATGGTGTGGAATAGAGTGTGGATTGAGGAGAACGACC。
4. the Multiplex real-time PCR kit of a kind of detection dengue virus and zika virus according to right 1, its Be characterised by, the negative control for autoclave sterilization pyrocarbonic acid diethyl ester processing water, the robust positive control be comprising The positive plasmid sample of the high concentration of DENV and ZIKV conserved region gene sequence, its plasmid copy number is 2 × 106copies/ ML, the weakly positive control is the positive plasmid sample of the low concentration of the conserved region gene sequence comprising DENV and ZIKV, its matter Grain copy number is 2 × 104copies/mL。
5. a kind of Multiplex real-time PCR reagent for detecting dengue virus and zika virus according to claim 1 Box, it is characterised in that primed probe mixing liquid pipe is brown pipe.
6. the Multiplex real-time PCR kit of a kind of detection dengue virus and zika virus according to right 1, its It is characterised by, described kit is stored in -20 DEG C.
7. the Multiplex real-time PCR kit of a kind of detection dengue virus and zika virus according to right 1 is in inspection The application surveyed in DENV and ZIKV nucleic acid.
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* Cited by examiner, † Cited by third party
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CN108676914A (en) * 2018-02-13 2018-10-19 连云港出入境检验检疫局检验检疫综合技术中心（江苏国际旅行卫生保健中心连云港分中心、连云港出入境检验检疫局口岸门诊部） A kind of dengue fever virus and zika virus rapid fluorescence PCR detection kit
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
CN110699491A (en) * 2019-11-13 2020-01-17 中国疾病预防控制中心病毒病预防控制所 Zika virus real-time fluorescent quantitative RT-RPA detection primer, probe and detection kit
CN110699491B (en) * 2019-11-13 2022-05-03 中国疾病预防控制中心病毒病预防控制所 Zika virus real-time fluorescent quantitative RT-RPA detection primer, probe and detection kit
CN111826463A (en) * 2019-12-31 2020-10-27 深圳市人民医院 Primer probe combination and kit for detecting five important arthropod/rodent-borne viruses and application of primer probe combination and kit
CN112899397A (en) * 2020-12-25 2021-06-04 中山大学 Primer and probe for detecting dengue virus
CN113337640A (en) * 2021-06-04 2021-09-03 深圳大学 Dengue virus and Zika virus detection reagent combination and detection method
CN116083655A (en) * 2023-02-23 2023-05-09 南方医科大学 DENV-crRNA for I-IV dengue virus detection, kit and application thereof
CN116083655B (en) * 2023-02-23 2024-03-01 南方医科大学 DENV-crRNA for I-IV dengue virus detection, kit and application thereof

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