CN106755572A - I types DHV and duck plague virus double fluorescent quantitative PCR method - Google Patents

I types DHV and duck plague virus double fluorescent quantitative PCR method Download PDF

Info

Publication number
CN106755572A
CN106755572A CN201611080993.3A CN201611080993A CN106755572A CN 106755572 A CN106755572 A CN 106755572A CN 201611080993 A CN201611080993 A CN 201611080993A CN 106755572 A CN106755572 A CN 106755572A
Authority
CN
China
Prior art keywords
dhv
quantitative pcr
fluorescent quantitative
double fluorescent
duck plague
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201611080993.3A
Other languages
Chinese (zh)
Inventor
赵丽丽
陈洪岩
韩凌霞
张圆圆
陆涛峰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harbin Veterinary Research Institute of CAAS
Original Assignee
Harbin Veterinary Research Institute of CAAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Harbin Veterinary Research Institute of CAAS filed Critical Harbin Veterinary Research Institute of CAAS
Priority to CN201611080993.3A priority Critical patent/CN106755572A/en
Publication of CN106755572A publication Critical patent/CN106755572A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Abstract

The present invention provides a kind of I types DHV and duck plague virus double fluorescent quantitative PCR method, a pair specific detection primer DHV I F, the DHV I R and a fluorescence probe DHV I P for I type DHVs are separately designed, it is directed to specific detection primer DPV F, DPV R and fluorescence probe DPV P of duck plague virus a pair, and the specific primer and the concentration of fluorescence probe of the double fluorescent quantitative PCR method for I types DHV and duck plague virus is determined.The double fluorescent quantitative PCR method that the present invention is provided can simultaneously detect and differentiate two kinds of viruses of I types DHV and duck plague virus, there is simple to operate, sensitiveness high, high specificity, reproducible, not only can with it is cost-effective, can also be as epidemic monitoring and epidemic situation control wins valuable time, and the research such as the Rapid＆Early diagnosis of infectiousness duck disease caused by both viruses, monitoring and epidemiology survey provides effective means.

Description

I types DHV and duck plague virus double fluorescent quantitative PCR method
Technical field
The invention belongs to technical field of biological, more particularly to a kind of I types DHV and duck plague virus it is dual glimmering Fluorescent Quantitative PCR method.
Background technology
Duck virus hepatitis is the disease of a kind of acute, strong, highly infective with hepatitis as principal character and lethal high Toxoinfection disease, cause of disease is DHV (DHV), mainly causes the duckling below 3 week old to fall ill.DHV mainly has 3 serotypes, respectively I type, II type, III type, these three serotypes are separate, in the mainly I type duck liver that China is popular Scorching virus (DHV-I).I type DHV is most easily popular in month in spring 3-4, the duckling below 3 week old catch an illness after death Rate is high, the duckling death rate within a week old also reaches 50% or so up to more than 80%, 2-3 week old, serious harm I The sound development of state's various regions duck culturing industry.
Duck plague is acute, the height death rate the infectious disease caused by duck plague virus (DPV), is also called duck viral intestines Inflammation, is commonly called as " infection with swollen head ".Duck plague virus is herpesviral, and virion is spherical in shape, and the duck of various ages and kind can infect, Occur in annual each Ji Junke, but with the end of spring and the beginning of summer most easily popular with autumn.This disease mainly by transmission, can also pass through Mating, eye conjunctiva and respiratory infectious, sick duck and by the apparatus of its manure contamination, means of transport so with epidemic-stricken area or epidemic disease people The dealing of member is all likely to result in the propagation of duck plague, and when duck plague is popular, the morbidity and death of the duck that grows up are more serious, below 1 monthly age Duckling morbidity it is less, often bring huge economic loss to duck culturing industry.
At present, the primarily discrete culture of detection method, Serologic detection, the routine of I type DHV and duck plague virus PCR and real-time fluorescence quantitative PCR are detected.Be separately cultured has requirement higher to culture environment, nutritional condition, lock out operation, And it is time-consuming more long；Serologic detection has result interpretation, and to there are subjective factor, sensitivity and specificity not high, easily and Chlamydia The problems such as there is cross reaction Deng other microorganisms；Standard PCR detection exist primer combine lack specificity, laboratory pollution and PCR mortifiers there are problems that causing in clinical sample, also need electrophoresis to be sequenced to verify after gene magnification, relative consumption When, cost is also higher；Real-time fluorescence probe PCR method distinguishes species according to sequence-specific probes, improves the spy of detection The opposite sex and sensitivity, such as CN200810198528.9 disclose a kind of molecular biology identification method of I type DHV, adopt I type DHV is identified with fluorescence quantifying PCR method, but the method can only detect I type DHV, it is impossible to while to I Type DHV and duck plague virus detected and differentiated exactly in time, easily delays epidemic monitoring and epidemic situation control System.
The content of the invention
In order to solve the above-mentioned technical problem, the present invention provides a kind of I types DHV and duck plague virus double fluorescent is fixed Amount PCR method, in methods described：
The specific primer of design detection I type DHVs and the sequence of probe are respectively：
Sense primer DHV-I-F：5 '-TGGGATACCCAGGAGTACACGTA-3 ' (as shown in SEQ ID No.1)；
Anti-sense primer DHV-I-R：5 '-TGTTAAGCTGGGAGGTGTCTTGT-3 ' (as shown in SEQ ID No.2)；
Fluorescence probe DHV-I-P：
5 '-VIC-CACCCACTGGCTTTGGAGCTGTGC-TAMRA-3 ' (as shown in SEQ ID No.3)；
The specific primer of design detection duck plague virus and the sequence of probe are respectively：
Sense primer DPV-F：5 '-CGGTTTTGGGAAGGCTTTCG-3 ' (as shown in SEQ ID No.4)；
Anti-sense primer DPV-R：5 '-ACCCTAACGGCTCCTGTAG-3 ' (as shown in SEQ ID No.5)；
Fluorescence probe DPV-P：
5 '-FAM-TCCACTGGCATTTGCTTGATTTCCGC-TAMRA-3 ' (as shown in SEQ ID No.6).
Further, methods described includes：
(1) design and synthetic primer and probe；
(2) positive plasmid standard items are prepared；
(3) optimization of double fluorescent quantitative PCR reaction condition is carried out, double fluorescent quantitative PCR detection architecture is set up；
(4) virus is detected using double fluorescent quantitative PCR detection architecture.
Further, the detailed process for preparing positive plasmid standard items includes：
A. the primer for preparing positive plasmid standard items is synthesized；
DHV primers：
Sense primer P1：5’-GTTTTGGAAGGGAAGATACAGTGGT-3’；
Anti-sense primer P2：5’-GTAGGGTAGGGAATAGTAAAGTAAA-3’；
Amplification purpose fragment is 516bp；
DPV primers：
Sense primer P3：5’-GCAGGGCATTTGTTAGACGATA-3’；
Anti-sense primer P4：5’-TGCCGCCAATTTGTAAAG-3’；
Amplification purpose fragment is 337bp；
B. I type DHV RNA and duck plague virus DNA are extracted respectively with commercial kit, is protected under the conditions of -70 DEG C Deposit standby；
C. the RNA to I type DHVs carries out reverse transcription reaction acquisition cDNA, and reaction system is to contain 0.2- 1000ng/ μ L template ribonucleic acids, 0.5 μM of anti-sense primer P2,1mM dNTP, 1U/ μ L Rnase Inhibition (reverse transcriptase suppression Agent), 5 × Primescript Buffer (5 times of reverse transcription buffers) and DEPC of 200U Prime Script reverse transcriptase The mixed liquor of water, wherein 5 × Primescript Buffer represent that its volume fraction in mixed liquor is 20%；
Concretely comprise the following steps：Water-bath 10min at RNA templates, anti-sense primer P2 are put into 65 DEG C, then ice bath 5min, of short duration Added after centrifugation dNTP, Rnase Inhibition, Prime Script reverse transcriptase, 5 × Primescript Buffer and DEPC water, water-bath 1h at 42 DEG C, water-bath 10min at 70 DEG C, ice bath 5min, -20 DEG C save backup；
D. pcr amplification reaction is carried out by template of the DNA of the cDNA of I type DHVs and duck plague virus respectively, then Recovery purifying amplified production；
Wherein, the reaction system of the pcr amplification reaction of the cDNA of I types DHV is to contain 0.5-1000ng/ μ L moulds Plate cDNA, 0.4 μM of sense primer P1,0.4 μM of anti-sense primer P2,0.8mM dNTP, 1.6mM MgCl2、0.1U/μL Ex Taq 10 × Buffer (10 times of buffer solutions) and ddH2The mixed liquor of O, wherein 10 × Buffer represents its body in mixed liquor Fraction is 10%；
Amplification reaction condition is：95 DEG C of predegenerations 5min, 94 DEG C of 60s, 51.8 DEG C of 40s, 72 DEG C of 60s, 30 circulations, 72 DEG C Extend 10min；
Duck plague virus DNA for template pcr amplification reaction reaction system be containing 0.2-1000ng/ μ L template DNAs, 0.4 μM of sense primer P3,0.4 μM of anti-sense primer P4,0.8mM dNTP, 1.6mM MgCl2, 0.1U/ μ L Ex Taq 10 × Buffer and ddH2The mixed liquor of O, wherein 10 × Buffer represents that its volume fraction in mixed liquor is 10%；
Amplification reaction condition is：95 DEG C of predegenerations 5min, 94 DEG C of 60s, 51.8 DEG C of 40s, 72 DEG C of 60s, 30 circulations, 72 DEG C Extend 10min；
E. I types DHV after purification and the PCR primer of duck plague virus are connected respectively on carrier pMD18-T； Vector introduction competent escherichia coli cell after connecting again, is converted and coated plate；Finally carry out extracting and obtain positive criteria Quality grain.
Further, the double fluorescent quantitative PCR detection architecture is specifically included：Conventional method extracts testing sample RNA and DNA, and reverse transcription reaction acquisition cDNA is carried out to RNA；Again double fluorescent quantitative PCR is carried out by template of cDNA and DNA Reaction.
Further, the reaction system of the reverse transcription reaction be containing 0.2-1000ng/ μ L template ribonucleic acids, 0.5 μM it is random Primer, 1mM dNTP, 1U/ μ L Rnase Inhibition, 200U Prime Script reverse transcriptase 5 × The mixed liquor of Primescript Buffer and DEPC water, wherein 5 × Primescript Buffer represent it in mixed liquor Volume fraction be 20%.
Further, the reaction condition of the reverse transcription reaction is：42 DEG C of water-baths 1h, 70 DEG C of water-bath 10min, ice bath 5min, obtains virus cDNA.
Further, the reaction system of the double fluorescent quantitative PCR reaction is to contain 0.5-1000ng/ μ L templates CDNA, 0.2-1000ng/ μ L template DNAs, 0.28 μM of sense primer DHV-I-F, 0.28 μM of anti-sense primer DHV-I-R, 0.36 μM Fluorescence probe DHV-I-P, 0.28 μM of sense primer DPV-F, 0.28 μM of anti-sense primer DPV-R, 0.36 μM of fluorescence probe DPV-P The mixed liquor of 2 × Premix Ex Taq and DEPC water, wherein 2 × Premix Ex Taq represent its volume integral in mixed liquor Number is 50%.
Further, the reaction condition of the double fluorescent quantitative PCR reaction is：95℃30s；95 DEG C of 5s, 60 DEG C of 50s, 40 circulations.
Further, the optimization of the double fluorescent quantitative PCR reaction condition includes：
A. annealing temperature optimization：With plasmid as template, gradient quantitative PCR is carried out at 53 DEG C -61 DEG C, according to amplified reaction Ct, the fluorescence signal intensity of amplification curve and melting curve screening optimum annealing temperature；
B. the optimization of primer, concentration and probe concentration：With plasmid as template, the concentration of primer and probe is 0.04-0.4 μM, is carried out Quantitative fluorescent PCR, determines the optium concentration of primer and probe.
The present invention set up I types DHV and duck plague virus double fluorescent quantitative PCR method can detect simultaneously and Differentiate two kinds of viruses of I types DHV and duck plague virus, with simple to operate, sensitiveness is high, high specificity, reproducible etc. Advantage, not only can with it is cost-effective, and can also be both as epidemic monitoring and epidemic situation control wins valuable time The Rapid＆Early diagnosis of the infectiousness duck disease caused by virus, monitoring and the research such as epidemiology survey provide effective means.
Brief description of the drawings
Fig. 1 is bent for the standard of the double fluorescent quantitative PCR method of the I type DHVs of the specific embodiment of the invention Line；
Fig. 2 is the standard curve of the double fluorescent quantitative PCR method of the duck plague virus of the specific embodiment of the invention；
Fig. 3 is the sensitiveness point of the double fluorescent quantitative PCR method of the I type DHVs of the specific embodiment of the invention The amplification curve of analysis；
Fig. 4 is the sensitivity analysis of the double fluorescent quantitative PCR method of the duck plague virus of the specific embodiment of the invention Amplification curve；
Fig. 5 is the specificity point of the double fluorescent quantitative PCR method of the I type DHVs of the specific embodiment of the invention The amplification curve of analysis；
Fig. 6 is the specificity analysis of the double fluorescent quantitative PCR method of the duck plague virus of the specific embodiment of the invention Amplification curve.
Specific embodiment
Commercial reagents employed in the specific embodiment of the invention mainly include：
RNA vitro extractions kit Total RNA kit (R6934) is purchased from OMEGA companies；
DNA virus kit (D3809-01) are purchased from OMEGA companies；
Ex taq enzymes are purchased from TAKARA companies；
RNA reverse transcription reagent box Prime script RT Kit (RR047) is purchased from TAKARA companies；
DNA fragmentation QIAquick Gel Extraction Kit (08214RE1) is purchased from AXY companies；
Small amount plasmid extraction kit (06313KA1) is purchased from AXY companies；
Fluorescent quantitation Premix Ex Taq enzymes (RR390A) is purchased from TAKARA companies.
If not specializing, following detailed description is according to conventional laboratory conditions.
A kind of I types DHV and duck plague virus double fluorescent quantitative PCR method, including：
(1) design and synthetic primer and probe；
With reference to the gene order of I types DHV in NCBI, using the softwares of Primer 5, design synthesis pair for amplification mesh Fragment for 516bp primer, its amplified production be used for prepare standard items, in the standard items sequence choose one section of conservative sequence Row, can expand the fluorescence quantification PCR primer and TaqMan of 78bp for a pair using the synthesis of the Software for Design of Beacon designer 7.5 Fluorescence probe, the specific primer of detection I type DHVs and the sequence of probe of design are respectively：
Sense primer DHV-I-F：5’-TGGGATACCCAGGAGTACACGTA-3’；
Anti-sense primer DHV-I-R：5’-TGTTAAGCTGGGAGGTGTCTTGT-3’；
Fluorescence probe DHV-I-P：
5’-VIC-CACCCACTGGCTTTGGAGCTGTGC-TAMRA-3’；
With reference to the conserved sequence in the UL6 and UL7 of duck plague virus, using the softwares of Primer 5, design synthesis pair for amplification Purpose fragment is the primer of 337bp, and its amplified production is used to prepare standard items, with the standard items as template, using Beacon The synthesis of the Software for Design of designer 7.5 can expand the fluorescence quantification PCR primer and TaqMan fluorescence probes of 78bp, design for a pair The detection specific primer of duck plague virus and the sequence of probe be respectively：
Sense primer DPV-F：5’-CGGTTTTGGGAAGGCTTTCG-3’；
Anti-sense primer DPV-R：5’-ACCCTAACGGCTCCTGTAG-3’；
Fluorescence probe DPV-P：
5’-FAM-TCCACTGGCATTTGCTTGATTTCCGC-TAMRA-3’；
(2) positive plasmid standard items are prepared；
A. the primer for preparing positive plasmid standard items is synthesized；
DHV primers：
Sense primer P1：5’-GTTTTGGAAGGGAAGATACAGTGGT-3’；
Anti-sense primer P2：5’-GTAGGGTAGGGAATAGTAAAGTAAA-3’；
Amplification purpose fragment is 516bp；
DPV primers：
Sense primer P3：5’-GCAGGGCATTTGTTAGACGATA-3’；
Anti-sense primer P4：5’-TGCCGCCAATTTGTAAAG-3’；
Amplification purpose fragment is 337bp；
B. I type DHV RNA and duck plague virus DNA are extracted respectively with commercial kit, is protected under the conditions of -70 DEG C Deposit standby；
C. the RNA to I type DHVs carries out reverse transcription reaction acquisition cDNA, and reaction system is to contain 20ng/ μ L moulds Plate RNA, 0.5 μM of anti-sense primer P2,1mM dNTP, 1U/ μ L Rnase Inhibition, 200U Prime Script reverse transcriptions The mixed liquor of 5 × Primescript Buffer and DEPC water of enzyme；
Concretely comprise the following steps：Water-bath 10min at RNA templates, anti-sense primer P2 are put into 65 DEG C, then ice bath 5min, of short duration Added after centrifugation dNTP, Rnase Inhibition, Prime Script reverse transcriptase, 5 × Primescript Buffer and DEPC water, water-bath 1h at 42 DEG C, water-bath 10min at 70 DEG C, ice bath 5min, -20 DEG C save backup；
D. pcr amplification reaction is carried out by template of the DNA of the cDNA of I type DHVs and duck plague virus respectively, then Recovery purifying amplified production；
Wherein, the reaction system of the pcr amplification reaction of the cDNA of I types DHV be containing 20ng/ μ L templates cDNA, 0.4 μM of sense primer P1,0.4 μM of anti-sense primer P2,0.8mMdNTP, 1.6mM MgCl2, 0.1U/ μ L Ex Taq 10 × Buffer and ddH2The mixed liquor of O；
Amplification reaction condition is：95 DEG C of predegenerations 5min, 94 DEG C of 60s, 51.8 DEG C of 40s, 72 DEG C of 60s, 30 circulations, 72 DEG C Extend 10min；
Duck plague virus DNA is containing on 20ng/ μ L template DNAs, 0.4 μM for the reaction system of the pcr amplification reaction of template Trip primer P3,0.4 μM of anti-sense primer P4,0.8mM dNTP, 1.6mM MgCl2, 0.1U/ μ L Ex Taq 10 × Buffer and ddH2The mixed liquor of O；
Amplification reaction condition is：95 DEG C of predegenerations 5min, 94 DEG C of 60s, 51.8 DEG C of 40s, 72 DEG C of 60s, 30 circulations, 72 DEG C Extend 10min；
Recovery purifying amplified production carries out glue reclaim using the DNA gel extraction kits of Axygen；
E. positive criteria product plasmid is prepared, is specifically included：By I types DHV and the PCR of duck plague virus after purification Product is connected respectively on carrier pMD18-T；Vector introduction competent escherichia coli cell after connecting again, is converted and is applied Plate；Finally carry out extracting and obtain positive criteria product plasmid；
(3) optimization of double fluorescent quantitative PCR reaction condition is carried out, double fluorescent quantitative PCR detection architecture is set up；
S1. the making of double fluorescent quantitative PCR detection architecture standard curve
With 108-102The I types DHV of copies/ μ L and the plasmid of duck plague virus enter performing PCR and expand respectively as template Increase reaction, obtain the double fluorescent quantitative PCR method of I type DHVs standard curve (Fig. 1) and duck plague virus it is dual The standard curve (Fig. 2) of fluorescence quantifying PCR method, as seen from the figure in each reaction system template copy numbers 108- 102There is good linear relationship in the range of copies/ μ L, coefficient correlation is the amplification efficiency of 0.999, I type DHVs It is E=100.8%, the amplification efficiency of duck plague virus is E=95.7%；
S2. the optimization of double fluorescent quantitative PCR reaction condition
A. annealing temperature optimization：With standard items plasmid as template, gradient quantitative PCR is carried out at 53 DEG C -61 DEG C, according to amplification Reaction Ct, the fluorescence signal intensity of amplification curve and melting curve screening optimum annealing temperature；
B. the optimization of primer, concentration and probe concentration：With standard items plasmid as template, the concentration of primer and probe is 0.04-0.4 μ M, carries out quantitative fluorescent PCR, determines the optium concentration of primer and probe；
Grope to show that the final concentration of the primer of I types DHV and duck plague virus is 0.28 μM, I type ducks through experiment The final concentration of the probe of hepatitis viruse and duck plague virus is 0.36 μM, and optimum annealing temperature is 60 DEG C；
S3. double fluorescent quantitative PCR detection architecture is set up, detection architecture is specifically included：
1. commercial reagents box is used, the RNA and DNA of product to be tested is extracted using conventional method, and it is anti-to carry out reverse transcription to RNA Should obtain cDNA, the reaction system of the reverse transcription reaction be containing 0.2-1000ng/ μ L template ribonucleic acids, 0.5 μM of random primer, 1mM dNTP, 1U/ μ L Rnase Inhibition, 5 × Primescript of 200U Prime Script reverse transcriptase The mixed liquor of Buffer and DEPC water；
Random primer, dNTP, Rnase Inhibition, Prime Script in the reaction system of reverse transcription reaction is anti- Transcriptase, 5 × Primescript Buffer, DEPC water are all from RNA reverse transcription reagent box Prime script RT Kit (RR047)；
Reaction condition：42 DEG C of water-baths 1h, 70 DEG C of water-bath 10min, ice bath 5min, obtain virus cDNA；
2. double fluorescent quantitative PCR reaction is carried out by template of cDNA and DNA, reaction system is to contain 0.5-1000ng/ μ L template cDNA, 0.2-1000ng/ μ L template DNAs, 0.28 μM of sense primer DHV-I-F, 0.28 μM of anti-sense primer DHV-I-R, 0.36 μM of fluorescence probe DHV-I-P, 0.28 μM of sense primer DPV-F, 0.28 μM of anti-sense primer DPV-R, 0.36 μM of fluorescence probe The mixed liquor of 2 × Premix Ex Taq and DEPC water of DPV-P；
Reaction condition is：95℃ 30s；95 DEG C of 5s, 60 DEG C of 50s (collection fluorescence signal), 40 circulations；
S4. the double fluorescent quantitative PCR detection architecture having built up is tested
1. the sensitivity analysis of double fluorescent quantitative PCR detection method
Plasmid standard is carried out into 10 times of doubling dilutions, 1 × 10 is taken8-1×102Each 2 μ L conducts of copies/ μ L standard plasmids Hybrid template carries out double fluorescent quantitative PCR detection, determines limit of identification and sets up negative control；
7 groups of experiments are set, respectively with the I types DHV of 10 times of doubling dilutions and the plasmid (1 × 10 of duck plague virus8- 1×102Copies/ μ L) enter performing PCR amplification as template, experiment group number is as shown in table 1 with the corresponding relation of plasmid concentration：
Table 1 tests the corresponding relation of group number and plasmid concentration
Experiment group number Plasmid concentration (copies/ μ L)
1 1×108
2 1×107
3 1×106
4 1×105
5 1×104
6 1×103
7 1×102
Susceptibility results are shown in Fig. 3 and Fig. 4 respectively, as seen from the figure, using 1 × 102The plasmid of copies/ μ L is used as template pair I types DHV and duck plague virus enter performing PCR detection still fluorescence curve, shows that the detection method susceptibility is 100copies/μL；
2. the specificity analysis of double fluorescent quantitative PCR detection method
Using the double fluorescent quantitative PCR detection architecture that the present invention is provided, respectively to including I types DHV, duck plague It is viral to be detected in interior various viral samples, verify I types DHV and duck plague virus double fluorescent quantitative PCR method Specificity；
The double fluorescent quantitative PCR detection method provided using the present invention detects that each group is sick to 12 groups of viral samples Contained viral species are as shown in table 2 in malicious sample：
Contained viral species in each group viral sample of table 2
As shown in Figure 5 and Figure 6, as seen from the figure, only I types DHV and duck plague virus have amplification glimmering to specific outcome Light curve, other etiology nucleic acids are detected without amplification fluorescent curve, therefore detection method specificity is preferably；
3. the repeatability analysis of double fluorescent quantitative PCR detection method
Replica test is divided into group repeating to test repeatedly to be tested and between group,
Repeat to test in group：With 10 in same experiment6The I types DHV of copies/ μ L and the matter of duck plague virus Grain standard items hybrid template does 5 parallel reactions, calculates the standard deviation and the coefficient of variation of each concentration fluorescence Ct values, verifies I types The stability of DHV and duck plague virus double fluorescent quantitative PCR method；
Result of the test is repeated in group as shown in table 3,5 parallel reactions, I types DHV and duck are done to same sample Repeatability is good in the group of pestivirus double fluorescent quantitative PCR method；
Replica test in the double fluorescent quantitative PCR group of table 3
Repeat to test between group：Examination is repeated after 3d and 7d with identical reaction condition to above-mentioned same hybrid template Test, calculate the standard deviation and the coefficient of variation of its Ct value, verify I types DHV and duck plague virus double fluorescent quantitative PCR side The stability of method；
Result of the test is repeated between group as shown in table 4, it is nucleic acid-templated using identical, with identical testing conditions, 3d and 7d After repeat experiment, the Ct value average coefficient of variation of I types DHV and duck plague virus is respectively 0.30% and 1.2%, I Repeatability is good between the group of type DHV and duck plague virus double fluorescent quantitative PCR method；
Replica test between the double fluorescent quantitative PCR group of table 4
(4) virus is detected using double fluorescent quantitative PCR detection architecture；
The 166 parts of anus swabs collected to Xuzhou area duck group using the double fluorescent quantitative PCR detection architecture set up Detected, as a result detected 13 parts of I type DHV (positive rate 7.8%), 75 parts of duck plague virus (positive rate 45%).
Finally it should be noted that embodiment of above is merely illustrative of the technical solution of the present invention and unrestricted, although ginseng The present invention has been described in detail according to better embodiment, it will be understood by those within the art that, can be to this hair Bright technical scheme is modified or equivalent, and without deviating from the spirit and scope of technical solution of the present invention, it all should contain Cover in the middle of scope of the presently claimed invention.
Sequence table
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture
<120>I types DHV and duck plague virus double fluorescent quantitative PCR method
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence
<400> 1
TGGGATACCC AGGAGTACAC GTA 23
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence
<400> 2
TGTTAAGCTG GGAGGTGTCT TGT 23
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence
<400> 3
CACCCACTGG CTTTGGAGCT GTGC 24
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
CGGTTTTGGG AAGGCTTTCG 20
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence
<400> 5
ACCCTAACGG CTCCTGTAG 19
<210> 6
<211> 26
<212> DNA
<213>Artificial sequence
<400> 6
TCCACTGGCA TTTGCTTGAT TTCCGC 26

Claims (7)

1. a kind of I types DHV and duck plague virus double fluorescent quantitative PCR method, it is characterised in that in methods described：
The specific primer of design detection I type DHVs and the sequence of probe are respectively：
Sense primer DHV-I-F：5’-TGGGATACCCAGGAGTACACGTA-3’；
Anti-sense primer DHV-I-R：5’-TGTTAAGCTGGGAGGTGTCTTGT-3’；
Fluorescence probe DHV-I-P：
5’-VIC-CACCCACTGGCTTTGGAGCTGTGC-TAMRA-3’；
The specific primer of design detection duck plague virus and the sequence of probe are respectively：
Sense primer DPV-F：5’-CGGTTTTGGGAAGGCTTTCG-3’；
Anti-sense primer DPV-R：5’-ACCCTAACGGCTCCTGTAG-3’；
Fluorescence probe DPV-P：
5’-FAM-TCCACTGGCATTTGCTTGATTTCCGC-TAMRA-3’。
2. I types DHV according to claim 1 and duck plague virus double fluorescent quantitative PCR method, its feature exist In methods described includes：
(1) design and synthetic primer and probe；
(2) positive plasmid standard items are prepared；
(3) optimization of double fluorescent quantitative PCR reaction condition is carried out, double fluorescent quantitative PCR detection architecture is set up；
(4) virus is detected using double fluorescent quantitative PCR detection architecture.
3. I types DHV according to claim 2 and duck plague virus double fluorescent quantitative PCR method, its feature exist In the double fluorescent quantitative PCR detection architecture is specifically included：Conventional method extracts the RNA and DNA of testing sample, and to RNA Carry out reverse transcription reaction and obtain cDNA；Again double fluorescent quantitative PCR reaction is carried out by template of cDNA and DNA.
4. I types DHV according to claim 3 and duck plague virus double fluorescent quantitative PCR method, its feature exist In the reaction system of, the reverse transcription reaction be containing 0.2-1000ng/ μ L template ribonucleic acids, 0.5 μM of random primer, 1mM dNTP, 5 × Primescript Buffer and DEPC of 1U/ μ L Rnase Inhibition, 200U Prime Script reverse transcriptase The mixed liquor of water.
5. I types DHV according to claim 4 and duck plague virus double fluorescent quantitative PCR method, its feature exist In the reaction condition of the reverse transcription reaction is：42 DEG C of water-baths 1h, 70 DEG C of water-bath 10min, ice bath 5min, obtain virus cDNA.
6. I types DHV according to claim 3 and duck plague virus double fluorescent quantitative PCR method, its feature exist In the reaction system of the double fluorescent quantitative PCR reaction is to contain 0.5-1000ng/ μ L template cDNA, 0.2-1000ng/ μ L Template DNA, 0.28 μM of sense primer DHV-I-F, 0.28 μM of anti-sense primer DHV-I-R, 0.36 μM of fluorescence probe DHV-I-P, 0.28 μM of sense primer DPV-F, 0.28 μM of anti-sense primer DPV-R, 2 × Premix Ex Taq of 0.36 μM of fluorescence probe DPV-P With the mixed liquor of DEPC water.
7. I types DHV according to claim 6 and duck plague virus double fluorescent quantitative PCR method, its feature exist In the reaction condition of the double fluorescent quantitative PCR reaction is：95℃30s；95 DEG C of 5s, 60 DEG C of 50s, 40 circulations.
CN201611080993.3A 2016-11-30 2016-11-30 I types DHV and duck plague virus double fluorescent quantitative PCR method Pending CN106755572A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611080993.3A CN106755572A (en) 2016-11-30 2016-11-30 I types DHV and duck plague virus double fluorescent quantitative PCR method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611080993.3A CN106755572A (en) 2016-11-30 2016-11-30 I types DHV and duck plague virus double fluorescent quantitative PCR method

Publications (1)

Publication Number Publication Date
CN106755572A true CN106755572A (en) 2017-05-31

Family

ID=58898136

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611080993.3A Pending CN106755572A (en) 2016-11-30 2016-11-30 I types DHV and duck plague virus double fluorescent quantitative PCR method

Country Status (1)

Country Link
CN (1) CN106755572A (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107287352A (en) * 2017-08-02 2017-10-24 山东省农业科学院家禽研究所 The probe primer group and its method of duck enteritis virus and duck hepatitis virus quick detection
CN107475452A (en) * 2017-09-21 2017-12-15 四川农业大学 A kind of primer and RT PCR and fluorescent quantitative PCR detection method for detecting duck plague virus ICP4 genes
CN107475453A (en) * 2017-09-21 2017-12-15 四川农业大学 A kind of primer and RT PCR and fluorescent quantitative PCR detection method for detecting duck plague virus UL48 genes
CN108531652A (en) * 2018-04-20 2018-09-14 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） A kind of preparation method and application of the important epidemic disease quantitative measurement standard product of aquatic bird

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103773899A (en) * 2014-01-26 2014-05-07 广西壮族自治区兽医研究所 GeXP (Gene Expression Profiler) detection kit for differentiating 11 kinds of duck viral diseases

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103773899A (en) * 2014-01-26 2014-05-07 广西壮族自治区兽医研究所 GeXP (Gene Expression Profiler) detection kit for differentiating 11 kinds of duck viral diseases

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
刘海燕等: "I型鸭肝炎病毒 TaqMan 荧光定量RT-PCR方法的建立及初步应用", 《中国比较医学杂志》 *
石建平等: "实时荧光定量PCR快速检测鸭病毒性肠炎标准方法的建立", 《中国兽医学报》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107287352A (en) * 2017-08-02 2017-10-24 山东省农业科学院家禽研究所 The probe primer group and its method of duck enteritis virus and duck hepatitis virus quick detection
CN107475452A (en) * 2017-09-21 2017-12-15 四川农业大学 A kind of primer and RT PCR and fluorescent quantitative PCR detection method for detecting duck plague virus ICP4 genes
CN107475453A (en) * 2017-09-21 2017-12-15 四川农业大学 A kind of primer and RT PCR and fluorescent quantitative PCR detection method for detecting duck plague virus UL48 genes
CN107475452B (en) * 2017-09-21 2021-03-23 四川农业大学 Primer for detecting duck plague virus ICP4 gene and RT-PCR and fluorescent quantitative PCR detection method
CN108531652A (en) * 2018-04-20 2018-09-14 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） A kind of preparation method and application of the important epidemic disease quantitative measurement standard product of aquatic bird

Similar Documents

Publication Publication Date Title
CN112094948B (en) Application of target gene combination in African swine fever virus detection and kit
CN107630109A (en) A kind of fluorescence quantification PCR primer and kit for detecting Novel pig acute diarrhea syndrome coronavirus
CN106755572A (en) I types DHV and duck plague virus double fluorescent quantitative PCR method
CN103614489B (en) Constant-temperature amplification detection kit for dengue viruses and detection method
CN1995386A (en) BCR-ABL gene fluorescence quantitative RT-PCR primer and probe and reagent kit
CN106167833B (en) A kind of while 8 kinds of entomophila encephalitis viruses of detection RT-PCR primers and probe combinations and kit
CN108866244A (en) Detect RPA primer and probe, kit and its method of prawn irido virus
CN104774953B (en) Fluorescence PCR detection reagent of African swine fever virus CP530R genes and preparation method thereof and purposes
CN104263857B (en) A kind of nano PCR kit of quick detection mink enteritis virus and its application
CN105112558B (en) The triple real time fluorescent quantitative RT PCR detection kits of the type of foot and mouth disease virus O, A and Asia I
CN109762932A (en) Identify the detection primer and probe, kit and application of HP-PRRSV and NADC30-like strain
CN106048097A (en) Specific primers and method for real-time fluorescent quantitative PCR detection of tomato chlorosis virus
CN103276099A (en) Primer and kit for fluorescent quatititive PCR (polymerase chain reaction) detection of helicobacter pylori
CN101386893B (en) Single cell real time fluorescent quantitative RT-PCR method for detecting foot-and-mouth disease virus genome RNA
CN101381767A (en) Universal real time fluorescent PCR detection method of trichinella
CN108841998A (en) Detect primed probe group, quick detection kit and the method for feline herpetovirus I type nucleic acid
CN108085413A (en) It is a kind of to be used for the high-throughput kit of three kinds of poultry diease viral diseases of detection and its detection method and application simultaneously
CN109913583A (en) A kind of primer and its method of quick detection Koi herpesvirus
CN116356079A (en) RPA-CRISPR-Cas12a based visual detection kit for detecting Gaota virus and application
CN106521038B (en) A kind of real-time fluorescence quantitative PCR detection methods of highly sensitive BHV 2 and kit
CN105400908B (en) A kind of primer, kit and detection method using pyrosequencing techniques detection channel catfish virus
CN111534603B (en) Method for identifying aedes albopictus by using fluorescent RPA
CN107267666A (en) A kind of fluorescent quantitation RT PCR detection kits based on pig atypia pestivirus raq gene
CN107227380A (en) The primer sequence and method of a kind of synchronous detection PCV2 and PRV infection
CN114381551A (en) Real-time fluorescent RAA primer, probe and kit for detecting iridovirus of micropterus salmoides

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20170531