CN105002297A - Multi-RT-PCR method for monitoring pollution of six viruses in pig farm environment through one system and application - Google Patents
Multi-RT-PCR method for monitoring pollution of six viruses in pig farm environment through one system and application Download PDFInfo
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Abstract
The invention provides a multi-RT-PCR method for monitoring pollution of six main viruses in a pig farm environment through one system and application. According to the method, primer design is conducted for CSFV, JEV, PRRSV, PCV-2, PRV and PPV, a multi-RT-PCR reaction system for the six viruses is established, air samples are collected through liquid collision, the viruses are subjected to separation concentration through ultrafiltration centrifugation, and the method for detecting the pollution of the main viruses in the pig farm environment is established. The method can be used for monitoring the pollution of the six viruses in air, fodder, drinking water, appliances, excrement and pig groups in the pig house and pig farm environment, and technical support is provided for establishing a main virus pollution early-warning mechanism and a main virus disease preventing, controlling and purifying system in the pig farm environment.
Description
Technical field
the present invention relates to the multiple RT-PCR technology of main viral pollution monitoring in a kind of piggery enviroment, belong to technical field of bioengineering.
Background technology
Pig industry is gradually to intensive, large-scale development, and intensive feeding manner is current main aquaculture model, and pig transmissible disease is mainly propagated using the air in environment, feed, drinking-water, utensil, ight soil etc. as medium.Pestivirus suis (CSFV), Japanese B encephalitis virus (JEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 type (PCV-2), porcine pseudorabies virus (PRV) and pig parvoviral (PPV) etc. are the main virus polluting large-scale pig farm.Existing technology is mainly used in the diagnosis after pig morbidity, and lacks the Monitoring techniques to virus contamination in environment, more can not set up early warning mechanism, can not shift to an earlier date prevention and control virus infection, can not monitor the purge cases of pig farm cause of disease.Therefore, set up above-mentioned 6 kinds of virus contamination multiple RT-PCR monitoring methods in piggery enviroment, above-mentioned 6 kinds of virus contamination of air, feed, drinking-water, utensil, ight soil and swinery in monitoring pig house and piggery enviroment, provide technical support for setting up main viral pollution prewarning mechanism and the prevention and control of main diseases viral disease and purification systems in piggery enviroment.
summary of the invention:
The object of this invention is to provide a kind of good test effect, an individual system easy to use monitors multiplex RT-PCR method and the application of 6 kinds of virus contamination in piggery enviroment simultaneously.
Technical scheme of the present invention is, 1, one individual system monitors the method for 6 kinds of virus contamination in piggery enviroment simultaneously, it is characterized in that comprising the following steps:
1) design of primers utilizes software to carry out design of primers in Pestivirus suis (CSFV), Japanese B encephalitis virus (JEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 type (PCV-2), porcine pseudorabies virus (PRV) and pig parvoviral (PPV) conserved regions.
CSFVF GCTCCCTGGGTGTTCTAAGT
CSFVR TGCTGTCCTTCTCATGCTCTT
PRRSVF GGCCAGCCAGTCAATCAG
PRRSVR GGCAAACTAAACTCCACAGTG
JEVF CAAACTGGCTCTGAAAGG
JEVR TGTCTCAGGTCCATCTACG
PCV-2F CAGCACCCTGTAACGTTTG
PCV-2R AGGAGTACCATTCCAACGG
PPVF ACATCTAAATATGCCAGAACACG
PPVR GTTTGCCATGAGTGAGTTAATTT
PRVF CTCCTTGAGCGTCTTCGTCG
PRVR CCTTCCTGTCCAACCCCTTC
Homologous segment be connected to carrier and be transformed in intestinal bacteria, after obtaining a large amount of amplification, according to the ratio of 1:5, protective material and DNA being mixed, make special marker, as reference standard.
2) foundation of multiple RT-PCR establishes an individual system and detects above-mentioned 6 kinds of viral multiplex RT-PCR methods simultaneously.
3) air sample collection to be gathered in pig house by liquid knockout method and air in piggery enviroment; Be that the super filter tube ultrafiltration of 10kD is centrifugal with molecular weight cut-off to carry out separation to virus and concentrate.
Involved by this monitoring method, the object fragment of 6 kinds of viruses (CSFV, JEV, PRRSV, PCV-2, PRV and PPV) is respectively 829bp, 1045 bp, 275 bp, 382 bp, 142 bp and 636bp.
This multiple RT-PCR adopts 50 μ L reaction systems: 2 × TaqMaster Mix is 25 μ L, 6 kinds of viral mix primer (20 μMs/L) totally 3 μ L, 6 kinds of virus mixing nucleic acid totally 3 μ L, sterile purified water 19 μ L.Reaction conditions is: 94 DEG C of 5min; 94 DEG C of 45s; 56 DEG C of 1m30s; 72 DEG C of 50s cycle numbers are 40 times, and last 72 DEG C extend 10min, 4 DEG C of preservations.
In pig house and piggery enviroment air sample collecting position and count for: in pig house the position of collected specimens be distance floor level 0.5-1m place of pig house passageway central authorities according to the area determination sampling number of pig house, 30m
21 sampling point in left and right, in pig farm the position of collected specimens be pig farm central authorities and courtyard wall around four corner totally 5 sampling points, distance ground 0.5-1m, each sampling point acquisition time is 40min, and outside pig farm, the position of collected specimens is each 3 sampling points in distance 200m place, upper and lower air port, pig farm.Feces collection gathers fresh faecal samples about 20g after selecting feeding in morning.
The method can be used for monitoring above-mentioned 6 kinds of virus contamination of air in pig house and piggery enviroment, feed, drinking-water, utensil, ight soil and swinery, provides technical support for setting up main viral pollution prewarning mechanism and the prevention and control of main diseases viral disease and purification systems in piggery enviroment.
Invention thinking of the present invention is:
1. according to the sequence of Genbank, by its conserved sequence of DNAstar compare of analysis, for its conserved sequence design primer.
2. obtain the Reference strains of Pestivirus suis (CSFV), parvovirus (PPV), Pseudorabies virus (PRV), PCV-II-2 type (PCV-2), porcine reproductive respiratory distress syndrome virus (PRRSV), Japanese B encephalitis virus (JEV),
3. extract the nucleic acid of virus; carry out PCR and RT-PCR of individual event; and be connected to pMD-18 carrier T; proceeded in competent cell and preserved; extract plasmid, after carrying out pcr amplification, glue reclaims each fragment and measures concentration and be diluted to its respective concentration; according to the ratio of 1:5, protective material and DNA are mixed, make special marker.
4. gather air in pig house and pig farm internal and external environment by liquid knockout method, be that the super filter tube of 10kD is centrifugal with molecular weight cut-off to carry out separation to virus and concentrate, other samples such as method of sampling of feed, drinking-water, utensil, ight soil and swinery adopts conventional collection method, and concentration method is identical.
5. multiple RT-PCR, adopts 50 μ L reaction systems: 2 × Taq MasterMix is 25 μ L, 6 kinds of viral mix primer (20 μMs/L) totally 3 μ L, 6 kinds of virus mixing nucleic acid totally 3 μ L, sterile purified water 19 μ L.Reaction conditions is: 94 DEG C of 5min; 94 DEG C of 45s; 56 DEG C of 1m30s; 72 DEG C of 50s cycle numbers are 40 times, and last 72 DEG C extend 10min, 4 DEG C of preservations.
6. detected by the agarose gel electrophoresis of 1.5%, with electrophoresis time about 30min under 1 × TAE damping fluid, 110V voltage, room temperature condition, when loading Buffer reaches the mid-way of glue, compare with special marker after electrophoresis, there is respective strap, be judged to be the positive.
7. any one sample detection result is positive, can judge that this is subject to virus contamination and threatens, point out this pig farm should take corresponding prevention and control measure.
8. early warning mechanism: any one sample of outside upper, lower air port, pig farm detects virus-positive, and pig farm, sample is negative in pig house, is one-level early warning; In pig farm, any one sample detects virus-positive, and in pig house, sample is negative, or vaccine immunity toxin expelling phase pig house intradermal vaccine poison is positive, is secondary early warning; In non-vaccine immunity toxin expelling phase pig house, any one sample of air, feed, drinking-water, utensil detects virus-positive, and the swinery of a same pig house or faecal samples are negative, are three grades of early warning; The non-vaccine immunity toxin expelling phase detects virus-positive at any one sample of swinery or ight soil, is level Four early warning.
Accompanying drawing illustrates:
Fig. 1 is the figure that reaction product of the present invention is identified with 1.5% agarose gel electrophoresis.
Drawing illustrates: Fig. 1 sepharose detects JEV, CSFV, PRRSV, PPV, PCV-2, PRV 6 multiplex PCR result of virus, M:DL2000 DNA Marker; 1: special Marker 2:JEV, CSFV, PRRSV, PPV, PCV-2, PRV multiple RT-PCR, 3:JEV, 4:CSFV, 5:PPV, 6:PCV-2,7:PRRSV, 8:PRV.
embodiment:
Following examples further illustrate content of the present invention, but should not be construed limitation of the present invention, to amendment or the replacement of the inventive method, step or condition, all belong to scope of the present invention.
Embodiment further describes the present invention below.
the preparation of embodiment 1, special Marker
The present invention utilizes the reference sequences of Genbank, carries out gene comparision, selects conserved sequence, utilize biology information technology for each primers, extract the nucleic acid of virus, carry out PCR or RT-PCR, and glue reclaims PCR primer, is connected to pMD-18T carrier, is transformed into DH
5 αpreserve in competent cell, be that template carries out pcr amplification with recombinant plasmid, glue reclaims each fragment; measure its concentration, be diluted to respective concentration and add protective material (36% glycerine, the tetrabromophenol sulfonphthalein of 0.05%; the EDTA of 0.05% diformazan cyanophenyl blue FF, 30mM).And be that 1:5 mixes in the ratio of protective material and DNA, namely make special Marker.Special Marker is made up of DNA fragmentation 1045bp, 829bp, 636bp, 382bp, 275bp and 142bp, totally six bands, and wherein the DNA amount of 636bp band is 30ng/ μ L, and all the other bands are 10 ng/ μ L.Preserve 1 year at-20 DEG C, avoid multigelation.
experimental example 2,6 kind of virus multiple RT-PCR method
The cDNA of DNA and JEV of extraction PCV-2, PPV, PRV, CSFV, PRRSV mixing is carried out the detection of multiple RT-PCR as template, multiplex PCR adopts the reaction system of 50 μ L: 2 × Taq MasterMix is 25 μ L, 6 kinds of viral mix primer (20 μMs/L) totally 3 μ L, 6 kinds of virus mixing nucleic acid totally 3 μ L, sterile purified water 19 μ L.Brief centrifugation mixes.Reaction conditions is: 94 DEG C of 5min; 94 DEG C of 45s; 56 DEG C of 1m30s; 72 DEG C of 50s cycle numbers are 40 times, and last 72 DEG C extend 10min, 4 DEG C of preservations.Reaction product 1.5% agarose gel electrophoresis is identified.
the monitoring of experimental example 3, piggery enviroment
Be connected with bubble receiving flask by portable gas sampling pump, add the PBS damping fluid (containing 5% glycerine) of 20 mL pH=7.2 in receiving flask, working flow is 6 L/min, and collection time is 30min, and sample was disposed in 24 hours.
Air sample concentrates and adopts the mode of ultrafiltration to concentrate, above-mentioned collection liquid is added in the super filter tube that molecular weight cut-off is (10kD), the centrifugal 30min of 7000rpm under 4 DEG C of conditions, concentrated solution is carried out the centrifugal 10min of 10000rpm again, get supernatant and extract nucleic acid, carry out multiple RT-PCR detection.
Ight soil, Feed Sample take sample 5g, add the 0.1M PBS liquid of 15mL, and after vortex makes suspension, 4 DEG C, the centrifugal 10min of 3000r/min, gets supernatant, and the centrifugal 30min of supernatant 10000r/min, gets supernatant; Swab elutriant got by utensil, swinery, and the centrifugal 30min of 10000r/min, gets supernatant; 20mL got by drinking-water, and the centrifugal 30min of 10000r/min, gets supernatant.Concentration method is identical with the concentration method of above-mentioned air sample.Sample is detected with multiplex RT-PCR method.
Monitoring air sample 113 parts, faecal samples 86 parts, multiple RT-PCR detects with individual event PCR result and compares, and coincidence rate is 100%.
Rear attached sequence table
Sequence table
<110> Henan Science and Technology College
<120> mono-individual system monitors the method for 6 kinds of virus contamination in piggery enviroment simultaneously
<130> 2015
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> japanese encephalitis virus (Japanese encephalitis virus, JEV)
<400> 1
caaactggct ctgaaagg 18
<210> 2
<211> 19
<212> DNA
<213> japanese encephalitis virus (Japanese encephalitis virus, JEV)
<400> 2
tgtctcaggt ccatctacg 19
<210> 3
<211> 20
<212> DNA
<213> Pestivirus suis (Classical swine fever virus)
<400> 3
gctccctggg tgttctaagt 20
<210> 4
<211> 21
<212> DNA
<213> Pestivirus suis (Classical swine fever virus)
<400> 4
tgctgtcctt ctcatgctct t 21
<210> 5
<211> 18
<212> DNA
<213> porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus)
<400> 5
ggccagccag tcaatcag 18
<210> 6
<211> 21
<212> DNA
<213> porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus)
<400> 6
ggcaaactaa actccacagt g 21
<210> 7
<211> 23
<212> DNA
<213> pig parvoviral (porcine parvovirus)
<400> 7
acatctaaat atgccagaac acg 23
<210> 8
<211> 23
<212> DNA
<213> pig parvoviral (porcine parvovirus)
<400> 8
gtttgccatg agtgagttaa ttt 23
<210> 9
<211> 19
<212> DNA
<213> porcine circovirus 2 type (Porcine circovirus type 2)
<400> 9
cagcaccctg taacgtttg 19
<210> 10
<211> 19
<212> DNA
<213> porcine circovirus 2 type (Porcine circovirus type 2)
<400> 10
aggagtacca ttccaacgg 19
<210> 11
<211> 20
<212> DNA
<213> Pseudorabies virus (Pseudorabies Virus)
<400> 11
ctccttgagc gtcttcgtcg 20
<210> 12
<211> 20
<212> DNA
<213> Pseudorabies virus (Pseudorabies Virus)
<400> 12
ccttcctgtc caaccccttc 20。
Claims (5)
1. an individual system monitors the method for 6 kinds of virus contamination in piggery enviroment simultaneously, it is characterized in that comprising the following steps:
1) design of primers utilizes software to carry out design of primers in Pestivirus suis (CSFV), Japanese B encephalitis virus (JEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 type (PCV-2), porcine pseudorabies virus (PRV) and pig parvoviral (PPV) conserved regions;
CSFVF GCTCCCTGGGTGTTCTAAGT
CSFVR TGCTGTCCTTCTCATGCTCTT
PRRSVF GGCCAGCCAGTCAATCAG
PRRSVR GGCAAACTAAACTCCACAGTG
JEVF CAAACTGGCTCTGAAAGG
JEVR TGTCTCAGGTCCATCTACG
PCV-2F CAGCACCCTGTAACGTTTG
PCV-2R AGGAGTACCATTCCAACGG
PPVF ACATCTAAATATGCCAGAACACG
PPVR GTTTGCCATGAGTGAGTTAATTT
PRVF CTCCTTGAGCGTCTTCGTCG
PRVR CCTTCCTGTCCAACCCCTTC
Homologous segment be connected to carrier and be transformed in intestinal bacteria, after obtaining a large amount of amplification, according to the ratio of 1:5, protective material and DNA being mixed, make special marker, as reference standard;
2) foundation of multiple RT-PCR establishes an individual system and detects above-mentioned 6 kinds of viral multiplex RT-PCR methods simultaneously;
3) air sample collection to be gathered in pig house by liquid knockout method and air in piggery enviroment; Be that the super filter tube ultrafiltration of 10kD is centrifugal with molecular weight cut-off to carry out separation to virus and concentrate.
2. an individual system according to claim 1 monitors the method for 6 kinds of virus contamination in piggery enviroment simultaneously, it is characterized in that: involved by this monitoring method, the object fragment of 6 kinds of viruses (CSFV, JEV, PRRSV, PCV-2, PRV and PPV) is respectively 829bp, 1045 bp, 275 bp, 382 bp, 142 bp and 636bp.
3. an individual system according to claim 1 monitors the method for 6 kinds of virus contamination in piggery enviroment simultaneously, it is characterized in that: this multiple RT-PCR adopts 50 μ L reaction systems: 2 × TaqMaster Mix is 25 μ L, 6 kinds of viral mix primer (20 μMs/L) totally 3 μ L, 6 kinds of virus mixing nucleic acid totally 3 μ L, sterile purified water 19 μ L, reaction conditions is: 94 DEG C of 5min; 94 DEG C of 45s; 56 DEG C of 1m30s; 72 DEG C of 50s cycle numbers are 40 times, and last 72 DEG C extend 10min, 4 DEG C of preservations.
4. an individual system according to claim 1 monitors the method for 6 kinds of virus contamination in piggery enviroment simultaneously, it is characterized in that: in pig house and piggery enviroment air sample collecting position and count for: in pig house the position of collected specimens be distance floor level 0.5-1m place of pig house passageway central authorities according to the area determination sampling number of pig house, 30m
21 sampling point in left and right, in pig farm the position of collected specimens be pig farm central authorities and courtyard wall around four corner totally 5 sampling points, distance ground 0.5-1m, each sampling point acquisition time is 40min, outside pig farm, the position of collected specimens is each 3 sampling points in distance 200m place, upper and lower air port, pig farm, and feces collection gathers fresh faecal samples about 20g after selecting feeding in morning.
5. an individual system according to claim 1 monitors the method for 6 kinds of virus contamination in piggery enviroment simultaneously, it is characterized in that: the method can be used for monitoring above-mentioned 6 kinds of virus contamination of air in pig house and piggery enviroment, feed, drinking-water, utensil, ight soil and swinery, providing technical support for setting up main viral pollution prewarning mechanism and the prevention and control of main diseases viral disease and purification systems in piggery enviroment.
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