CN105010235B - The method that pig farm purifies epidemic disease - Google Patents
The method that pig farm purifies epidemic disease Download PDFInfo
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- CN105010235B CN105010235B CN201510503742.0A CN201510503742A CN105010235B CN 105010235 B CN105010235 B CN 105010235B CN 201510503742 A CN201510503742 A CN 201510503742A CN 105010235 B CN105010235 B CN 105010235B
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of method of pig farm purification epidemic disease, and swinery is divided into negative antibody by immune antiboidy detection and divides group and antibody positive to divide group；The negative antibody divides group to carry out booster immunization inoculation, detects immune antiboidy after inoculation again, antibody positive pig includes the antibody positive and divides group；The body fluid when antibody positive divides Farrowing in group in piglet umbilical cord is gathered as detection sample, CSFV and PRV are synchronously detected by PCR method, it is negative pig to retain CSFV and PRV；Periodically sampling observation and complete detection are carried out to the pig of reservation, CSFV is eliminated and PRV is positive or one of them is positive pig, to realize the synchronous purification of CSFV and PRV.This method synchronous purification swine fever and PRV, efficiency high, stress be small, easy to operate.
Description
Technical field
The present invention relates to disease field of purification, particularly relates to a kind of method of pig farm purification epidemic disease.
Background technology
Swine fever is commonly called as " rinderpest ", be a kind of acute, heating caused by the CSFV belonged to as flaviviridae CSFV,
Contagious disease, there is highly infectious and lethal.Porcine pseudorabies be swinery multiple infectious disease primary disease it
One.It is residual that the pig infection porcine pseudorabies or swine fever of large-scale pig farm are caused directly or indirectly delivery room grice diarrhoea, child care pig height
Secondary rate, in big porcine respiratory disease the problems such as, cause high mortality and high incidence, huge economic damage brought to pig farm
Lose.
At present in large-scale pig farm, the method purified respectively is taken for swine fever and porcine pseudorabies.For the net of swine fever
Change generally use collection tonsillotome carries out swine fever purification, then carries out the net of porcine pseudorabies by gene-deleted vaccine and discriminating
Change.Its specific embodiment is：First, immunity inoculation is carried out to kind of swinery, collection sample carries out detection immune antiboidy, gathers almond
Body, hog cholera immune fluorescence or Nest RT-PCR or fluorescence quantitative PCR detection CSFV are carried out, detects positive sow, it is naughty to give
Eliminate.Then, purified for PRV, i.e., it is immune to combine antidiastole, detect that wild poison is positive and eliminate, pig is pseudo- at present
The antidiastole sensitivity of rabies viruses virus is relatively low, generally needs 2-3 to monitor superseded with positive boar, finally
Reach the purpose of purification.
Because different virus has different preferendums and the method for purification.The method of existing pig farm purification epidemic disease is not
The purification work of porcine pseudorabies and swine fever can synchronously be carried out, need to be carried out respectively.Also, because boar body weight is big, no matter collection
Vena cava anterior blood or collection tonsillotome are relatively complicated, and excessive stress easily cause farrowing sow miscarriage, while blood sample is adopted
Collection needs more people to assist, and needs special installation, and gatherer process is relatively time-consuming, and cross pollution easily occurs in collection tonsillotome.
The content of the invention
In view of this, it is an object of the invention to propose a kind of method of pig farm purification epidemic disease, synchronous purification swine fever and pig
Pseudorabies virus, efficiency high, stress be small, easy to operate.
Based on the method for above-mentioned purpose purification epidemic disease in pig farm provided by the invention, detected by immune antiboidy and be divided into swinery
Negative antibody divides group and antibody positive to divide group；The negative antibody divides group to carry out booster immunization inoculation, and detection is exempted from again after inoculation
Epidemic disease antibody, antibody positive pig include the antibody positive and divide group；Gather piglet when the antibody positive divides Farrowing in group
Body fluid in umbilical cord synchronously detects CSFV and PRV by PCR method, retains hog cholera as detection sample
Poison and PRV are negative pig；Periodically sampling observation and complete detection are carried out to the pig of reservation, eliminate hog cholera
Poison and PRV are positive or one of them is positive pig, to realize the same of CSFV and PRV
Step purification.
Preferably, the body fluid gathered when the antibody positive divides Farrowing in group in piglet umbilical cord is used as detection sample,
DNA and RNA is extracted respectively, wherein the RNA extracted detects CSFV by Nest RT-PCR method；The DNA of extraction passes through PCR
Method detects PRV.
Preferably, the DNA of the extraction expands the gD genetic fragments and gE gene pieces of PRV by PCR method
Section, is identified through agarose gel electrophoresis.
Preferably, the size of the gD genetic fragments is 217bp, the size of the gE genetic fragments is 350bp.
Preferably, the Nest RT-PCR method detection CSFV genetic fragment, wherein overcoat PCR obtain 806bp's
Genetic fragment, it is 512bp that inner sleeve PCR, which obtains genetic fragment size,.
Optionally, the body fluid during detection sample Farrowing in piglet umbilical cord is 3-5mL.
Preferably, the immune antiboidy detection is antibody against swine fever virus, PRV gB antibody, the type of pig annulus 2
Antibody and blue ear antiviral antibody, the detection of four kinds of immune antiboidies, which is all positive pig, to be included antibody positive and divides group, one of which be present
Or more the negative pig of immune antiboidy include negative antibody and divide group.
Preferably, the negative antibody divides group to carry out booster immunization inoculation, it is inoculated with it and is detected as the epidemic disease antibody pair of feminine gender
The vaccine answered once, is inoculated with the latter moon, carries out the immune antiboidy detection again, detects positive pig and includes the antibody sun
Property divides group, and negative pig is eliminated.
Preferably, it is described periodically sampling observation with complete detection in, quarterly by 20-30% sampling Detections antibody against swine fever virus,
PRV gB antibody, the type antibody of pig annulus 2 and blue ear antiviral antibody are once.
Preferably, it is described periodically sampling observation with complete detection in, every half a year whole piglet break umbilical cord when gather umbilical cord in body fluid,
CSFV and PRV are detected by PCR method, and it is that positive or one of which is to eliminate two kinds of epidemic diseases of detection
Positive pig.
From the above it can be seen that the method for purification epidemic disease in pig farm provided by the invention can synchronously reach purification swine fever
With two kinds of important diseases of porcine pseudorabies, easy to operate, quick, no cross contamination and stress be small.Reduce due to swine fever and pig
Lost caused by pseudoabies.
Brief description of the drawings
Fig. 1 is the schematic flow sheet for the method that pig farm of the embodiment of the present invention purifies epidemic disease.
Embodiment
For the object, technical solutions and advantages of the present invention are more clearly understood, below in conjunction with specific embodiment, and reference
Accompanying drawing, the present invention is described in more detail.
Fig. 1 is the schematic flow sheet for the method that pig farm of the embodiment of the present invention purifies epidemic disease.As shown in figure 1, in the present embodiment
In, swinery is divided into negative antibody by immune antiboidy detection and divides group and antibody positive point by the method for the pig farm purification epidemic disease
Group；The negative antibody divides group to carry out booster immunization inoculation, detects immune antiboidy after inoculation again, antibody positive pig includes institute
State antibody positive and divide group；Gather the body fluid when antibody positive divides Farrowing in group in piglet umbilical cord and be used as detection sample,
CSFV and PRV are synchronously detected by PCR method, it is feminine gender to retain CSFV and PRV
Pig；Periodically sampling observation and complete detection are carried out to the pig of reservation, it is the positive to eliminate CSFV and PRV
Or one of them is positive pig, to realize the synchronous purification of CSFV and PRV.It can be seen that the present embodiment pig farm
The method for purifying epidemic disease detects the umbilical cord body fluid of pig farm Farrowing by PCR method, and synchronous purification CSFV and pig puppet are mad
Dog disease poison, stress small, efficiency high, easy to operate.
Embodiment one：Immune antiboidy detects and tentatively divides group
Clinical appearance inspection is carried out to the boar of the whole audience, eliminates boars more than old, weak and 8 tires of farrowing, composition is false
Fixed health kind swinery.
The vena cava anterior blood system of above-mentioned hypothesis health kind swinery is gathered from serum, (Enzyme-linked Immunosorbent Assay is real by ELISA
Test) method detection assumes the health kind CSFV of swinery, PRV, pig circular ring virus, the immune antiboidy of blue otopathy
(antibody against swine fever virus, PRV gB antibody, the type antibody of pig annulus 2 and the indigo plant of biological Co., Ltd's research and development before the section of Wuhan
Otopathy poison antibody ELISA kit) expression, it is the swinery that positive pig is successful immunization to choose four kinds of antibody, is included
Antibody positive divides group.
Criterion：
CSFV：S/P (sample OD630nmValue/positive control OD630nmValue) value >=0.17, it is judged to the positive；S/P values ＜
0.17, it is judged to feminine gender.
PRV gB antibody：S/N (sample OD630nmValue/negative control OD630nmValue) value >=0.7, it is judged to feminine gender；
S/N value≤0.6 is the positive；If 0.6 ＜ S/N value≤0.7, to be suspicious, sample detects again, if result is identical, is spaced two
Zhou Zaici sample detectings.
Porcine circovirus 2 type antibody：Sample (S-N)/(P-N) value >=0.16, then be judged to the positive；If sample (S-N)/
(P-N) value ＜ 0.16, then be judged to feminine gender.Wherein, S is sample OD630nmValue；P is positive control OD630nmValue；N is negative control
OD630nmValue.
Blue ear antiviral antibody：Sample KQ value >=20, are judged to the positive；Sample KQ values ＜ 20, is judged to feminine gender.Wherein, KQ=(samples
This OD630nmValue/positive control average value) * 100.
According to testing result, it will be assumed that health kind swinery is divided into antibody positive and divides group to divide group, two groups of isolation with negative antibody
Raising.Wherein, antibody positive divides group to be all positive pig for the detection of above-mentioned immune antiboidy, and negative antibody divides group to be deposited for above-mentioned detection
In the negative pig of immune antiboidy.
Embodiment two：Negative antibody divides group's booster immunization
According to a kind of antibody test result of embodiment, negative antibody divides the pig booster immunization of group to be inoculated with it to be detected as feminine gender
Epidemic disease antibody corresponding to vaccine once, according to corresponding vaccine operation instruction dose operation.
Corresponding vaccine is inoculated with after 1 month, carries out corresponding immune antiboidy detection again, will based on the criterion of embodiment one
Negative pig is eliminated, and positive pig includes antibody positive and divides group.
Embodiment three：Antibody positive divides group synchronization to purify
In the present embodiment, the qualified pig of the antibody of above-mentioned antibody test result, i.e. antibody positive divide group's pig, in sow
During childbirth, collection piglet is broken the body fluid in umbilical cord, delivers to laboratory, the body in umbilical cord that piglet during the Farrowing of collection is broken
Liquid, DNA and RNA is extracted respectively.Wherein, the RNA of extraction detects CSFV by Nest RT-PCR method；The DNA of extraction leads to
Cross PCR method detection PRV.The DNA of the extraction expands the gD gene pieces of PRV by PCR method
Section and gE genetic fragments, are identified through agarose gel electrophoresis.
Positive (carrier) pig of detection, eliminates, piglet isolated rearing, booster immunization, no longer stays immediately after sow wean
Kind, big porker sale.
Test in laboratory comprises the following steps that：
1) sample treatment
Body fluid 3-5mL during Farrowing in the disconnected umbilical cord of piglet is as sample.Preferably, collect the body fluid 4mL in umbilical cord
As sample, 1500rpm/min centrifugation 15min, the μ L of supernatant 200 are taken, extract DNA and RNA respectively.
2) RNA is extracted
Body fluid total serum IgE slightly carries, and positive-virus is as positive control.
1. taking the μ L of pre-treatment Supernatant samples 200, it is added in 1.5mL Eppendorf pipes, adds 1000 μ L RNA
Trizol vibrations mix, and are stored at room temperature 10min；
2. add 200 μ L chloroforms, on vortex mixer vibration mix 5s (can not be excessively strong, can also in order to avoid produce emulsion layer
Mixed with hand is reverse), 4 DEG C of centrifugation 10min of ice bath 10min, 12000rpm/min；
3. take supernatant in another new Eppendorf pipes, add isometric isopropanol (4 DEG C of precoolings) mix, room temperature or 4 DEG C it is quiet
Put 15min；
4. 4 DEG C, 12000rpm/min centrifugations 10min (Eppendorf tube openings keep placing towards centrifugal basket direction of principal axis),
Supernatant carefully is removed, is inverted on blotting paper and is stained with dry liquids or blots residual liquid with rifle, add the ethanol of 1mL 75%, overturn
Washing；
5. 7500rpm/min centrifuges 5min, Eppendorf ramp downs are put, carefully exhaust liquid with rifle tube mouth, normal temperature is done
Dry 20min or so.
6. in being dissolved in the water that 25 μ L pollute without RNase, -20 DEG C of short-term preservations, -70 DEG C long-term preserve or inverted immediately
Record.
In the present embodiment, all RNA extracts reagents, various sample-adding pipette tips, Eppendorf pipes and other contact samples
Utensil all must be the pollution of no RNase, should wear disposable glove and often change during operation.
RNA reversion cDNA systems (20 μ L)
System is placed in PCR instrument after preparing reacts, 42 DEG C, 60min extension, 70 DEG C, the inactivation of 5min reverse transcriptase.Instead
It should terminate, sample is dispensed, -20 DEG C of preservations are put into after mark is good.
3) DNA extraction
Blood sample, cell, the tissue gene group DNA extraction kit specification provided with reference to Tiangeng company is carried out, set up it is cloudy,
Positive control.4 DEG C of the DNA extracted storages is standby, if -20 DEG C of preservations are placed in storage for a long time.
4) RT-PCR and PCR amplifications：
1. CSFV Nest RT-PCR detection method mainly uses Nest RT-PCR swine fever detection method, specific as follows：
Primer：
First, it is respectively upstream and downstream primer using Pup-1 and Pdown-1, with above-mentioned steps 2) body fluid that obtains of extraction is total
RNA after reverse transcription obtains cDNA, carries out overcoat PCR reactions as template.
Reaction system：
Amplification condition：95 DEG C of 3min → (95 DEG C of 45s, 55 DEG C of 1mim, 72 DEG C of 1mim) 35 circulate → 72 DEG C of extension 5min.
Then, it is respectively upstream and downstream primer using Pup-2 and Pdown-2, the PCR primer obtained using overcoat PCR is mould
Plate, carry out inner sleeve PCR reactions.
Reaction system：
Amplification condition：95 DEG C of 3min → (95 DEG C of 45s, 55 DEG C of 1mim, 72 DEG C of 1mim) 35 circulate → 72 DEG C of extension 5min.
2. PRV PCR detection method
First, PRV gD genetic fragments are expanded with reference to pseudo- mad dog detection method (GB/T 18641-2002),
Clip size is 217bp.
Primer：
Sense primer Up1：5'-CACggAggACgAgCTggggCT-3'
Anti-sense primer Up2：5'-gTCCACgCCCCgCTTgAAgCT-3'
Reaction system：
Reaction condition：
95 DEG C of 5min → (94 DEG C of 1min, 57 DEG C of 1min, 72 DEG C of 1min) 30 circulate → 72 DEG C of extension 10min, last 4 DEG C
Protection, terminate reaction.
Secondly, according to the PRV Becker pnca gene group sequences (GenBank announced:JF797219) gene order designs
Antidiastole PRV gE genes, purpose fragment 350bp.
Primer：
Sense primer gEup:5'-ATG CGG CCC TTT CTG CTG CY-3'
Anti-sense primer gEdown:5'-CGA CAC GGC GTC GCA GCC GC-3'
Reaction system：
Reaction condition：
95 DEG C of pre-degenerations 5min, 94 DEG C of 1min, 60 DEG C of 1min, 72 DEG C of extensions, 30 circulations, last 72 DEG C of 10min.
5) agarose gel electrophoresis, analyses and comparison result
The μ l of product 10 for taking PCR and Nest RT-PCR to expand, in electrophoresis detection on 0.8% Ago-Gel.
Positive control and negative control are set up, and then represent positive amplifying purpose fragment.
1. PRV：
It is detected simultaneously by 217bp gD genetic fragments and the 350bp gE fragment positives is eliminated for wild virus infection；
GE genetic fragments are only detected, in order to during safety, quarantine, collection blood sample carries out antidiastole；
Only detect that gD genetic fragments are then shown to be vaccine strain, do not eliminate.
2. CSFV：Overcoat PCR obtains 806bp genetic fragment, and it is 512bp that inner sleeve PCR, which obtains genetic fragment size,
Then it is judged as the positive.
Culling level：Detect PRV gE and swine fever is positive or one of them is positive, sow wean is washed in a pan
Eliminate, piglet isolated rearing, no longer reserve seed for planting, eliminate.
Example IV：Periodically sampling observation and complete detection
The kind swinery after the above method purifies is maintained, is quarterly sampled by 20%-30%, is detected by the method for embodiment one
The immune antiboidies such as CSFV, PRV, blue otopathy and PCV-II 1 time.Antibody test feminine gender booster vaccine is exempted from
Epidemic disease is inoculated with, and antibody test positive swinery adheres to periodic detection and feeding management.
Preferably, quarterly by 20% sampling.
Every half a year whole piglet break umbilical cord when, by the method for embodiment three, gather body fluid in umbilical cord, and detect CSFV
And PRV, eliminate positive pig.Culling level：Detect PRV and swine fever is positive or one of them
For the positive, sow wean is eliminated, piglet isolated rearing, no longer reserved seed for planting, eliminates.
By 1-2, carry out-superseded-the monitoring purification work such as immune of the detection of swine fever and porcine pseudorabies, you can completely
Control swine fever, PRV, it is established that healthy and stable kind swinery.
Embodiment five：
When implementing purification techniques, implementing for supporting measures is also carried out, takes Synthetical prevention technology.
To do the purification of swine fever well, following items supplementary measures should be carried out：
1) pig farm will strictly carry out isolated rearing, the feeding management system of " all-in and all-out ".Except doing hog cholera vaccine, pig well
Pseudorabies virus disease vaccine it is immune outer, also to carry out the immunity inoculation of aftosa, blue otopathy and parvovirus.
2) " five fixed work " is carried out, be i.e. periodically sterilization, regular expelling parasite, is periodically killed mouse, regular desinsection, regular medicinal health-care.
Every bio-security is implemented comprehensively.
3) do immunoprophylaxis well by the immune programme for children of science, improve the non-specific immunity of pig.
4) full price safe feed is fed, forbids to feed the feed of moldy metamorphism.
5) improve feeding environment, pay attention to animal welfare.
6) to the boar newly introduced, strict quarantine is carried out, porcine pseudorabies viral disease is introduced and antibody against swine fever virus is cloudy
Property or wild malicious negative antibody pig.After introducing, isolated rearing 2 months, sample inspection of drawing blood, antibody or wild virus infection antibody are negative patient
Group feeding is mixed with this other pig again to support.As other swinerys, every half a year once checks, for the infection positive pig detected
Strictly to isolate, after vaccinating, make fattening swine rearing and sell.
7) bio-safety depth is established
In the present embodiment, pig farm planted agent be additionally provided with disinfection station, terminal, ozone generator, wheel barnyard, specially draw pig
Car, air filtration boar station, independent feed factory.
Wherein, the construction of disinfection station and the development first step measure that application is diseases prevention；Terminal by between each pig farm, it is outer
It is relatively isolated between portion client and pig farm, forms bio-safety first of safety curtain of depth；Ozone generator is marched into the arena for pig farm
Guarding for mouth, more thoroughly eliminates the important infection sources；Wheel barnyard is provided and can shifted for the processing of sudden illness and control and prevention of disease
Space；It is special to draw pig car to cut off the transmission of pathogen between several fields；Air filtration boar station provides high-quality seminal fluid；Independent
Feed factory reduces the risk due to using outside feed.
8) reasonable immune programme for children is formulated
According to pig farm antibody test situation, with reference to pig farm Growth Results, clinical health degree suitably adjusts pig farm immune programme for children,
Ensure that pig farm production is stable and swinery is healthy.
Embodiment six：
The kind pig farm of one scale, 2000 sows, epidemic disease is purified by carrying out pig farm of the present invention since in April, 10
Method, carry out according to aforesaid operations, by the purification of 1.5 years, in October, 11, Pathogen test total negative, reached expected net
Change effect.And early stage generally needs more than 2-3 using conventional method, and purification process stress be big, causes farrowing sow miscarriage etc.
Problem.
Specific Growth Results change see the table below：
Because vertical transmission can occur for CSFV and PRV, and umbilical cord is that connection parent and fetus are unique
Passage, in addition fetus developing immune system imperfection, therefore after fetal infection virus, virus can be bred in fetus body, navel
The body fluid stayed in when with interior body fluid being Farrowing in the fetus body in umbilical cord.Therefore the method for the pig farm purification epidemic disease of the present invention,
Swine fever and PRV virus infection have been can decide whether by detecting body fluid in umbilical cord.A kind of sample is only gathered, i.e.,
Two kinds of diseases can be purified simultaneously, improve efficiency.
Meanwhile purified for pseudoabies, conventionally employed ELISA antidiastole technology for detection vena cava anterior serum gE resists
Body is horizontal, and the problems such as false positive easily occurs in this method, using round pcr it is possible to prevente effectively from.
It can be seen that the method choice piglet of purification epidemic disease in pig farm provided by the invention is broken, the body fluid of umbilical cord does CSFV and pig
Pseudorabies virus is detection sample, is differentiated using the Nest RT-PCR technology and PRV PCR vaccines of detection swine fever and examined
Disconnected technology, detects positive pig, and piglet isolated rearing is no longer reserved seed for planting, and sow wean is eliminated, by 1-2 detections-immune-
Eliminate program and realize purification.The shortcomings that conventional method need to distinguish sample detecting purification is avoided, gather same sample can be simultaneously
The purification of CSFV and PRV is carried out, improves purification efficiency.Also, the operability of sample collection is good, more often
Advise that acquisition operations are easier, quick, no cross contamination and stress be small.Avoid tonsillotome and the collection of forced venous blood sample is multiple
Miscellaneous, easy pollution, stress be big, easily causes the problems such as farrowing sow is miscarried.
Those of ordinary skills in the art should understand that：The discussion of any of the above embodiment is exemplary only, not
It is intended to imply that the scope of the present disclosure (including claim) is limited to these examples；Under the thinking of the present invention, above example
Or can also be combined between the technical characteristic in different embodiments, step can be realized with random order, and exist such as
Many other changes of upper described different aspect of the invention, for simplicity, they are not provided in details.Therefore, it is all
Within the spirit and principles in the present invention, any omission for being made, modification, equivalent substitution, improvement etc., it should be included in the present invention's
Within protection domain.
Claims (6)
- A kind of 1. method of pig farm purification epidemic disease, it is characterised in that swinery is divided into by negative antibody point by immune antiboidy detection Group and antibody positive divide group；The negative antibody divides group to carry out booster immunization inoculation, detects immune antiboidy, antibody after inoculation again Positive pig includes the antibody positive and divides group；Gather the body fluid in piglet umbilical cord when the antibody positive divides Farrowing in group As detection sample, CSFV and PRV are synchronously detected by PCR method, retain CSFV and pseudorabies Virus is negative pig；Periodically sampling observation and complete detection are carried out to the pig of reservation, eliminate CSFV and pseudorabies Virus is positive or one of them is positive pig, to realize the synchronous purification of CSFV and PRV；The body fluid when antibody positive divides Farrowing in group in piglet umbilical cord is gathered as detection sample, extracts DNA respectively With RNA, wherein the RNA extracted pass through Nest RT-PCR method detect CSFV；Overcoat primer pair in Nest RT-PCR method For Pup-1 and Pdown-1, inner sleeve primer pair is Pup-2 and Pdown-2, and the nucleotide sequence of primer is respectively：Pup-1： Ctagtaactggggcacaag, Pdown-1：Gttgtgttgccgatcaagc, Pup-2：Gtcacagcacttaatgtggtc, Pdown-2：cacttacctatggggtagtgtg；The DNA of extraction detects PRV by PCR method；The extraction DNA gD genetic fragments and the gE genetic fragments of PRV are expanded by PCR method, reflected through agarose gel electrophoresis It is fixed；The primer pair of amplification PRV gD genetic fragments is sense primer Up1 and anti-sense primer Up2, the nucleotides of primer Sequence is：Sense primer Up1：5'-CACggAggACgAgCTggggCT-3', anti-sense primer Up2：5'- gTCCACgCCCCgCTTgAAgCT-3'；The primer pair of PRV gE genetic fragments is that sense primer gEup and downstream are drawn Thing gEdown, the nucleotides sequence of primer are classified as：Sense primer gEup:5'-ATGCGGCCCTTTCTGCTGCY-3', anti-sense primer gEdown:5'-CGACACGGCGTCGCA GCCGC-3'；The size of the gD genetic fragments is 217bp, and the size of the gE genetic fragments is 350bp；It is detected simultaneously by 217bp's GD genetic fragments and the 350bp gE fragment positives are eliminated for wild virus infection；GE genetic fragments are only detected, in order to during safety, Quarantine, collection blood sample carries out antidiastole；Only detect that gD genetic fragments are then shown to be vaccine strain, do not eliminate；The Nest RT-PCR method detects CSFV genetic fragment, and wherein overcoat PCR obtains 806bp genetic fragment, interior It is 512bp to cover PCR to obtain genetic fragment size；When overcoat PCR obtains 806bp genetic fragment, inner sleeve PCR obtains genetic fragment Size is 512bp, then is judged as the positive.
- 2. the method for purification epidemic disease in pig farm according to claim 1, it is characterised in that during the detection sample Farrowing Body fluid in piglet umbilical cord is 3-5mL.
- 3. the method for purification epidemic disease in pig farm according to claim 1, it is characterised in that the immune antiboidy detection is pig Pestivirus antibody, PRV gB antibody, the type antibody of pig annulus 2 and blue ear antiviral antibody, four kinds of immune antiboidy detections are complete Antibody positive is included for positive pig and divides group, the negative pig of one of which or more immune antiboidy is present and is included negative antibody point Group.
- 4. the method for purification epidemic disease in pig farm according to claim 1, it is characterised in that the negative antibody divides group to be added Strong immunity inoculation, it is inoculated with it and is detected as vaccine corresponding to the epidemic disease antibody of feminine gender once, the latter moon is inoculated with, again described in progress Immune antiboidy detects, and detects positive pig and includes the antibody positive and divide group, negative pig is eliminated.
- 5. the method for purification epidemic disease in pig farm according to claim 1, it is characterised in that periodically sampling observation and the complete detection In, quarterly by 20-30% sampling Detections antibody against swine fever virus, PRV gB antibody, the type antibody of pig annulus 2 and blue ear Antiviral antibody is once.
- 6. the method for purification epidemic disease in pig farm according to claim 1, it is characterised in that periodically sampling observation and the complete detection In, every half a year whole piglet break umbilical cord when gather body fluid in umbilical cord, pass through PCR method and detect CSFV and porcine pseudorabies Poison, and it is pig of the positive or one of which for the positive to eliminate two kinds of epidemic diseases of detection.
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