CN107988436A - A kind of PCR system and detection method for detecting pig virus Reproduction Disorder - Google Patents
A kind of PCR system and detection method for detecting pig virus Reproduction Disorder Download PDFInfo
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Abstract
The invention discloses a kind of PCR system and detection method for detecting pig virus Reproduction Disorder, include the following steps：Viral DNA and RNA are extracted, and RNA is subjected to reverse transcription, is carried out at the same time CSFV, PPV, PRRSV, JEV specific primer design.Then, the optimization of PCR reaction conditions is carried out to the primer of design, so that it is determined that optimal multiplex PCR pattern.Tested according to optimal multi-PRC reaction condition, verify the specificity, sensitiveness and stability of multiplex PCR, be carried out at the same time clinical sample detection to verify the feasibility of the method operation.In addition, the present invention also disclosed for while detect the primer sequence of CSFV, PPV, PRRSV, JEV.The multiplex PCR system and detection method of four kinds of pig virus Reproduction Disorders of the present invention, not only remain high specific, the sensitiveness of Standard PCR, reduce operating procedure again, the purpose of multiple-microorganism can be detected at the same time by realizing once amplification, the time is greatly saved, has saved expenditures.
Description
Technical field
The present invention relates to agricultural and animal quarantine field, is specifically a kind of detection pig virus Reproduction Disorder
PCR system and detection method.
Background technology
Continuous with China's scale livestock farming expands, and intensive feeding manner considerably increases connecing between swinery
Probability is touched, while the increase of contact probability accelerates transmission, also so that mixed infection phenomenon is often presented in diseased pig, wherein
Pig breeding dysfunction disease becomes one of most important epidemic disease in large and medium-sized pig farm, and is in global distribution, China currently still with
Swine fever (CSF), pig breeding with respiratory disorder syndrome (PRRS), Porcine circovirus desease (PCVDA), porcine parvovirus (PPI),
Breeding difficulty caused by pig Japanese B encephalitis (JE) is the most universal and serious, and huge economic loss is caused to pig breeding industry.
The detection method of existing pig breeding dysfunction disease has serological method and molecular biology method etc..Serological method bag
Include neutralization test, enzyme-linked immunosorbent assay (ELISA), latex agglutination test, hemagglutination test and hemagglutination-inhibition test, colloidal gold
Immunochromatography, wherein neutralization test are using relatively broad.Molecular biology method high sensitivity, specificity are good, and this method is main
Including nucleic acid probe, genetic chip, PCR detections etc..And for genetic chip, traditional chip platform is because depending on fluorescence
Labelling technique is higher to technical requirements, it is difficult to accomplish base, it is necessary to expensive scanning device is scanned and operating process is complicated
Layer is promoted.
Multiplex PCR (multiplex polymerase chain reaction, mPCR) is sent out on the basis of Standard PCR
The technology that exhibition is got up, it is 2 pairs or more than 2 pairs primers of addition in same reaction system, expands the micro- life of Different Kinds of Pathogens respectively
The template of thing, can obtain different purpose fragments, and this method had not only remained the specificity and sensitiveness of conventional PCR method, but also
Reduce operating procedure and reagent dosage, the specific gene of multiple pathogenic microorganisms can be detected in same reaction, is examined at the same time
Survey identifies multiple pathogens, no matter in terms of Diagnosis of Infectious Diseases caused by pathogenic microorganisms, micro- life of still causing a disease in the environment
The inspection field of thing, mPCR technologies all have wide application prospect.
The content of the invention
It is an object of the invention to provide a kind of PCR system for detecting pig virus Reproduction Disorder and detection side
Method, to solve the problems mentioned in the above background technology.
To achieve the above object, the present invention provides following technical solution：
A kind of PCR system and detection method for detecting pig virus Reproduction Disorder, comprises the following steps：
(1) extraction of PPV viral DNAs；
(2) extraction and reverse transcription of CSFV, PRRSV, JEV viral RNA；
(3) specific primer design and screening；
(4) PCR reaction condition optimizations are carried out to the specific primer designed by step (3)；
(5) the optimal multi-PRC reaction system established according to step (4) carries out multiplexed PCR amplification specificity verification；
(6) the optimal multi-PRC reaction system established according to step (4) carries out multiplex PCR sensitiveness verification；
(7) the optimal multi-PRC reaction system established according to step (4) carries out multiplex PCR stability verification；
(8) according to detection of the multiple PCR method of foundation to clinical sample.
Further：In step (1), the extracting method of the PPV viral DNAs is：Appropriate PPV virus liquids are taken, are placed in
In 1.5mL Eppendorf pipes, with reference to the E.Z.N.A of OMEGA companiesTMTissue DNA Kit extracts kits specifications into
OK, the genomic DNA of extraction is placed in -20 DEG C to save backup.
Further：In step (2), the extraction of CSFV, PRRSV, JEV viral RNA and reverse transcription method are：Take CSFV,
PRRSV and JEV virus liquids, with reference to the MiniBEST ViralRNA/DNA Extraction Kit specifications extraction of TaKaRa companies
Viral RNA, by the reverse transcription of viral RNA of extraction into cDNA；According to Magen companies M-MLV H-First-Stand
Synthesis Kit reverse transcription reagent box specification carries out, and the cDNA of reverse transcription is placed in -20 DEG C and is saved backup.
Further：In step (3), design and the screening of the specific primer are specially：Applied biology software pair
CSFV genes, PPV genes, PRRSV genes and JEV genes carry out homology analysis, screen and determine primer, it is ensured that designed primer
Specific primer is necessary for, pair of primers can only amplify the sequence fragment of corresponding virus, and not have between other primers
Interference, i.e., it is no homologous, complementary, do not form hairpin structure；The primer sequence of the design is：
The specific primer of CSFV：
F:5′-TGCAACTGAATGACGGGAC-3′
R:5′-GCCAAATACCTCCTACTGACCAC-3′
The specific primer of PPV：
F:5′-AAAAACAATCCACCAGGACAAC-3′
R:5′-CTGGATCTCATTTTTGCTGTGA-3′
The specific primer of PRRSV：
F:5′-CGGGCGGTATGTCCTAAGTA-3′
R:5′-ACCTCAACTTTGCCCCTTTT-3′
The specific primer of JEV：
F:5′-AGCTTGTTGGACGGCAGAG-3′
R:5′-GCCACATGATTGAGCCTTCA-3′
Further：In step (4), the PCR reaction condition optimization specific methods are：The PCR of 25 μ L is selected to react
System, system include 10 × PCR buffer (Mg2+Plus) 2.5 μ L, 2.5mmol/L dNTP, 3 μ L, 5U/ μ L Taq polymerases
0.4 μ L, DNA and cDNA hybrid template, 1 μ L, primer working concentration are 10 μm of ol/L, according to the bright of multiplex PCR band, suitably
Adjust the ratio of 3 pairs of primer additions；Annealing temperature according to primer Tm scope carry out 50 DEG C, 51.7 DEG C, 52.9 DEG C, 54.3
DEG C, 55.4 DEG C, 56.6 DEG C, 57.8 DEG C, the experiment of 59.9 DEG C of annealing temperature gradients, finally determine optimal multiplex PCR condition.
Further：In step (4), optimal multiplex PCR condition is specially：10 × buffer (Mg in 25 μ L reaction systems2 +) 2.5 μ L, dNTP 3 μ L (2.5mmol), 4 couples of 2.4 μ L of primer upstream and downstream mix primer, 0.4 μ L of Taq polymerase (5U/ μ L),
1 μ L of DNA/cDNA hybrid templates, add sterilizing distilled water to 25 μ L；Optimal multi-PRC reaction condition is：94℃5min；94℃
1min, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72℃10min.
Further：In step (5), multiplexed PCR amplification specificity verification is specially：At the same time with 4 couples of spies in step (3)
Specific primer, respectively to distilled water, Escherichia coli, PRV and CSFV, PPV, PRRSV, JEV template various combination, according to step
(4) the optimal PCR reaction conditions in are expanded, to verify its specificity.
Further：In step (6), multiplex PCR sensitiveness verification is specially：First by CSFV, PPV, PRRSV and
The standard DNA or cDNA samples of JEV viruses measure original template concentration with ultramicron nucleic acid-protein analyzer, then continuous with 10 times
The template of CSFV, PPV, PRRSV and JEV 4 is diluted to 7 concentration gradients by dilution respectively, according to the optimal PCR in step (4)
Reaction condition is expanded, and measures the sensitiveness of multiplex PCR.
Further：In step (7), multiplex PCR stability verification is specially：It is more with what is established in step (4)
Weight PCR method repeats detection 9 times to CSFV, PPV, PRRSV and JEV, with the reliability of verification result.
Further：In step (8), clinical sample detection is specially to verify its feasibility：By doubting for collection
DNA and RNA is extracted according to step (1) and step (2) like pathological material of disease, the multi-PCR detection method established according to step (4) is expanded
Increase, to verify the feasibility of this method.
Compared with prior art, the beneficial effects of the invention are as follows：
(1) it is simple, directly perceived：4 pairs of primers that the present invention designs, G/C content is close, and Tm values are close, and this guarantees identical
Annealing temperature under, CSFV, PPV, PRRSV and JEV can be expanded well.Its amplified fragments be respectively 91bp,
152bp, 1988bp and 238bp, this to carry out on same running gel when detecting, by observing PCR product band
Position just can judge.
(2) high specificity and rapid reaction, time-consuming short：The detection method that the present invention establishes shows that either single PCR is also
It is multiplex PCR, energy specific amplification goes out CSFV, PPV, PRRSV and JEV band；Compared with regular-PCR detection method, this reality
Testing the multiplex PCR of use has sensitivity and the accuracy of higher；Compared with using immunology ELISA detection method, multiplex PCR flower
Take that small, detection time is short, experiment condition requirement is relatively also less strict.
The present invention dissolves synthetic specific primer with TE Buffer by designing and synthesizing specific primer, according to
The PCR reaction systems and PCR response procedures set carries out primer amplification, and observes result with agarose gel electrophoresis.The party
Method can effectively solve existing detection device and method, and time-consuming, puts into the problems such as big.This method is established as from now on using multiple
PCR detection method carries out animal epidemics clinical diagnosis and research provides theoretical foundation, has also established base with elimination epidemic situation in order to control
Plinth.
Brief description of the drawings
Fig. 1 is individual event PCR and multiplexed PCR amplification as a result, wherein M：Marker DL500；1：Negative control；2：CSFV；；
3：PPV；4：PRRSV；5：JEV；6：JEV+PRRSV+PPV+CSFV.
Fig. 2 be different annealing temperature carry out multi-PRC reaction, wherein M：Marker DL500；1：Negative control；2：50
℃；3：51.7℃；4：52.9℃；5：54.3℃；6：55.4℃；7：56.6℃；8：57.8℃；9：59.9℃.
Fig. 3 is the specific test of multiplex PCR as a result, wherein M：Marker DL500；1：Distilled water；2：JEV+PRRSV+
PPV+CSFV；3：Escherichia coli；4：PCV2.
Fig. 4 is multiplex PCR sensitivity tests as a result, wherein M：Marker DL500；1：100Dilution；2：101Dilution；3：
102Dilution；4：103Dilution；5：104Dilution；6：105Dilution；7：106Dilution；8：107Dilution；9：Negative control.
Fig. 5 is multiplex PCR repetitive test as a result, wherein M：Marker DL500；1：Negative control；2-10：Same
The amplification of multiplex PCR under part.
Embodiment
Below in conjunction with the attached drawing in the embodiment of the present invention, the technical solution in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other without making creative work
Embodiment, belongs to the scope of protection of the invention.
Please refer to Fig.1~5, in the embodiment of the present invention, a kind of PCR system for detecting pig virus Reproduction Disorder and
Detection method, comprises the following steps：
(1) extraction of PPV viral DNAs：150 μ l of PPV cytopathies venom are taken into centrifuge tube (1.5ml), add 25 μ l respectively
After mixing, during which the water-bath 10min in 60 DEG C of water-baths is carried out shaking and is allowed to fully react OB Protease.Water-bath is complete
Into rear to centrifuge tube 10000 × g high speed centrifugation 5min, precipitation is discarded, takes supernatant separately to put in a new centrifuge tube, adds 220 μ l
Buffer BL are mixed, 70 DEG C of water-bath 10min of water-bath.After the completion of water-bath, 220 μ l ethanol solutions (rooms are added into centrifuge tube
Temperature), it is quick to mix, liquid is then moved into adsorption column and puts on collecting pipe, after 8000 × g high speed centrifugations 1min, discards liquid.
A collecting pipe is replaced, 500ul Buffer HB, 8000 × g centrifugation 1min are added into adsorption column, abandon liquid.One is replaced again
A collecting pipe, adds DNA Wash Buffer (adding alcohol in advance to final concentration of 25mg/ml) 700ul in adsorption column, and 8000
× g high speed centrifugation 1min, discard liquid, and again plus DNA Wash Buffer carry out DNA secondary washing and centrifuge, and outwell receipts
Liquid in collector, 14000 × g high speed centrifugations 2min drying adsorption columns.Collecting pipe is replaced with sterile centrifugation tube (1.5ml),
150 μ l are added in adsorption column and shift to an earlier date 70 DEG C of preheated Elution Buffer, after being stored at room temperature 3min, 10000 × g at a high speed from
Heart 1min, DNA is eluted out from adsorption column, then adds 150 μ l Elution Buffer to carry out secondary elution to DNA, will be molten
Liquid moves to sterile centrifugation tube (1.5ml), and -20 DEG C save backup.
(2) extraction and reverse transcription of CSFV, PRRSV, JEV viral RNA：200 μ l virus stock solution useds are taken to add 200 μ l's
The Carrier RNA of Buffer VGB, the Proteinase K of 20 μ l and 1.0 μ l, fully mix and divide in 56 DEG C of water-bath warm bath 10
Clock, 200 μ l absolute ethyl alcohols are added into lysate, are fully inhaled and are played mixing；Spin Column are placed in Collection
On Tube, solution is moved in Spin Column, and 12,000rpm centrifugations 2 minutes, abandon filtrate；The Buffer RWA of 500 μ l are added
Enter into Spin Column, 12,000rpm centrifugations 1 minute, abandon filtrate；The Buffer RWB of 700 μ l are added to Spin
In Column, 12,000rpm centrifugations 1 minute, abandon filtrate；Repeat aforesaid operations step；Spin Column are placed in
On Collection Tube, 12,000rpm centrifugations 2 minutes；Spin Column are placed in new 1.5mlRNase free
On collection tube, add the RNase free dH2O of 30-50 μ l in the centre of Spin Column films, be stored at room temperature 5
Minute；12,000rpm centrifugation 2min eluted dnas/RNA.By the reverse transcription of viral RNA of extraction into cDNA, according to Magen companies M-
MLV H-First-Stand Synthesis Kit reverse transcription reagent box specification carries out.Reaction system is 20 μ L, wherein RNA
6 20 1 μ L, d NTPs of μ L, Oligo (dT) of solution, 1 μ L, Nuclease Free Water complement to 13 μ L, and mixed liquor is placed in
65 DEG C of incubation 5min, rapid take out are placed in cooled on ice 1min, 5 × M-MLV First Stand are added in backward reaction solution
4 μ L, 0.1M DTT of Buffer, 1 μ L, M-MLV RNase H-RT 1 μ L, RNase Inhibitor (40U/ μ L) 1 μ L, softly
Moment centrifuges after piping and druming mixes.Reacted 45 DEG C, 30min, 85 DEG C, moment centrifuges after 10s, -20 DEG C of product saves backup.
(3) specific primer design and screening：According to CSFV, PPV, PRRSV, JEV gene logged in Gene Bank
Reference sequences, carry out CSFV genes, PPV genes, PRRSV genes and JEV genes using DNA DNAStar or DNAMAN software
Homology analysis, according to analysis result, using 5.0 softwares of Primer Premier for respective conserved regions design 4 to primer,
And its specificity is identified by NCBI Blast.Screening determines final the primer, it is ensured that designed primer can
Corresponding target product is amplified, and does not occur any intersect instead in multi-PRC reaction is implemented, between multipair primer
Should, i.e., it is no homologous, complementary, hairpin structure etc. is not formed.Primer sequence determined by final see the table below.
(4) PCR reaction condition optimizations：PCR reagent used in the present invention produces for TAKARA companies.PCR reaction conditions
Optimization is specially：The PCR reaction systems of 25 μ L are selected, system includes 10 × PCR buffer (Mg2+Plus) 2.5 μ L,
3 0.4 μ L, DNA and cDNA hybrid templates of μ L, 5U/ μ L Taq polymerases of 2.5mmol/L dNTP, 1 μ L, primer working concentration are 10
μm ol/L, according to the bright of multiplex PCR band, suitably adjusts the ratio of 3 pairs of primer additions.Annealing temperature is according to primer Tm
Scope carries out 50 DEG C, 51.7 DEG C, 52.9 DEG C, 54.3 DEG C, 55.4 DEG C, 56.6 DEG C, 57.8 DEG C, 59.9 DEG C of annealing temperature gradient examinations
Test.Finally determine optimal multiplex PCR pattern.Finally determine that optimal multi-PRC reaction system is：10 in 25 μ L reaction systems
×buffer(Mg2+) 2.5 μ L, dNTP 3 μ L (2.5mmol), 4 couples of primer upstream and downstream mix primers 2.4 μ L, 0.4 μ of Taq polymerase
L (5U/ μ L), 1 μ L of DNA/cDNA hybrid templates, add sterilizing distilled water to 25 μ L.Optimal multi-PRC reaction condition is：94℃、
5min；94 DEG C, 1min, 55 DEG C, 30s, 72 DEG C, 30s, 30 circulation；72℃、10min.
(5) multiplexed PCR amplification specificity verification：With 4 pairs of primers of CSFV, PPV, PRRSV and JEV, respectively to distilled water,
The template of Escherichia coli, the DNA of PRV, CSFV, PPV, PRRSV and JEV and cDNA carries out PCR reactions under the same conditions respectively.
After multiplexed PCR amplification, product is detected through 2% agarose gel electrophoresis, it is seen that CSFV, PPV, PRRSV and JEV are amplified respectively
91bp, 152bp, 198bp, 238bp, are consistent with expected purpose band, and 4 couples of primer pair distilled waters, Escherichia coli, PRV do not go out
As a result what incumbent amplified band, the multiplex PCR specific amplification products of CSFV, PPV, PRRSV and JEV are shown after sequencing analysis
Show consistent with expected results.As it can be seen that to confirm that this 4 pairs of primers have very strong special for the specificity verification experiment of multiplex PCR
Property, CSF, PPI, PRRS, JE can be detected at the same time.
(6) multiplex PCR sensitiveness is verified：Through ultramicron nucleic acid-protein analyzer measure CSFV, PPV, PRRSV and JEV
Template original concentration is respectively 310ng/ μ L, 112ng/ μ L, 232ng/ μ L, 189ng/ μ L, and DNA profiling is pressed 10-1-10-7 times
Serial dilution carries out PCR amplification into 7 concentration gradients by above PCR reaction systems and condition.The results show that this mPCR pairs
The minimum nucleic acid concentration detection limit of CSFV, PPV, PRRSV and JEV is respectively：8.31pg, 3.22pg, 1.12pg, 19.6pg.Can
See, the sensitiveness that multiplex PCR sensitivity tests demonstrates this 4 pairs of primer pair template concentrations is higher.
(7) multiplex PCR stability is verified：CSFV, PPV, PRRSV and JEV are repeated to detect with the multiple PCR method of foundation
9 times, amplification is consistent, and the multiple PCR method for showing to establish has preferable stability.
(8) detection of clinical sample：The sample being collected is passed through into milled processed, is carried out according to above-mentioned (1) and (2) step
The extraction of DNA, RNA, then carry out PCR amplification, 11 parts of pathological material of diseases of the results show according to the multi-PRC reaction system having had built up
In have 8 parts of PRRSV infections, positive rate is up to 72.7% (8/11), and 2 parts of infection CSFV, positive rate is 18.1% (2/11), with
Individual event PCR coincidence rates are up to 100%.The detection of clinical sample demonstrates the feasibility of this method.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.Any reference numeral in claim should not be considered as to the involved claim of limitation.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each embodiment is only wrapped
Containing an independent technical solution, this narrating mode of specification is only that those skilled in the art should for clarity
Using specification as an entirety, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
It is appreciated that other embodiment.
Claims (10)
1. a kind of PCR system and detection method for detecting pig virus Reproduction Disorder, it is characterised in that including following step
Suddenly：
(1) extraction of PPV viral DNAs；
(2) extraction and reverse transcription of CSFV, PRRSV, JEV viral RNA；
(3) specific primer design and screening；
(4) PCR reaction condition optimizations are carried out to the specific primer designed by step (3)；
(5) the optimal multi-PRC reaction system established according to step (4) carries out multiplexed PCR amplification specificity verification；
(6) the optimal multi-PRC reaction system established according to step (4) carries out multiplex PCR sensitiveness verification；
(7) the optimal multi-PRC reaction system established according to step (4) carries out multiplex PCR stability verification；
(8) according to detection of the multiple PCR method of foundation to clinical sample.
2. the PCR system and detection method of detection pig virus Reproduction Disorder according to claim 1, its feature
It is, in step (1), the extracting method of the PPV viral DNAs is：Appropriate PPV virus liquids are taken, are placed in 1.5mL
In Eppendorf pipes, with reference to the E.Z.N.A of OMEGA companiesTMTissue DNA Kit extracts kits specification carries out, and will carry
The genomic DNA taken is placed in -20 DEG C and saves backup.
3. the PCR system and detection method of detection pig virus Reproduction Disorder according to claim 1, its feature
It is, in step (2), the extraction of CSFV, PRRSV, JEV viral RNA and reverse transcription method are：Take CSFV, PRRSV and JEV sick
Venom, with reference to TaKaRa companies MiniBEST ViralRNA/DNA Extraction Kit specifications extraction viral RNA, will carry
The reverse transcription of viral RNA taken is into cDNA；According to the M-MLV H-First-Stand Synthesis Kit reverse transcriptions of Magen companies
Kit specification carries out, and the cDNA of reverse transcription is placed in -20 DEG C and is saved backup.
4. the PCR system and detection method of detection pig virus Reproduction Disorder according to claim 1, its feature
It is, in step (3), design and the screening of the specific primer are specially：Applied biology software is to CSFV genes, PPV
Gene, PRRSV genes and JEV genes carry out homology analysis, screen and determine primer, it is ensured that designed primer is necessary for specificity
Primer, pair of primers can only amplify the sequence fragment of corresponding virus, and not interfered between other primers, i.e., without same
Source, complementation, do not form hairpin structure；The primer sequence of the design is：
The specific primer of CSFV：
F:5′-TGCAACTGAATGACGGGAC-3′
R:5′-GCCAAATACCTCCTACTGACCAC-3′
The specific primer of PPV：
F:5′-AAAAACAATCCACCAGGACAAC-3′
R:5′-CTGGATCTCATTTTTGCTGTGA-3′
The specific primer of PRRSV：
F:5′-CGGGCGGTATGTCCTAAGTA-3′
R:5′-ACCTCAACTTTGCCCCTTTT-3′
The specific primer of JEV：
F:5′-AGCTTGTTGGACGGCAGAG-3′
R:5′-GCCACATGATTGAGCCTTCA-3′。
5. the PCR system and detection method of detection pig virus Reproduction Disorder according to claim 1, its feature
It is, in step (4), the PCR reaction condition optimization specific methods are：The PCR reaction systems of 25 μ L are selected, system includes
10×PCR buffer(Mg2+Plus) 0.4 μ L, DNA of 2.5 μ L, 2.5mmol/L dNTP, 3 μ L, 5U/ μ L Taq polymerases with
1 μ L of cDNA hybrid templates, primer working concentration are 10 μm of ol/L, according to the bright of multiplex PCR band, suitably adjust 3 pairs of primers
The ratio of addition；Annealing temperature according to primer Tm scope carry out 50 DEG C, 51.7 DEG C, 52.9 DEG C, 54.3 DEG C, 55.4 DEG C,
56.6 DEG C, 57.8 DEG C, 59.9 DEG C of annealing temperature gradient experiments, finally determine optimal multiplex PCR condition.
6. the PCR system and detection method of pig virus Reproduction Disorder are detected according to claim 1 or 5, its
It is characterized in that, in step (4), optimal multiplex PCR condition is specially：10 × buffer (Mg in 25 μ L reaction systems2+) 2.5 μ L,
3 μ L (2.5mmol) of dNTP, 4 couples of 2.4 μ L of primer upstream and downstream mix primer, 0.4 μ L of Taq polymerase (5U/ μ L), DNA/cDNA is mixed
1 μ L of shuttering, add sterilizing distilled water to 25 μ L；Optimal multi-PRC reaction condition is：94℃5min；94 DEG C of 1min, 55 DEG C
30s, 72 DEG C of 30s, 30 circulations；72℃10min.
7. the PCR system and detection method of detection pig virus Reproduction Disorder according to claim 1, its feature
It is, in step (5), multiplexed PCR amplification specificity verification is specially：At the same time with 4 pairs of specific primers in step (3), divide
It is other to distilled water, Escherichia coli, PRV and CSFV, PPV, PRRSV, JEV template various combination, according to optimal in step (4)
PCR reaction conditions are expanded, to verify its specificity.
8. the PCR system and detection method of detection pig virus Reproduction Disorder according to claim 1, its feature
It is, in step (6), multiplex PCR sensitiveness verification is specially：First by the mark of CSFV, PPV, PRRSV and JEV virus
Quasi- DNA or cDNA samples measure original template concentration with ultramicron nucleic acid-protein analyzer, then respectively will with 10 times of serial dilutions
The template of CSFV, PPV, PRRSV and JEV 4 is diluted to 7 concentration gradients, according to the optimal PCR reaction conditions in step (4) into
Row amplification, measures the sensitiveness of multiplex PCR.
9. the PCR system and detection method of detection pig virus Reproduction Disorder according to claim 1, its feature
It is, in step (7), multiplex PCR stability verification is specially：With the multiple PCR method pair established in step (4)
CSFV, PPV, PRRSV and JEV repeat detection 9 times, with the reliability of verification result.
10. the PCR system and detection method of detection pig virus Reproduction Disorder according to claim 1, it is special
Sign is, in step (8), clinical sample detection is specially to verify its feasibility：By the doubtful pathological material of disease of collection according to
Step (1) and step (2) extraction DNA and RNA, the multi-PCR detection method established according to step (4) is expanded, with verification
The feasibility of this method.
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