Establishment And Application Of A Six-plex PCR Method For Simultaneous Detection Of Viral Diseases Of Porcine Reproductive Disorder

Porcine reproductive disorder are becoming more and more common in recentyears in pig industry in China, causing great economic losses to our country. Porcinereproductive disorder is caused by one or more pathogens, including pseudorabiesvirus, porcine parvovirus, porcine cricovirus, reproductive and respiratory syndromevirus, swine fever virus and Japanese encephalitis virus. Infection by multiplepathogens in pigs is becoming more and more poplar nowadays, leading to highmorbidity and mortality in pigs and making more difficult in the diagnosis andtreatment of diseases. To provide reference for rapid identification of pathogens inmixed infection, Six-plex PCR method is established and applied for the rapiddetection of these pathogens. The main research contents are as follows:1.Establishment of the multiple PCR method of PRV, PPV, PCV2, PRRSV,CSFV and JEVAccording to the PRV gE （NC₀06151）, PPV NS1（NC₀01718）, PCV2ORF2（NC₀05148）, PRRSV ORF7（NC₀01961）, CSFV E2（X87939）and JEV E （M55506）gene sequences published in Genbank, six pairs of primers were designed andsynthesized to amplify part fragments of the specific genes of PRV gE, PPV NS1,PCV2ORF2, PRRSV ORF7, CSFV E2and JEV E respectively.The results of the diagnosis method of the single PCR showed that:（1） The optimalreaction systems as follows: the primers of PPV, PCV2, PRRSV and JEV were2μL（10μ mol/L）, while the PRV and CSFV2.5μL（15μ mol/L）; The template of PPV,PCV2，PRRSV and JEV were3μL, while the PRV and CSFV4μL; The optimal totalreaction system was50μL.（2） The optimum reaction conditions of single PCR: theoptimal annealing temperature of PRV, PPV, PCV2, PRRSV, CSFV and JEV were 57℃,52℃,56℃,57℃,56℃and57℃respectively.（3） The sensitivity test results asfollows: the minimum amount of nucleic acid detection of PRV, PPV, PCV2, PRRSV,CSFV and JEV were2.5pg,7.3pg,4.8pg,3.9pg,3.8pg and0.4pg respectively. Inaddition, the specificity test showed that both the test results of swine influenza virus（SIV） and Escherichia coli （E.coli） were negative.The results of the established Six-plex PCR method showed:（1） The optimalreaction system was the primers of PPV, PCV2, PRRSV and JEV were1μL （10μmol/L）, while the PRV and CSFV1.5μL （15μ mol/L）; The template amount of PPV,PCV2, PRRSV and JEV were1μL, while the PRV and CSFV2μL; the optimalreaction system was50μL.（2） The optimum reaction conditions of multiplex PCRwas：one cycle at50℃40min;then another cycle at95℃for5min; followed by35cycles at94℃for30s,56.4℃for30s, and72℃for30s; with a final extension stepat72℃for10min.（3） the sensitivity and specificity test results as follows：theminimum amount of nucleic acid detection of PRV, PPV, PCV2, PRRSV, CSFV andJEV were6.6pg,96pg,12.9pg,10.5pg,51pg and46pg respectively, and both the testresults of swine influenza virus （SIV） and Escherichia coli （E.coli） were negative.2. Application of Six-plex PCR diagnostic method in clinical samplesThe results of107clinical samples tested by Six-plex PCR showed that the positiverate of PCV2infection was the highest, as high as83.1%（89/107）; The positive rateof PCV2and PRRSV co-infection was51.4%（55/107）; The positive rate of PPV,PCV2, PRRSV and CSFV mixed infection was4.7%（5/107）. Different degrees ofmixed infection of PCV2and JEV, PRV and JEV, PPV, PCV2and PRRSV were alsodetected. The results above indicated that the six viral diseases commonly exist in pigfarms in Guizhou province, and the mixed infection was very serious. The Six-plexPCR method established in this study not only could detect the infection of PRV, PPV,PCV, PRRSV, CSFV and JEV simultaneously, but also could detect DNA and RNAvirus in one reaction system synchronouslyt, it provided a more convenient andreliable method for routine diagnosis of multiple viral infections in swine clinicalspecimens. 3. Isolation and identification of Porcine circovirus2Guizhou strain andsequence analysis of the complete genomeIn order to obtain three PCV2Guizhou strains and their complete genomes, threesamples detected PCV2positive by Six-plex PCR method were treated, and theninoculated PK-15cells by synchronous inoculation method. Subsequently, thecomplete genes of these three isolated PCV2strains were specific amplified, clonedand sequenced. The isolated strains were respectively named GZ-CS1, GZ-QZ1andGZ-RH1strain and the complete genome length of these isolates was1768bp,1767bpand1767bp respectively. The sequence of isolated strains had been submitted toGenBank, and the accession numbers were JQ809462, JQ809463and JQ809464respectively.The sequence analysis results showed that: The complete genome nucleotidesimilarities between the three PCV2Guizhou strains above and LG strain were95.3%～96.2%.There was one base missing between GZ-QZ1/GZ-RH1and LG strain,causing LG strain amino acid of C terminal ranged from LKP to LNPR;There was nomissing between GZ-CS1and LG strain, but there were certain variations mainlycollected from1160to1350genomes, indicating that the PCV2strains prevailing inGuizhou province exist a certain degree variations, but these variations impact onvirulence and vaccine immune of PCV2still need further study.