The Establishment And Application Of Multiplex PCR For Rapid Detection Of PCV2, PPV, PRV, PRRSV And CSFV

Two multiplex PCR assays were developed and subsequently evaluated for their effectiveness as means to simultaneously detect multiple viral infections of swine. Specific primers for DNA and RNA viruses, namely, porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) and classical swine fever virus (CSFV), were synthesized and used by DNAStar and Oligo 6.0 software. By the optimization of PCR reaction factors, such as the concentration of primers, the annealing temperature, the concentration of Mg2+ et al. two multiplex PCR assays, namely, PCV2, PPV, PRV, PRRSV multiplex PCR and CSFV, PRRSV multiplex RT-PCR were established, respectively. The relative efficiency and sensitivity of the two multiplex PCRs developed in this study seemed to meet their potential application for routine molecular diagnostic purposes.179 clinical samples were collected from Xinjiang, Shandong, Jilin and Heilongjiang areas and detected for the five viruses by two multiplex PCRs, and the results showed that 158 cases were PRRSV positive, with the positive rate 88.3 %; 149 cases were PCV2 positive, with the positive rate 83.2 %; PCV2/PRRSV co-infection rate was 71.5 % (128 of 179). PPV, PRV and CSFV were't detected in clinical samples.The open reading frame 2 (ORF2) of PCV2 isolated and the open reading frame 7 (ORF7) of PRRSV isolated were amplified by PCR and sequenced. The sequences were compared with sequences of PCV2 published in GenBank. The results showed that the PCV2 isolated strain was exihibited 98 %( PCV2 1767bp), 96 % (PCV2 1768 bp) and 85% (PCV1) nucleotide sequence identities. There was 90 % nucleotide identity with other American genotype PRRSV in GenBank by compared sequences of ORF7 of PRRSV isolates, and only had 70.2 % nucleotide sequence identities in compare to M96262(LV).In the study reported here, two multiplex PCR methods can be used to effectively detect and differentiate PCV2, PPV, PRV, PRRSV and CSFV single or co-infection in samples from diseased porcine, thus can be used on the clinical detection and the epidemiologic monitoring.