Development and optimization of Multiplex- PCR for simultaneous detection of Porcine Pseudorabies Virus, Porcine Parvovirus, and Porcine Circovirus Type 2

Multiple infections by pathogens are currently the most serious problems in pig herds. Clinically, accurate diagnosis is difficult due to similarity of the symptoms of porcine pseudorabies virus (PRV), porcine parvovirus (PPV), and porcine circovirus type 2 (PCV2). A multiplex polymerase chain reaction (multiplex-PCR) was developed and optimized for the simultaneous detection of the three DNA viral infections in pigs. Four pairs of specific primers were designed for each of the three viruses. Each of the four target fragments produced a specific amplicon 657 bp (PPV, NS1), 490 bp (PCV2, ORF2), 372 bp (PRV, gB), and 298 bp (PRV, gE) in a single PCR. The optimal parameters, individual reaction component concentrations (the concentrations of primers, MgCl2, dNTP, and Taq DNA polymerase), and annealing temperature, of the multiplex PCR were defined based on single PCR conditions. The sensitivity of the multiplex PCR for NS1, ORF2, gB, and gE in a 20 μl mixture using purified recombinant plasmids containing the viral target genes was 10-5 (1.375 × 10-4 ng). The specificity of primer pairs for the classical swine fever virus, as well as porcine reproductive and respiratory syndrome virus was analyzed by multiplex PCR. The PCR products tested negative. The multiplex-PCR method is a convenient diagnostic tool for the routine surveillance of viral co-infections for the simultaneous detection of PCV2, PRV, and PPV.