Multiplex PCR for detection and typing of porcine circoviruses

Sets of oligonucleotide primers were designed according to the sequences of the open reading frames (ORFs) ORF1 and ORF2 of the prototype nonpathogeni c PK-15 strain of porcine circovirus (PCV) type 1 (PCV-I), By the PCR perfo rmed with the various primer sets, genomic DNA or RNA from other bacterial or viral pathogens of the respiratory tracts of pigs could not be amplified . A positive amplification reaction could be visualized with DNA extracted from a viral suspension containing as few as 10 viral particles per mi. No DNA fragment could be amplified from lysates of continuous porcine cell lin es (PT, ST, and PFT cells) known to be negative for PCV. When tested with c linical samples from pigs, the results of the single PCR method showed near ly 93% (13 of 14 samples) correlation with histopathological and immunohist ochemical findings. Interestingly, subclinical PCV infections could be dete cted by single PCR with clinical samples that have been submitted from anim als with irrelevant cases of respiratory and/or enteric problems. On the ba sis of the nucleotide sequences of PCV strains (PCV-2) recently associated with outbreaks of postweaning multisystemic wasting syndrome (PWMS) in Queb ec, Canada, pig farms, other primers were designed from the PCV-I genome, a nd these primers failed to amplify genomic fragments specific to the ORF1 o r ORF2 genes of clinical isolates associated with PWMS but amplified DNA fr om the PCV-1 strain. Two rapid multiplex PCR (mPCR) methods have been devel oped to distinguish between both genotypes of PCV. By those two mPCR method s, (i) species-specific primer pairs were used to amplify a DNA fragment of 488 bp specific for the ORF2 genes of both genotypes, whereas a 375-bp fra gment was amplified from the ORF1 gene of the PCV-1 strain only, or (ii) sp ecies-specific primer pairs were used to amplify a DNA fragment of 646 bp s pecific for the ORF1 genes of both genotypes, whereas a 425-bp fragment was amplified from the ORF2 gene of the PCV-1 strain only. By both mPCR method s, a PCV-2 infection was demonstrated in tissues of 94.2% (33 of 35) of the sick pigs tested, in agreement with previous findings showing the close as sociation of this new genotype of PCV with outbreaks of PMWS in Europe and North America. On the other hand, a PCV-1 infection was confirmed in only 5 .7% (2 of 35) of the pigs, and confirmation of a mixed infection with PCV-2 was obtained by a single PCR with PCV-2-specific primers.