Multiplex PCR for detection and typing of porcine circoviruses

Abstract

Sets of oligonucleotide primers were designed according to the sequences of the open reading frames (ORFs) ORF1 and ORF2 of the prototype nonpathogenic PK-15 strain of porcine circovirus (PCV) type 1 (PCV-1). By the PCR performed 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 ml. No DNA fragment could be amplified from lysates of continuous porcine cell lines (PT, ST, and PFT cells) known to be negative for PCV. When tested with clinical samples from pigs, the results of the single PCR method showed nearly 93% (13 of 14 samples) correlation with histopathological and immunohistochemical findings. Interestingly, subclinical PCV infections could be detected by single PCR with clinical samples that have been submitted from animals with irrelevant cases of respiratory and/or enteric problems. On the basis of the nucleotide sequences of PCV strains (PCV-2) recently associated with outbreaks of postweaning multisystemic wasting syndrome (PWMS) in Quebec, Canada, pig farms, other primers were designed from the PCV-1 genome, and these primers failed to amplify genomic fragments specific to the ORF1 or ORF2 genes of clinical isolates associated with PWMS but amplified DNA from the PCV-1 strain. Two rapid multiplex PCR (mPCR) methods have been developed to distinguish between both genotypes of PCV. By those two mPCR methods, (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 fragment was amplified from the ORF1 gene of the PCV-1 strain only, or (ii) species-specific primer pairs were used to amplify a DNA fragment of 646 bp specific 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 methods, 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 association 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.

FIG. 1

Electrophoretic profiles of DNA amplicons obtained by single PCR with genomic DNA extracted from CsCl gradient-purified PCV-1. The six primer pairs used to amplify ORF1 of the PCV-1 strain yielded DNA amplicons of the expected size. Lane 1, primers ORF1.PCV1.S1 and ORF1.PCV1.AS5 (865 bp); lane 2, primers ORF1.PCV1.S2 and ORF1.PCV1.AS5 (830 bp); lane 3, ORF1.PCV1.S1 and ORF1.PCV1.AS2 (500 bp); lane 4, primers ORF1.PCV1.S1 and ORF1.PCV1.AS1 (415 bp); lane 5, primers ORF1.PCV1.S2 and ORF1.PCV1.AS2 (465 bp); and lane 6, primers ORF1.PCV1.S2 and ORF1.PCV1.AS1 (375 bp); lane L, 1-kb DNA ladder.

FIG. 2

Sensitivity of the single PCR method in detecting virus-specific DNA extracted from 10-fold dilutions (10⁻¹ to 10⁻⁶; lanes 1 to 6, respectively) of purified viral preparation of PCV-1 (preadjusted to 10⁶ viral particles per ml) (A and C) or from PK-15 cells infected with PCV-1 (total of 10⁶ cells) (B). The primer pairs used were chosen in order to permit amplification of ORF1 fragments of either 375 bp (primers ORF1.PCV1.S2 and ORF1.PCV1.AS1) or 865 bp (primers ORF1.PCV1.S1 and ORF1.PCV1.AS5) in length. Lane l, 1-kb DNA ladder.

FIG. 3

PCR products resulting from enzymatic amplification of PCV genomic DNA extracted from infected porcine lung tissue. Clinical samples with histopathological lesions suggestive of a PCV infection (presence of basophilic inclusion bodies) (Table 4) were treated as described in the Materials and Methods section, and then extracted DNA was tested by the single PCR method with primers ORF1.PCV1.S2 and ORF1.PCV1.AS2, which yielded DNA amplicons of 465 bp in length. Lanes 1 to 22, samples from 22 pigs, respectively; lane L, 1-kb DNA ladder; +, DNA from a purified PCV-1 strain.

FIG. 4

Strain specificity of the oligonucleotide primers used in both mPCR methods. As expected from the genomic sequences of PCV-1 and PCV-2, primer pair ORF1.PCV1.S3 and ORF1.PCV1.AS2 and primer pair ORF2.PCV1.S and ORF2.PCV1.AS which were used in the first mPCR method (A) yielded DNA amplicons of 488 bp specific for the ORF2 genes of both genotypes, whereas a 375-bp fragment was amplified for the ORF1 gene of the PCV-1 strain only. In the second mPCR-2 method (B), primer pair ORF1.PCV1.S2 and ORF1.PCV1.AS6 and primer pair ORF2.PCV1.S1 and ORF2.PCV1.AS1 yielded DNA amplicons of 646 bp specific for the ORF1 genes of both genotypes, whereas a 407-bp fragment was amplified for the ORF2 gene of the PCV-1 strain only. Lane L, 1-kb DNA ladder; lanes 1 and 2, genotypes 1 and 2 of PCV, respectively.

FIG. 5

Detection and typing of PCV in a panel of positive clinical specimens (lung tissue; lanes 1 to 9) by both mPCR methods. As described in the Materials and Methods section, in both mPCR methods, two sets of primers were used simultaneously with DNA extracted from lungs of the affected pigs to amplify fragments of the ORF1 and ORF2 genes of PCV-1 but only the ORF1 or ORF2 fragment of PCV-2. (A and B) For only one of the clinical samples tested, an mPCR profile of a PCV-1 strain was observed. As expected, by the mPCR1 method (A), a fragment of 488 bp specific for the ORF2 genes of strains of both genotypes could be amplified for all positive samples, whereas for only one sample (lane 3), the ORF1 fragment (375 bp) could be amplified. By the mPCR2 method (B), a fragment of 646 bp specific for the ORF1 genes of both genotypes could be amplified for all positive samples, whereas for only the same sample described in panel A, the 407-bp fragment specific for the ORF1 of PCV-1 could be amplified (lane 3). Lane L, 1-kb DNA ladder; lane +, DNA extracted from purified PCV-1.

FIG. 6

Differentiation of PCV-1 and PCV-2 strains by single PCR with universal and type 2-specific primer pairs. Primer pair ORF1.PCV1.S2 and ORF1.PCV1.AS6 (lanes 1 to 4) yielded a DNA amplicon of 646 bp specific for the ORF1 genes of both genotypes, whereas primer pair ORF2.PCV2.S4 and ORF2.PCV2.AS4 (lanes 5 to 8) yielded a DNA amplicon of 493 bp specific for the ORF2 gene of PCV-2 only. Lanes 1 and 5, DNA extracted from PK-15 cells persistently infected with the PCV-1 strain; lanes 2 and 6, clinical specimen positive for PCV-1 by the mPCR; lanes 3 and 7, Quebec PCV-2 strain IAF-614; lanes 4 and 8, Quebec PCV-2 strain IAF-4370; lanes L, 1-kb DNA ladder; lane +, DNA extracted from purified PCV-1.