Development and validation of a real-time PCR for the detection and quantification of porcine circovirus type 2

Porcine circovirus type 2 (PCV2) is a spherical and non-enveloped virus belonging to the genus Circovirus of the Circoviridae family with a single stranded circular DNA genome. This virus, already detected worldwide, has been associated to several diseases and was implicated as the etiological agent of a disease named postweaning multisystemic wasting syndrome. Several methods have been described for the detection of PCV2, being real-time PCR the most simple and reliable. As far as we know, all the real-time PCR systems described until now are based on ORF2 gene, that exhibit the highest variability. This paper reports the development and validation of a real-time PCR targeted to ORF1 and based on a TaqMan probe for the detection of porcine circovirus type 2 DNA in swine samples. Due to the lack of PCV1 samples, the ability of the test to discriminate between PCV1 and PCV2 positive samples was evaluated in silico. Estimations of 100% specificity and 100% sensitivity were obtained based on the qPCR results with panel of 81 swine samples (known PCV2-positive (n = 50); known PCV2-negative (n = 17); samples positive to other common swine viral pathogens (n = 13) and one sample from a BFDV-positive parrot (n = 1)). Intra- and inter-assay coefficients of variation obtained with three positive samples of different viral charges in five replicates or in five independent assays were below the acceptance threshold. The limit of detection determined with a recombinant plasmid containing the amplicon, led to conclude that this assay can detect at least three plasmid copies.