Comparative evaluation of a competitive polymerase chain reaction (PCR) and a SYBR Green–based real-time PCR to quantify Porcine circovirus-2 DNA in swine tissue samples

Abstract

Porcine circovirus-2 (PCV-2) is considered the major etiological agent of post-weaning multisystemic wasting syndrome (PMWS) in pigs. The clinical manifestations of the disease are correlated with moderate to high amounts of PCV-2 DNA in biological samples of affected pigs. A threshold of 10(7) DNA copies/ml is suggested as the trigger factor for symptoms. A comparative study was conducted to determine which quantitative method would be more suitable to estimate the PCV-2 DNA load. Two polymerase chain reaction (PCR) assays were developed: a competitive PCR (cPCR) and a SYBR Green-based real-time PCR. The assays were compared for their capacity to detect PCV-2 in DNA samples extracted from liver, lung, spleen, mesenteric lymph nodes, and kidney of PMWS-affected (n = 23) or non-PMWS-affected pigs (n = 9). Both assays could successfully quantify PCV-2 DNA in all tissue samples and were able to detect significant differences between the numbers of PCV-2 DNA copies found in tissues of PMWS-affected and non-PMWS-affected pigs (≥ 10(2.5)). The highest mean viral loads were detected by the SYBR Green real-time PCR, up to 10(7.0 ± 1.5) copies/100 ng of total DNA sample, while the cPCR detected up to 10(4.8 ± 1.5). A mean difference of 10(1.8) was found between the amounts of PCV-2 DNA detected, using the SYBR Green real-time PCR and the cPCR, suggesting that the viral load threshold for PMWS should be determined for each particular assay.