Simultaneous detection of porcine parvovirus and porcine circovirus type 2 by duplex real-time PCR and amplicon melting curve analysis using SYBR Green.

Abstract

The development of a SYBR Green-based duplex real-time PCR is described for simultaneous detection of porcine parvovirus (PPV) and porcine circovirus type 2 (PCV-2) genomes. Viral genomes were identified in the same sample by their distinctive melting temperature (T(m)) which is 77.5°C for PPV VP2 313bp amplicon and 82.3°C for PCV-2 ORF2 171bp amplicon, respectively. The detection limit of the method was 0.01TCID(50)/mL for PPV and PCV-2, about 10 times more sensitive than conventional PCR. In addition, PPV and PCV-2 viral load were measured in 126 field samples, confirming the sensitivity and specificity, and the result showed that 70/126 samples were positive for PPV and 92/126 samples were positive for PCV2 by the duplex real-time PCR. This method may be a useful alternative rapid and reliable method for the detection of PPV/PCV-2 co-infection.