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

J Virol Methods. 2013 Jan;187(1):15-9. doi: 10.1016/j.jviromet.2012.06.024. Epub 2012 Jul 4.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzothiazoles
  • Circoviridae Infections* / diagnosis
  • Circoviridae Infections* / veterinary
  • Circoviridae Infections* / virology
  • Circovirus / genetics*
  • Coinfection
  • DNA Primers
  • DNA, Viral / analysis
  • Diamines
  • Limit of Detection
  • Nucleic Acid Denaturation
  • Organic Chemicals
  • Parvoviridae Infections* / diagnosis
  • Parvoviridae Infections* / veterinary
  • Parvoviridae Infections* / virology
  • Parvovirus, Porcine / genetics*
  • Quinolines
  • Real-Time Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Swine
  • Swine Diseases / diagnosis
  • Swine Diseases / virology
  • Viral Load

Substances

  • Benzothiazoles
  • DNA Primers
  • DNA, Viral
  • Diamines
  • Organic Chemicals
  • Quinolines
  • SYBR Green I