A SYBR Green-based real-time RT-PCR assay for simple and rapid detection and differentiation of highly pathogenic and classical type 2 porcine reproductive and respiratory syndrome virus circulating in China

Arch Virol. 2013 Feb;158(2):407-15. doi: 10.1007/s00705-012-1504-7. Epub 2012 Oct 16.

Abstract

SYBR Green coupled to melting curve analysis has been suggested to detect RNA viruses showing high genomic variability. Here, a SYBR Green-based real-time RT-PCR assay was developed for simultaneous detection and differentiation of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and classical type 2 PRRSV (C-PRRSV). The different strains were identified by their distinctive melting temperatures: 82.98 ± 0.25 °C and 85.95 ± 0.24 °C for HP-PRRSVs or 82.74 ± 0.26 °C for C-PRRSVs. Specificity was tested using nine other viral and bacterial pathogens of swine. The detection limit was 1 TCID(50) for HP- or C-PRRSV. Furthermore, the detection results for samples from an animal trial with HP- or C-PRRSV infections showed that the SYBR Green-based real-time RT-PCR was more sensitive than the conventional RT-PCR. Additionally, an analysis of 319 field samples from North China, Central China and Northeast China showed that HP- and C-PRRSVs co-circulated in pig herds. Thus, the SYBR Green-based real-time RT-PCR, which can be performed within one hour, is a rapid, sensitive and low-cost diagnostic tool for rapid differential detection and routine surveillance of HP- and classical type 2 PRRSVs in China.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzothiazoles
  • China
  • Diamines
  • Molecular Diagnostic Techniques / economics
  • Molecular Diagnostic Techniques / methods*
  • Organic Chemicals / metabolism
  • Porcine Reproductive and Respiratory Syndrome / diagnosis*
  • Porcine Reproductive and Respiratory Syndrome / virology
  • Porcine respiratory and reproductive syndrome virus / genetics
  • Porcine respiratory and reproductive syndrome virus / isolation & purification*
  • Quinolines
  • Real-Time Polymerase Chain Reaction / economics
  • Real-Time Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Staining and Labeling / economics
  • Staining and Labeling / methods
  • Swine
  • Time Factors
  • Transition Temperature
  • Veterinary Medicine / economics
  • Veterinary Medicine / methods*
  • Virology / economics
  • Virology / methods*

Substances

  • Benzothiazoles
  • Diamines
  • Organic Chemicals
  • Quinolines
  • SYBR Green I