Diagnosis of Barmah Forest virus infection by a nested real-time SYBR green RT-PCR assay

PLoS One. 2013 Jul 23;8(7):e65197. doi: 10.1371/journal.pone.0065197. Print 2013.

Abstract

Barmah Forest virus (BFV) is a mosquito borne (+) ssRNA alphavirus found only in Australia. It causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. We developed a real-time PCR assay to detect BFV in an effort to improve diagnosis early in the course of infection. The limit of detection was 16 genome equivalents with a specificity of 100%. Fifty five serum samples from BFV-infected patients were tested by the PCR. 52 of 53 antibody-positive samples were PCR negative. Two culture-positive (neutralizing antibody negative) samples were positive on first round PCR, while one sample (IgM and neutralizing antibody strongly positive, IgG negative) was positive on second round PCR, suggesting that viral RNA is detectable and transiently present in early infection. PCR can provide results faster than culture, is capable of high throughput and by sequencing the PCR product strain variants can be characterized.

MeSH terms

  • Alphavirus / genetics*
  • Alphavirus / isolation & purification*
  • Alphavirus Infections / diagnosis*
  • Alphavirus Infections / virology*
  • Animals
  • Benzothiazoles
  • Cell Line
  • Diamines
  • Humans
  • Limit of Detection
  • Organic Chemicals*
  • Polymerase Chain Reaction / methods*
  • Quinolines
  • Real-Time Polymerase Chain Reaction / methods*
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity

Substances

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

Grants and funding

The authors have no support or funding to report.