Evaluation of the sensitivity and specificity of primer pairs and the efficiency of RNA extraction procedures to improve noroviral detection from oysters by nested reverse transcription-polymerase chain reaction

J Microbiol. 2010 Oct;48(5):586-93. doi: 10.1007/s12275-010-0047-4. Epub 2010 Nov 3.

Abstract

Noroviruses (NoV) are the key cause of acute epidemic gastroenteritis, and oysters harvested from NoV-polluted sea areas are considered as the significant vectors of viral transmission. To improve NoV detection from oyster using nested reverse transcription-polymerase chain reaction (RT-PCR), we evaluated the sensitivity and specificity of previously published primer pairs and the efficiency of different RNA extraction procedures. Among the primer pairs used for RT-PCR, the sensitivity of GIF1/GIR1-GIF2/GIR1 and GIIF1/GIIR1-GIIF2/GIIR1 was higher than that of other primer pairs used in nested RT-PCR for the detection of NoV genogroup I (NoV GI) and NoV GII from both NoV-positive stool suspension and NoV-seeded oyster concentrates, respectively; the resulting products showed neither unspecific bands in the positive samples nor false-positive bands in the negative controls. The extraction of NoV RNA from oyster samples using a QIAamp® Viral RNA Mini kit with a QIAshredder™ Homogenizer pretreatment afforded more efficient recovery (mean recovery for NoV GI and GII, 6.4%) and the procedure was less time consuming (<30 min) than most other RNA extraction procedures. The results of RNA extraction procedure and primer pairs evaluated by nested RT-PCR assay in this study can be useful for monitoring NoV contamination in oysters, which is an indicator of possible public health risks.

Publication types

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

MeSH terms

  • Animals
  • DNA Primers* / genetics
  • Feces / virology
  • Humans
  • Norovirus / isolation & purification*
  • Ostreidae / virology*
  • Polymerase Chain Reaction / methods
  • RNA / isolation & purification*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Specimen Handling / methods
  • Virology / methods*

Substances

  • DNA Primers
  • RNA