Norovirus Detection in Shellfish Using a Rapid, Sensitive Virus Recovery and Real-Time RT-PCR Detection Protocol

Abstract

The development and in-house validation of a method for the detection of noroviruses in shellfish are described. The method comprises a simple virus recovery step using proteinase K digestion and extraction of viral ribonucleic acid (RNA) followed by norovirus detection in separate two-step genogroup I (GI) and II (GII) real-time reverse transcription polymerase chain reaction assays. The assay can be completed within 8 h. An internal armored RNA control, which detects the presence of reverse transcription PCR inhibitors, is an integral part of the assay. The method was found to be reliable, fast, robust, reproducible, and sensitive. Sixty New Zealand and imported shellfish samples were tested using the new method, of which 29 (48.3%) samples were associated with recent New Zealand gastroenteritis outbreaks. The nonoutbreak-related samples were submitted for environmental surveillance related to possible sewage contamination events. Norovirus was detected in 30 of 60 (50%) samples, of which 20 were implicated in gastroenteritis outbreaks, and ten were noncommercial shellfish samples from environmental sites. Of the 30 positive samples, 18 (30%) were positive for both GI and GII norovirus, and a further 12 samples (20%) were positive for GII norovirus only. No samples were positive for GI norovirus only. The method is now validated and accredited under ISO 17025. It can be used to monitor norovirus contamination of shellfish in different situations, including outbreak investigations, relaying, environmental monitoring, and following environmental contamination events.