Applied technique for increasing calicivirus detection in shellfish extracts.

Abstract

Optimal detection of enteric RNA viruses in clinical, environmental, and food products using reverse transcription-PCR (RT-PCR) when inhibitory substances in extracted sample materials are present. We adapted a device for detection of RNA viruses in plant tissues and insects to detect a calicivirus strain (San Miguel sea lion virus, serotype 17) in water and oyster tissue extracts. This single, compartmentalized tube-within-a-tube (TWT) device for RT-PCR-nested PCR was compared to a conventional protocol of RT-PCR-nested PCR. In the presence of 100 mg of shellfish tissue extract equivalent, this TWT device decreases the calicivirus assay detection limit 10-fold over that of conventional RT-PCR-nested PCR while maintaining an identical detection limit of viral nucleic acid suspended in water. Both the conventional and TWT methods estimated the total particle-to-infectious particle ratio for this strain of calicivirus at approximately 40 : 1. We believe that the TWT device with appropriate RT-PCR primers will decrease the detection limit for other calicivirus strains and RNA viruses in shellfish tissue extracts. We believe that the TWT approach is applicable to other situations where RT and/or PCR inhibitory materials are present or nucleic acid targets of bacteria or viruses are at low levels in extracts of food products or clinical specimens.