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Applied technique for increasing calicivirus detection in shellfish extracts
W Burkhardt1, G M Blackstone, D Skilling
1US Food and Drug Administration, Gulf Coast Seafood Laboratory, Dauphin Island, AL 36528-0158, USA. Wburkhar@cfsan.fda.gov
Aims:
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.
Methods And Results:
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.
Conclusions:
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.
Significance And Impact Of The Study:
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.