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Detection Methods for Human Enteric Viruses in Representative Foods

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Abstract:

Although viral foodborne disease is a significant problem, foods are rarely tested for viral contamination, and when done, testing is limited to shellfish commodities. In this work, we report a method to extract and detect human enteric viruses from alternative food commodities using an elution-concentration approach followed by detection using reverse transcription-polymerase chain reaction (RT-PCR). Fifty-gram lettuce or hamburger samples were artificially inoculated with poliovirus type 1 (PV1), hepatitis A virus (HAV), or the Norwalk virus and processed by the sequential steps of homogenization, filtration, Freon extraction (hamburger), and polyethylene glycol (PEG) precipitation. To reduce volumes further and remove RT-PCR inhibitors, a secondary PEG precipitation was necessary, resulting in an overall 10- to 20-fold sample size reduction from 50 g to 3 to 5 ml. Virus recoveries in secondary PEG concentrates ranged from 10 to 70% for PV1 and 2 to 4% for HAV as evaluated by mammalian cell culture infectivity assay. Total RNA from PEG concentrates was extracted to a small volume (30 to 40 μl) and subjected to RT-PCR amplification of viral RNA sequences. Detection limit studies indicated that viral RNA was consistently detected by RT-PCR at initial inoculum levels ≥102 PFU/50-g food sample for PV1 and ≥103 PFU/50-g food sample for HAV. In similar studies with the Norwalk virus, detection at inoculum levels ≥ 1.5 × 103 PCR-amplifiable units/50-g sample for both food products was possible. All RT-PCR amplicons were confirmed by subsequent Southern hybridization. The procedure reported represents progress toward the development of methods to detect human enteric viral contamination in foods other than shellfish.

Document Type: Research Article

Affiliations: Department of Food Science, College of Agriculture and Life Science, Box 7624, North Carolina State University, Raleigh, North Carolina 27695-7624, USA

Publication date: 2000-12-01

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