Development of methods to detect "Norwalk-like viruses" (NLVs) and hepatitis A virus in delicatessen foods: application to a food-borne NLV outbreak

Abstract

"Norwalk-like viruses" (NLVs) and hepatitis A virus (HAV) are the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for the detection of NLVs and HAV in foodstuffs. Although reverse transcription (RT)-PCR methods have been useful in detecting NLVs and HAV in bivalve mollusks implicated in outbreaks, to date such methods have not been available for other foods. To address this need, we developed a method to detect NLVs and HAV recovered from food samples. The method involves washing of food samples with a guanidinium-phenol-based reagent, extraction with chloroform, and precipitation in isopropanol. Recovered viral RNA is amplified with HAV- or NLV-specific primers in RT-PCRs, using a viral RNA internal standard control to identify potential sample inhibition. By this method, 10 to 100 PCR units (estimated to be equivalent to 10(2) to 10(3) viral genome copies) of HAV and Norwalk virus seeded onto ham, turkey, and roast beef were detected. The method was applied to food samples implicated in an NLV-associated outbreak at a university cafeteria. Sliced deli ham was positive for a genogroup II NLV as determined by using both polymerase- and capsid-specific primers and probes. Sequence analysis of the PCR-amplified capsid region of the genome indicated that the sequence was identical to the sequence from virus detected in the stools of ill students. The developed method is rapid, simple, and efficient.

FIG. 1

Flow chart for detection of viruses in foods.

FIG. 2

Detection of NV RNA and internal control RNA by RT-PCR and oligoprobing of 10-fold (−1) and 100-fold (−2) dilutions of RNA extracts from ham samples seeded with decreasing concentrations of NV and processed by method 2. Lanes 9 and 10 are negative sample controls. Other lanes include an NV RT-PCR positive control (pos), a negative reagent control (neg), 50 copies of an internal standard control (int std), and a digoxigenin-labeled molecular size marker (MarkerVIII; Boehringer Mannheim) (marker). (A) Ethidium bromide-stained agarose gel; (B) Southern blot of gel in panel A. Numbers at the right are molecular sizes, in base pairs.

FIG. 3

Detection of NLV RNA and internal control RNA by RT-PCR and oligoprobing of 10-fold (−1) and 100-fold (−2) dilutions of RNA extracts from outbreak food samples processed by both methods. (A) Ethidium bromide-stained agarose gel; (B) Southern blot of gel in panel A. Results were obtained with NLV GII capsid primer pair Mon441-Mon443. (C) Southern blot for each food extract dilution from a separate RT-PCR seeded with 50 copies of NV internal standard RNA, using the primer pair NVp35-NVp36 to identify sample inhibition. Other lanes include a negative reagent control (neg) and a digoxigenin-labeled molecular size marker (M). NLV-specific amplicons (268 bp) are seen in lanes 2, 8, and 9 of panel B. Internal standard control-specific amplicons (347 bp) are seen in lanes 2, 4 to 6, and 8 to 13 of panel C. The internal standard control is absent from lanes 1 and 3 of panel C due to sample inhibition.