Detection methods for human enteric viruses in representative foods.
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 microl) 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 > or =102 PFU/50-g food sample for PV1 and > or =10(3) PFU/50-g food sample for HAV. In similar studies with the Norwalk virus, detection at inoculum levels > or =1.5 X 10(3) 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.
Leggitt
P R
PR
Department of Food Science, College of Agriculture and Life Science, North Carolina State University, Raleigh 27695-7624, USA.
Jaykus
L A
LA
D005506:Food Contamination; D005516:Food Microbiology; D017957:Hepatitis A Virus, Human / Q000302:isolation & purification*; D018545:Lactuca / Q000821:virology*; D008460:Meat / Q000821:virology*; D009663:Norwalk virus / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D017955:Poliovirus / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D012367:RNA, Viral / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015345:Oligonucleotide Probes; D012367:RNA, Viral; D011092:Polyethylene Glycols