title: Methods for recovery of hepatitis A virus (HAV) and other viruses from processed foods and detection of HAV by nested RT-PCR and TaqMan RT-PCR.
abstract: Enteric viruses are important agents of foodborne disease. Unfortunately, robust, quantitative methods for sampling and analysis of enteric and other viruses in processed or complex foods are not well-established. As a result, epidemiologically determined etiologies or pathogen sources in foodborne outbreaks are rarely confirmed by virological analysis. In this study, an acid-adsorption elution concentration (AEC) method previously used to monitor virus occurrence and investigate enteric virus outbreaks in shellfish was adapted for examination of processed food items, namely tomato sauce and blended strawberries. Hepatitis A virus (HAV), poliovirus, and coliphage MS2 (MS2) were seeded in 10 or 30 g samples of tomato sauce or blended strawberries, recovered by AEC, and quantified by cell culture infectivity assay. In addition, nested reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan RT-PCR assays were used to detect HAV RNA. Viruses were efficiently adsorbed to foods as an initial concentration step, with infectious HAV and MS2 adsorption of 67% and 93%, respectively, to tomato sauce, and 89% and 99%, respectively, to blended strawberries. Forty-three to 65% of HAV and poliovirus were subsequently eluted and recovered from tomato sauce using 0.5 M threonine, pH 7.2. The lower limits of HAV detection were at initial seeding levels of 14 PFU/g of tomato sauce and 33 PFU/g of blended strawberries. Unlike TaqMan RT-PCR, nested RT-PCR was not inhibited by undiluted final RNA extracts of tomato sauce or blended strawberries. The successful adaptation of the AEC method for enteric and other virus recovery, quantitation and detection in processed foods demonstrates its potential for use in the investigation of foodborne outbreaks of viral etiology and for validation of virus disinfection and sanitary processing procedures used by the food industry.
authors.affiliation: Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, United States. davelove@berkeley.edu
authors.forename: David C
authors.initials: DC
authors.lastname: Love
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Casteel
authors.forename: John S
authors.initials: JS
authors.lastname: Meschke
authors.forename: Mark D
authors.initials: MD
authors.lastname: Sobsey
mesh_terms: D000327:Adsorption; D000818:Animals; D003257:Consumer Product Safety; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D031985:Fragaria / Q000821:virology*; D017957:Hepatitis A Virus, Human / Q000302:isolation & purification*; D006801:Humans; D017909:Levivirus / Q000302:isolation & purification; D018551:Solanum lycopersicum / Q000821:virology*; D017955:Poliovirus / Q000302:isolation & purification; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000821:virology
chemical_list: D012367:RNA, Viral