title: Development of a rapid total nucleic acid extraction method for the isolation of hepatitis A virus from fresh produce.
abstract: Recently, there have been increasing reports of foodborne illnesses associated with the consumption of fresh produce. Among these, hepatitis A virus (HAV) remains epidemiologically important and has been continually implicated in several outbreaks. We describe a rapid method (<8h) for the isolation and subsequent detection with real-time quantitative PCR (RT-qPCR) of the HAV HM-175 cytopathic strain seeded onto baby spinach and sliced tomatoes using a total RNA extraction method, utilizing a high concentration (4M) guanidine thiocyanate buffer. Consistent detection of HAV genome from both produce items was achieved at an inoculation level of 3×10³ PFU/25 g of food, with less consistent detection achieved at 3×10² PFU/25g. Initial studies revealed that a final precipitation of recovered RNA with potassium acetate to reduce carryover of polysaccharides and the addition of polyvinylpyrrolidone to remove polyphenolics in spinach were essential. For tomatoes, virus isolation was achieved with the incorporation of either an elution step with a high pH Tris-glycine-beef extract (TGBE) buffer or with an enzymatic digestion with pectinase. We also describe the development of a protocol for the detection of HAV from tomatoes utilizing a Luminex® microbead-based suspension array. The results correlated well with the RT-qPCR assay suggesting the feasibility of the Bioplex® as a detection platform for viruses isolated from foods.
authors: Division of Molecular Biology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD 20708, USA.
authors: Kaoru
authors: K
authors: Hida
authors: Michael
authors: M
authors: Kulka
authors: Efstathia
authors: E
authors: Papafragkou
mesh_terms: D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology / Q000379:methods*; D030041:Hepatitis A virus / Q000302:isolation & purification*; D018551:Solanum lycopersicum / Q000821:virology; D012367:RNA, Viral / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D018724:Spinacia oleracea / Q000821:virology
chemical_list: D012367:RNA, Viral