Development of an extraction and concentration procedure and comparison of RT-PCR primer systems for the detection of hepatitis A virus and norovirus GII in green onions

Abstract

Vegetables can be considered as a vector of transmission for human hepatic and enteric viruses such as hepatitis A virus (HAV) and noroviruses when contaminated by spoiled irrigation water or when prepared by infected food handlers. Recently, outbreaks of HAV have been reported in the USA involving fresh green onions. A viral elution-concentration method was developed for the detection of HAV and norovirus contaminated green onions by RT-PCR. Repeated pipetting/washings of the surface with a pH 9.5 glycine-buffered solution allowed the elution of viruses from the vegetables. Concentration of the viral load was performed by a polyethylene glycol (PEG) precipitation procedure. Viral RNAs were extracted and purified using a combination of Trizol–chloroform and poly(dT) magnetic beads methods. Different sets of primers, including two newly designed primers sets for HAV RT-PCR, were tested in order to achieve the best analytical sensitivity. Using the new primer design, it was possible to detect 10 0 TCID 50%/25 g of HAV in fresh green onions, while 1 RT-PCRU/25 g was detected for noroviruses GII using previously described primers. This method, based on molecular tools, would be useful for diagnostic laboratories in order to perform viral analyses of such commodities as fresh vegetables in cases of foodborne infections.