Concentration and detection of hepatitis A virus and rotavirus in spring water samples by reverse transcription-PCR

Every year, enteric viruses such as hepatitis A virus (HAV), rotaviruses, and noroviruses are responsible for viral gastro-enteritis and hepatitis reported worldwide. These viruses are mostly transmitted via the faecal-oral route, from direct contact between people, or by ingestion of contaminated food and water. Since only a few viral particles may cause disease, detection of low concentration of these viruses in food matrices is usually complex. The development of methods to concentrate viruses from food matrices is crucial in collecting data for the development of control strategies or for diagnostic purposes. In the present study, samples of bottled spring water were inoculated with known amounts of HAV (strain HM-175), and rotaviruses (strain Wa) viral particles and filtered through positively charged membranes. Elution of viruses attached to the membranes was achieved with a tryptose phosphate broth-glycine buffer. Eluates were further concentrated using Microsep 100. Finally, RNA was extracted using the Qiagen RNeasy kit followed by an evaporation step with a SpeedVac instrument. The detection limit by reverse-transcription (RT-PCR) was at least 10(-1) TCID50%/ml and at least 10(-3) TCID50%/ml for HAV and rotavirus, respectively.