Centers for Disease Control (CDC) reports that at least 50% of all foodborne outbreaks of gastroenteritis are due to noroviruses (NoV). Since NoV is mainly transmitted through the fecal-oral route and the infectious dose may be as low as 10 viral particles, the risk of infection after consumption of raw or improperly cooked seafood or after exposure to contaminated water is considered high. Although highly sensitive methods to detect NoV using RT-PCR are already available, isolation of either NoV RNA or virions from shellfish remains a cumbersome process.
We developed a new hybridization method to extract NoV RNA from contaminated shellfish that is much faster compared to existing methods. Using the new method, NoV detection includes three basic steps: an initial extraction of total RNA using TRIZol, followed by isolation of NoV RNA using biotinylated DNA probe hybridization and then NoV detection by TaqMan RT-PCR. With oyster (Crassostrea virginica) homogenate spiked with 100 PCR detection units (PDU) of NoV, the virus can be detected with CT values at about 30. Compared to published methods that require an initial virus purification step, the new method is much faster, requiring approximately 3 hr compared to at least 8 hr using conventional methods. Coupled with TaqMan RT-PCR, the new method can be used to detect NoV in contaminated oysters and clams (Corbicula fluminea) within 8 hr. The detection limit was 100 PDU of NoV in spiked oyster tissue samples. The method has been successfully used to detect NoV in oysters artificially contaminated in the laboratory and in rare cases, oysters collected from the field.
Ye, Xunyan; Ellender, R.D.; and Wang, Shiao Y., "Development of a Probe Hybridization Method to Facilitate Detection of Noroviruses in Oysters" (2010). Presentations. 1.