Title: Development of rapid hemocyte-based extraction methods for detection of hepatitis A virus and murine norovirus in contaminated oysters Authors
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: October 14, 2010
Publication Date: October 16, 2010
Citation: Dancho,B.,Kingsley,D.2010.Development of rapid hemocyte-based extraction methods for detection of hepatitis A virus and murine norovirus in contaminated oysters [abstract].Interstate Seafood Seminar.Ocean City,MD.p.1. Technical Abstract: The human enteric pathogens, hepatitis A virus and human norovirus, have been shown to contaminate molluscan shellfish and cause foodborne disease in consumers. Rapid viral extraction methods are needed to replace current time consuming methods, which use whole oysters or dissected tissues. In our laboratory, we showed that Eastern oyster (Crassostrea virginica) hemocytes are a site of viral persistence. Therefore, hemocytes might be utilized in screening for contaminated shellfish instead of processing oyster tissues. In this study, we compared RNA extraction efficiencies using two methods starting with hemocyte samples and the laboratory standard method starting with whole tissues, the GPTT protocol (Kingsley, D.H., and G.P. Richards. 2001. Appl. Environ. Microbiol. 67:4152-7). Hemocyte and whole oyster tissue samples were collected from laboratory-contaminated oysters as well as from oysters seeded with virus. Laboratory-contamination studies were performed by placing oysters in natural seawater containing various concentrations (1x10**4- 1x10**5 PFU) of hepatitis A virus and murine norovirus, a surrogate for human norovirus, per oyster and allowed to bioaccumulate the viruses for 16 hours at room temperature. Studies were also performed using oyster hemocyte and whole tissue samples seeded with 10-fold serial dilutions of HAV and MNV from 10**4 to 10-2 PFU. RNA extracts were prepared from oyster hemocytes using Dynabeads Oligo(dT)NO25 (Invitrogen) and the RNeasy Mini Kit (Qiagen), and were compared to RNA extracts prepared from whole oysters using the GPTT protocol. Duplex real time RT-PCR was used to detect hepatitis A virus and murine norovirus RNA in the hemocyte and whole oyster RNA extracts. We were able to detect virus-contamination in oysters with both hemocyte extraction methods evaluated in a fraction of the time necessary to perform the GPTT protocol on whole oysters. These data also showed that the sensitivity of the hemocyte-based assays are similar or better than the GPTT assay. These results suggest that the analysis of oyster hemocytes may be a rapid and useful tool to monitor shellfish safety.