Studies On Molecular Detection And Investigation Of Foodborne Viruses In Water And Shellfish

Rotavirus (RV), norovirus (NV), astrovirus (AV) and hepatitis A virus (HAV)are well known to be the most common foodborne viruses throughout the world.Shellfish, being aquatic filter feeders, are notorious as a source of foodborne viralinfectious because they actively concentrate viruses from contaminated water. Thelinkage between viral diseases and consumption of oysters has been reportedinternationally for many years. As a result, consumption of virus-contaminatedoysters represents a significant health threat to shellfish consumers, as well as aneconomic threat to the seafood industry. However, There are no detection methodsabout viruses in sanitary quality standards. Therefore, it is very significant todevelop new methods to detect foodborne viruses in shellfish and water in order toguarantee the people's health and economic developing.The major aim of the study was to develop fast and sensitive methods fordetection and concentration of four foodborne viruses. Then, we have detectedfoodborne viruses from the naturally contaminated shellfish water with these newdeveloped methods. The major results as follows.1. The results of this study demonstrated that monoplex and multiplex-RT-PCRwere sensitive and specific in detecting foodborne viruses during routine monitoringand risk assessment, as well as for large-scale screening during disease outbreaks andin marine products. The study results demonstrated that the primers and the optimizedreaction conditions required for this test were highly specific. In the sensitivity assays, the detection limits were 5pg for NV and AV, 10pg for RV and HAV in monoplex RT-PCR.The steady detection limits were 100pg for HAV, 50pg for RV, NV and AV inmultiplex RT-PCR.2. We developed an NASBA method to detect NV and RV. The results of thisstudy demonstrated that NASBA was a rapid and sensitive alternative to RT-PCR fordetecting foodbome viruses. The NASBA products were analysed by denaturingagarose gel electrophoresis and Northern hybridization. The high specificity ofNASBA was confirmed because no nonspecific band appeared even the highconcentration of external RNA mixed into template. The detection limit of NASBAwas 5pg. The sensitivity of NASBA is the same with that of RT-PCR even higher. Itsadvantage over the traditional RT-PCR technique is its sensitivity, rapidity, simplicityand can be applied to the departments where is short of special equipments.3. We compared the efficacy of the method of A1Cl₃ sedimentation andpositive-charged membrane filtration for concentrating viruses. The recovery rate ofthe sedimentation method of AlCl₃ and the positive-charged membrane was 96.04ï¼…and 91.98ï¼…respectively when tap water served as samples. The recovery rate of thesedimentation method of AlCl₃ and the positive-charged membrane was 93.85ï¼…and69.9ï¼…respectively when sewage served as samples. Compared the two concentrationmethods, there was no obvious difference in tap water but significant difference insewage. The results showed that the positive-charged membrane was adapt to thewater samples with little impurity. According to the experiment results, the twomethods could be used to effectively concentrate viruses in tap water. However, thesedimentation method of AlCl₃ was recommended to the primary method in otherwater samples.4. The Artificially contaminated assay effectively confirmed the validity of thedeveloped methods. In addition, we confirmed that the digestive tissues of shellishwere the major tissues for concentrating viruses. These findings effectively decreasedthe false-negative results and improved the efficiency of detection.5. It was the first survey of viruses contaminated status of Zhujiang River inGuangzhou city and shellfish in Guangdong province. A total of 111 water samples were collected from Zhujiang River in 10 sampling site of Guangzhou city, of these42 (37.8ï¼…) were viruses positive. Out of a total of 42 shellfish that collected inGuangdong province, 2 were positive for RV and 1 was positive for HAV. A specialphenomenon in the assay was that all the positive samples were detected from oysters.The results indicated that the oysters concentrated more viruses than other shellfishspecies.