The development of a sensitive and reliable molecular method for the detection of human pathogenic viruses in bivalve molluscs
The overall aim of this project was to develop a sensitive, specific and reliable molecular assay for the detection of human pathogenic viruses in shellfish. In initial studies, 'viral surrogates' were used to evaluate two different assay formats, the reverse transcriptase-polymerase chain reaction-enzyme-linked immunosorbent assay (RT-PCR-ELISA) and the enzyme-labelled deoxynucleic acid-enzyme-linked coagulation assay (EDNA-ELCA). Each format was tested for ease of use, reliability and sensitivity, when compared with ethidium bromide gel detection. The RT-PCR-ELISA proved to be a successful alternative to ethidium bromide gel electrophoresis and studies involving virus detection in contaminated environmental samples were performed. The newly developed ELISA method was able to successfully detect enterovirus (EV) and Norwalk-like viruses (NLVs) in artificially, and naturally contaminated shellfish. In shellfish studies the ELISA had a detection sensitivity of 10-100, and 100-1000 TCID50 PV respectively, when a traditional elution/precipitation, and an immunocapture procedure were employed. The assay could also successfully detect virus in trout kidney samples, artificially contaminated with infectious pancreatic necrosis virus (IPNV), with detection sensitivities of 105, and 107 pfu reported when the elution/precipitation, and immunocapture procedures were used. In naturally contaminated shellfish samples, positive NLV and EV detection were achieved using the ELISA method. The ability of the ELISA to positively detect virus in environmental samples was compared to the TaqMan quantitative PCR system, an alternative detection method. Both methods were used to screen various contaminated environmental samples, including faeces and shellfish. The ELISA performed well, and unlike the TaqMan system no optimisation for each sample matrix tested was required. Overall the ELISA was shown to be a very robust and sensitive method. The technique was easily established in a new laboratory and no specialised equipment was required to perform the assay. The method has a high sample throughput, capable of screening 96 samples per run. Each sample takes only approximately 50 s to screen, making the technique extremely time efficient. The ELISA is a safe, quick, reliable technique, which has great potential for use as a standard virus detection method in a standard equipped laboratory.