A Sensitive and Reliable Reverse Transcriptase PCR–Enzyme-Linked Immunosorbent Assay for the Detection of Human Pathogenic Viruses in Bivalve Molluscs

A colorimetric method, reverse transcriptase PCR with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was evaluated for ease of use, reliability, and sensitivity when detecting known human pathogenic virus present in shellfish, using a traditional polyethylene precipitation or immunocapture virus concentration method. The newly developed ELISA method could successfully detect enteroviruses and noroviruses in artificially and naturally contaminated shellfish. Overall, ELISA was shown to be a robust and sensitive method, which had a detection limit of 10 to 100 50% tissue culture infective dose enterovirus per gram of Crassostrea gigas (Pacific oyster) digestive gland and whole Mytilus edulis (common blue mussel). The technique was easily established in a new laboratory and required no specialized equipment. The method had a high sample throughput capable of screening 96 samples per run, making the technique extremely time efficient. RT-PCR-ELISA is a safe, quick, reliable technique, which has the potential for use as a standard virus detection method.