Detection and differentiation of Norwalk virus by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay

Abstract

We have developed a reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay (RT-PCR-ELISA), using genetic cluster-specific probes in a microtiter plate format, for the detection and differentiation of Norwalk virus (NV) in stool samples. The specificity of the RT-PCR-ELISA was confirmed by testing 76 stool specimens and 15 tissue culture fluids derived from growths of unrelated viruses. The sensitivity of the RT-PCR-ELISA was compared with conventional PCR and Southern hybridization by testing the four cDNA clones derived from the RNA-dependent RNA polymerase region of the NV68 (NV/GI) virus and viruses in the NV/GII/P1B, the NV/GII/P2A, and the NV/GII/P2B cluster. This assay was as sensitive as the conventional RT-PCR with Southern hybridization regardless of primer pairs and probes used in the experiments. However, the actual sensitivity of this method was higher when clinical stool samples were examined because this assay examines all the samples irrespective of the RT-PCR results. The RT-PCR-ELISA format is simple, time saving, and suitable for testing many samples. It should be reliable for large-scale epidemiological studies of NV.