Confirmation of Norwalk-like virus amplicons after RT-PCR by microplate hybridization and direct sequencing

A large number of Norwalk-like viruses (NLVs) have been identified from stool samples by RT-PCR by amplifying part of the polymerase-coding gene. A set of probes were selected based on sequence analysis of the viruses circulating in Finland during the years 1996-97 for confirmation of the findings by hybridization. A microplate hybridization test, which provides a rapid semi-automatic detection for PCR products, was designed and compared with agarose gel electrophoresis. From the material of 210 stool samples, mainly from diarrheal outbreaks during years 1997-1998, three probes, one for NLV genogroup GGI and one for each of the two GGII subgroups (Toronto-like and Lordsdale-like), were sufficient to detect 87.8% (36/41) of GGI and 89.0% (49/55) of GGII samples positive by gel electrophoresis. Amplicon sequencing of the strains not detected by the above probes revealed genetic variability in the sequences. Biotin-streptavidin binding was used both for microplate hybridization assays and for direct sequencing to identify the amplicons. Based on the sequences three more probes for the hybridization panel were added so that all the different NLVs of this study could be recognized.