Sensitive detection and differentiation of Sapporo virus, a member of the family Caliciviridae, by standard and booster nested polymerase chain reaction

Norwalk virus and Sapporo virus (SV) were approved as type species of the g enus Norwalk-like viruses and the genus Sapporo-like viruses, respectively, in the family Caliciviridae. Nested polymerase chain reaction (PCR), using newly designed primers in the RNA-dependent RNA polymerase region, was dev eloped to detect and differentiate viruses in the three genetic groups of S V based on the relative size of the PCR products obtained. In addition, a b ooster nested PCR that performs nested PCR in a single tube was introduced to reduce the chance of contamination during the procedure of standard nest ed PCR. The specificity of the newly developed PCR was confirmed by testing 77 stool specimens and 16 tissue culture fluids derived from growth of unr elated viruses. The sensitivity of the nested PCR was compared with the con ventional PCR using Sapp35/Sapp36 primer pair by testing the three cDNA clo nes obtained from viruses in the SV/SV82, the SV/London92, and the SV/Parkv ille virus, respectively. This assay can detect SV in a more sensitive way than the conventional PCR and Southern hybridization. Sensitive and suitabl e methods to detect and differentiate SV are required to obtain accurate ep idemiological data on these viruses and the standard and booster nested PCR should be a very useful tool for this purpose. (C) 2001 Wiley-Liss, Inc.