DOT-BLOT HYBRIDIZATION WITH A CDNA PROBE DERIVED FROM THE HUMAN CALICIVIRUS SAPPORO-1982 STRAIN

A dot blot hybridization;assay was developed for detection of human ca licivirus/Sapporo/82/J (HuCV/Sa/132) or strains closely related to HuC V/Sa/82 in stool specimens. The cDNA derived from the RNA-dependent RN A polymerase (RDRP) region of HuCV/Sa/82 was used as a positive probe and the pBR322 DNA as a negative control probe. Both probes were label ed with digoxigenin and the products of hybridization reaction were de tected with an anti-digoxigenin antibody-alkaline phosphatase conjugat e. This assay was specific for HuCV/Sa/82 and for HuCV antigenically r elated to HuCV/Sa/82. The lower limit of sensitivity of this assay was estimated to be about 10(5) physical particles or 10 pg of cDNA, simi lar to that of the previously developed ELISA for HuCV. In 1 273 stool specimens obtained from children with acute gastroenteritis in Sappor o, Japan, 110 (8.6%) contained small round structured viruses by EM an d 23 (1.8%) were positive for HuCV antigenically related to HuCV/Sa/82 by either the hybridization assay or :ELISA. A higher positive rate w as obtained with the dot blot assay (21%) than by ELISA (10%), suggest ing that the dot blot assay either detects HuCV more broadly than the ELISA or detects HuCV covered with fecal antibodies which interrupt an tigen-antibody reactions in the ELISA. Negative results for detection of Norwalk virus (NV) cDNA and feline calicivirus (FCV) RNA by both th is assay and the ELISA indicated that the HuCV/Sa/82 strain is distinc t antigenically and genetically from NV and FCV.