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 calicivirus/Sapporo/82/J (HuCV/Sa/82) or strains closely related to HuCV/Sa/82 in stool specimens. The cDNA derived from the RNA-dependent RNA 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 labeled with digoxigenin and the products of hybridization reaction were detected with an anti-digoxigenin antibody-alkaline phosphatase conjugate. This assay was specific for HuCV/Sa/82 and for HuCV antigenically related 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, similar to that of the previously developed ELISA for HuCV. In 1273 stool specimens obtained from children with acute gastroenteritis in Sapporo, Japan, 110 (8.6%) contained small round structured viruses by EM and 23 (1.8%) were positive for HuCV antigenically related to HuCV/Sa/82 by either the hybridization assay or ELISA. A higher positive rate was obtained with the dot blot assay (21%) than by ELISA (10%), suggesting that the dot blot assay either detects HuCV more broadly than the ELISA or detects HuCV covered with fecal antibodies which interrupt antigen-antibody reactions in the ELISA. Negative results for detection of Norwalk virus (NV) cDNA and feline calicivirus (FCV) RNA by both this assay and the ELISA indicated that the HuCV/Sa/82 strain is distinct antigenically and genetically from NV and FCV.
Kogawa
K
K
Department of Pediatrics, Sapporo Medical University School of Medicine, Japan.
Nakata
S
S
Ukae
S
S
Adachi
N
N
Numata
K
K
Matson
D O
DO
Estes
M K
MK
Chiba
S
S
D000208:Acute Disease; D002139:Caliciviridae / Q000235:genetics / Q000302:isolation & purification*; D017250:Caliciviridae Infections / Q000453:epidemiology / Q000821:virology*; D015342:DNA Probes; D018076:DNA, Complementary; D004279:DNA, Viral; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000453:epidemiology / Q000821:virology*; D006801:Humans; D007564:Japan / Q000453:epidemiology; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012680:Sensitivity and Specificity
D015342:DNA Probes; D018076:DNA, Complementary; D004279:DNA, Viral