PCR detection of group A bovine rotaviruses in feces.
A polymerase chain reaction (PCR) protocol has been developed for identification of bovine group A rotavirus infection in feces. Primers (20mers) complementary to 3' ends of double-stranded RNA genome segment 6 of bovine rotavirus NCDV strain were synthesized and used in PCR. Bovine rotavirus RNA from infected cell culture was employed to optimize the PCR protocol. Rotavirus-negative fecal samples were spiked with known quantities of bovine rotavirus, and the sensitivity of the PCR assay was determined. Fecal samples were extracted with phenol and treated to eliminate unidentified PCR inhibitor(s) in feces, and PCR was performed. PCR products were either visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the assay was 6 x 10(4) viral particles/ml of feces with ethidium bromide-stained agarose gel visualization or 6 x 10(2) viral particles/ml of feces with chemiluminescent hybridization. The PCR assay was applied to 18 fecal specimens from clinical cases. All 16 clinical samples that were positive for rotavirus by enzyme-linked immunosorbent assay (ELISA) or by ELISA and electron microscopy (EM) were positive by PCR. The 2 samples that were rotavirus negative by ELISA or by ELISA and EM were also negative on PCR analysis.
Chinsangaram
J
J
Department of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis 95616.
Akita
G Y
GY
Castro
A E
AE
Osburn
B I
BI
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis*; D002460:Cell Line; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005243:Feces / Q000382:microbiology*; D007668:Kidney; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012330:RNA, Double-Stranded / Q000032:analysis; D012367:RNA, Viral / Q000302:isolation & purification; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis / Q000662:veterinary*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012330:RNA, Double-Stranded; D012367:RNA, Viral