Use of in situ hybridization for the detection of foot-and-mouth disease virus in cell culture.
Biotinylated complementary DNA (cDNA) and RNA probes were prepared from a specific and highly conserved section of the foot-and-mouth disease virus (FMDV) genome coding for the RNA-dependent RNA polymerase. Hybridization was conducted on FMDV-infected, bovine enterovirus (BEV)-infected, and noninfected swine kidney cell cultures. The detection system utilized the enzyme system streptavidin-alkaline phosphatase, the substrate phosphate, and the chromogen nitroblue tetrazolium. Intense cytoplasmic granular staining was present at 2 and 4 hr postinfection (hpi), with less staining observed at 24 hpi. The staining was specific for FMDV, as indicated by a lack of staining of noninfected cells and BEV-infected cells. With the RNA probe, positive cells were detected up to the highest viral dilution assayed, which was approximately 96 TCID50. The cDNA probe was slightly less sensitive, detecting positive cells at 10-fold lower dilutions. This technique could prove useful in the diagnosis of foot-and-mouth disease in animals or in the detection of FMDV in biologics submitted for importation.
Meyer
R F
RF
Foreign Animal Disease Diagnostic Laboratory, USDA-APHIS, Greenport, NY 11944.
Brown
C C
CC
Molitor
T W
TW
Vakharia
V N
VN
D000818:Animals; D005537:Aphthovirus / Q000235:genetics / Q000302:isolation & purification*; D001710:Biotin; D002999:Clone Cells; D015342:DNA Probes; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization; D015347:RNA Probes
D015342:DNA Probes; D015347:RNA Probes; D001710:Biotin; D012321:DNA-Directed RNA Polymerases