Rapid detection of rabies and rabies-related viruses by RT-PCR and enzyme-linked immunosorbent assay.
A rapid detection method for the six established genotypes of rabies and rabies-related viral RNA using RT-PCR-ELISA is described. The detection of digoxigenin-labelled amplified products is performed by solution hybridization to two specific, biotin-labelled, capture probes, which are complementary to the inner region of the amplification products. The capture probe and amplified product hybrid are then immobilised on a streptavidin-coated microtitre plate, bound products are detected by an anti-DIG Fab fragment conjugated to peroxidase, and colorimetric reaction automatically measured. This method was up to 100-fold more sensitive than Southern blot hybridization, detecting 0.00002 TCID50/ml of a genotype 1, classical rabies virus strain. The complete detection methodology from RT-PCR to PCR-ELISA detection could be completed within 10 h. Using this procedure, we were 100% successful in detecting 60 isolates from a representative selection of the six established genotypes from all over the world. This test is a useful additional tool for the detection of the rabies and rabies-related viruses, which is easy to perform, rapid and highly sensitive.
Whitby
J E
JE
Rabies Research and Diagnostic Group, Department of Virology, Veterinary Laboratories Agency (Weybridge), Addlestone, Surrey, UK.
Heaton
P R
PR
Whitby
H E
HE
O'Sullivan
E
E
Johnstone
P
P
D000818:Animals; D015139:Blotting, Southern; D004797:Enzyme-Linked Immunosorbent Assay; D005838:Genotype; D006801:Humans; D006824:Hybridization, Genetic; D018114:Lyssavirus / Q000145:classification / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011818:Rabies / Q000175:diagnosis* / Q000662:veterinary; D011820:Rabies virus / Q000145:classification / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D015203:Reproducibility of Results; D018353:Rhabdoviridae Infections / Q000175:diagnosis / Q000662:veterinary; D012680:Sensitivity and Specificity; D013045:Species Specificity