The detection of bovine leukemia virus proviral DNA by PCR-ELISA.
A sensitive non-radioactive microplate hybridization assay for the detection of proviral DNA of bovine leukemia virus (BLV)-specific polymerase chain reaction (PCR) product is described. The PCR products are labeled by adding digoxigenin-dUTP to the nested PCR reaction and are captured by a microtitre plate coated with oligonucleotide probe, which is complementary to the inner region of the amplification product. Captured products are reacted with an anti-DIG Fab fragment conjugated to peroxidase, and detected using a colorimetric reaction. The PCR-enzyme linked immunosorbent assay (ELISA), detecting as low as 10(-4) ng of proviral DNA in a background of 1 microg of BLV-negative DNA, was up to 100-fold more sensitive than ethidium bromide staining, and showed equal sensitivity to Southern blot hybridization. Using this method it was possible to monitor the presence of proviral DNA in four sheep infected experimentally with BLV, over a 10 months postinfection period, as well as in 29 cattle infected naturally. The test is rapid and highly sensitive and is a useful additional tool for the detection of BLV-infected animals.
Rola
M
M
National Veterinary Research Institute, Al.Partyzantow 57, 24-100, Pulawy, Poland.
Kuzmak
J
J
D000818:Animals; D002417:Cattle; D004279:DNA, Viral / Q000032:analysis*; D016583:Enzootic Bovine Leukosis / Q000175:diagnosis* / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay; D001909:Leukemia Virus, Bovine / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000821:virology
D004279:DNA, Viral