Detection of DNA viruses by radioactive and non radioactive DNA probes: application to African swine fever virus.
A molecular hybridization technique using radioactive and non radioactive DNA probes, has been used to detect ASFV DNA immobilized on nitrocellulose paper. It is based on the use of plasmid pRPEL-2 as a hybridization probe. This plasmid contain the H-ClaI DNA fragment (size 5.6 Kbp) from the Spain-70 strain of ASFV. The sensitivity of detection using radioactive 32P-probes (specific activity about 2 X 10(8) cpm per microgram) was about 20 pg of viral DNA. The 32P-pRPEL-2 DNA probe can detect about 100 infected MS cells and failed to hybridize to DNA from HSV-2, MS cells or salmon sperm. The sensitivity with non radioactive probes was about 4 ng of viral DNA for a sulfonated DNA probe and 400 pg for a biotinylated DNA probe. The efficiency of DNA fixation to the filter, the effect of EDTA and of ultrasonic treatment of the sample were also investigated.
D000358:African Swine Fever Virus / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis*; D000882:Haplorhini; D007497:Iridoviridae / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization
D004279:DNA, Viral