A non-radioactive method for identifying enzyme-amplified products of the reticuloendotheliosis proviral env and LTR genes using psoralen-biotin labelled probes.
A novel polymerase chain reaction (PCR) system based on the env gene of reticuloendotheliosis virus (REV) strain REV-A for the detection of proviral DNA is described. The designed PCR product of 807 bp was identified using an internal probe of 278 bp produced by nested PCR from REV-infected DNA CEF. The env-gene PCR was then compared with the previously described PCR for proviral REV-long terminal repeat and the PCR product served also as the probe. The probes were labelled with the psoralen-biotin system by photoactivation and the southern blot hybridization signal was detected colorimetricaly. The advantages of using a non-radioactive means of probe labelling were demonstrated clearly in that study, as well as the effective labeling of probes with psoralen-biotin and the simple colorimetric method of detection. The env-gene PCR detected all eleven REV strains used in the study. These included three REV prototype strains and eight Israeli REV isolates. Both PCR systems had similar levels of sensitivity.
Davidson
I
I
Division of Avian Diseases, Kimron Veterinary Institute, Bet Dagan, Israel.
Malkinson
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M
D000818:Animals; D001710:Biotin; D002642:Chick Embryo; D002645:Chickens; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D004372:Ducks; D005363:Ficusin; D015686:Gene Products, env / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D001356:Reticuloendotheliosis virus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014421:Turkey
D015342:DNA Probes; D004279:DNA, Viral; D015686:Gene Products, env; D001710:Biotin; D005363:Ficusin