Comparison and verification of quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR) and real time RT-PCR for avian leukosis virus subgroup J.
Avian leukosis virus subgroup J (ALV-J) infections cause significant economic losses because of increased mortality, tumor production, decreased production, and cost for eradication. Current quantification methods for ALV-J expressed by TCID(50) are difficult to determine because of the lack of cytopathic effect in cell cultures and non-specificity of currently available antigen-capture ELISA tests. In this study, a one-tube fluorescent probe based real time RT-PCR method was developed for quantification of ALV-J and compared with available quantification methods. Cell lysates with different TCID(50)s determined by cell culture and antigen capture ELISA (ag-ELISA) were used for one-tube real time RT-PCR using fluorogenic probe and quantitative competitive RT-PCR (QC-RT-PCR). The results of QC-RT-PCR and real time RT-PCR were highly correlated to the TCID(50)s determined by conventional culture methods. They were also very specific, sensitive, easy to perform, reproducible, and rapid compared with conventional methods. These RT-PCR based quantification methods of ALV-J viral RNA will be useful for virological and pathogenesis studies.
Kim
Yongbaek
Y
Department of Veterinary Pathology and Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.
Gharaibeh
Saad M
SM
Stedman
Nancy L
NL
Brown
Tom P
TP
D000818:Animals; D001354:Avian Leukosis Virus / Q000235:genetics* / Q000276:immunology; D002642:Chick Embryo; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012367:RNA, Viral