Comparison and verification of quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR)...

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.