Development of quantitative competitive-reverse transcriptase-polymerase chain reaction for detection and...

Infection with avian leukosis virus subgroup J (ALV-J) causes severe economic losses in the broiler industry by increasing mortality, producing tumors, and decreasing weight gain in chickens. The quantitation of ALV-J is difficult because of its failure to produce a cytopathic effect in cell culture systems and the nonspecificity of antigen-capture enzyme-linked immunosorbent assay (ELISA) tests. This study was performed to develop a quantitative competitive-reverse transcriptase-polymerase chain reaction (QC-RT-PCR) method based on coamplification of ALV-J genomic RNA and a known amount of a synthesized RNA competitor. The 369 bp RNA competitor was constructed by restriction enzyme treatment of an ALV-J specific 545 bp PCR product, ligation, transformation into Escherichia coli, and in vitro transcription. The competitor contained the same amplification primer annealing sites and sequence as the original viral RNA, except that it had a 176 bp internal deletion. Coamplified RT-PCR products were visualized by electrophoresis and ethidium bromide staining, and fluorescences were quantified using computer-assisted image analysis. The sensitivity of this new QC-RT-PCR method was 25 fg of viral RNA, and 10-fold dilutions were differentiable. This method allowed absolute and relative quantification of ALV-J RNA copy numbers and was simpler than previously published methods for ALV-J quantification.