duplex real-time polymerase chain reaction assay for the simultaneous detection of long terminal repeat regions and envelope protein gene sequences of Reticuloendotheliosis virus in avian blood samples

• Published in: Journal of veterinary diagnostic investigation Vol. 23; no. 5; pp. 937 - 941
• Main Authors: Sun, Feng, Ferro, Pamela J, Lupiani, Blanca, Kahl, Janell, Morrow, Michael E, Flanagan, Joseph P, Estevez, Carlos, Clavijo, Alfonso
• Format: Journal Article
• Language: English
• Published: United States 01.09.2011
• Subjects: Animals; blood; Chemical Fractionation - methods; chickens; diagnostic sensitivity; DNA; DNA, Viral - genetics; Ducks; Galliformes; geese; genes; quails; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - veterinary; Reticuloendotheliosis virus; Reticuloendotheliosis Viruses, Avian - isolation & purification; Reticuloendotheliosis, Avian - blood; Reticuloendotheliosis, Avian - diagnosis; Reticuloendotheliosis, Avian - virology; Sensitivity and Specificity; terminal repeat sequences; Terminal Repeat Sequences - genetics; turkeys; Viral Envelope Proteins - genetics
• ISSN: 1040-6387; 1943-4936
• DOI: 10.1177/1040638711416631

The Reticuloendotheliosis virus (REV) group of retroviruses infects a wide range of avian species, including chickens, turkeys, ducks, geese, quail, and prairie chickens. The objective of the present study was to develop a highly sensitive and specific diagnostic test for the detection of REV in whole blood samples. In order to increase the diagnostic sensitivity, a duplex real-time polymerase chain reaction (PCR) that detects both the envelope protein gene (env) and the long terminal repeat (LTR) region of REV was designed. This assay demonstrated greater analytical and diagnostic sensitivity than the gel-based PCR assay when using DNA extracted from whole blood by both phenol-chloroform and magnetic bead methods. In general, threshold cycle values in the duplex real-time PCR assay were lower from DNA extracted using the magnetic bead system compared to DNA extracted by the phenol-chloroform method. Data presented herein show the successful development of a rapid and accurate test procedure, with high-throughput capability, for the diagnosis of REV infection using avian blood samples.