ABSTRACT
A simple PCR method was developed for the detection of Marek's disease (MD) and reticuloendotheliosis (RE) in formalin-fixed paraffin-embedded (FFPE) tissues, and for the detection of MD in tissues only preserved in 10% neutral buffered formalin. MD virus (MDV) and RE virus proviral DNA were detected in FFPE tissues stored for over 20 yr. MDV was also detected in tissues only preserved in formalin for up to 6 mo. The data indicate that PCR of formalin-fixed and FFPE tissues is a simple and valuable tool that can be used to identify MD and RE infection. The method described in this paper is a good alternative to any biologic or immunohistochemical assay to confirm the detection of MD and RE, as it does not require shipping frozen tissues to the diagnostic laboratory.
Subject(s)
Chickens , DNA, Viral/genetics , Herpesvirus 1, Meleagrid/isolation & purification , Polymerase Chain Reaction/methods , Poultry Diseases/diagnosis , Proviruses/genetics , Reticuloendotheliosis virus/isolation & purification , Animals , DNA, Viral/metabolism , Formaldehyde/chemistry , Marek Disease/diagnosis , Marek Disease/virology , Neoplasms/diagnosis , Neoplasms/veterinary , Paraffin/chemistry , Paraffin Embedding/veterinary , Polymerase Chain Reaction/veterinary , Poultry Diseases/virology , Proviruses/metabolism , Reticuloendotheliosis, Avian/diagnosis , Reticuloendotheliosis, Avian/virologyABSTRACT
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.
