Detection of contamination of vaccines with the reticuloendotheliosis virus by reverse transcriptase polymerase chain reaction (RT-PCR)

Abstract

The reverse transcriptase polymerase chain reaction (RT-PCR) was applied to detect contamination of Marek's disease (MD) vaccine with eticuloendothelios s virus (REV). The env primers were used for the 1st RT-PCR to amplify the DNA fragments of REV-A and -T. The rel and env primers were used for nested-PCR to confirm the sites deleted from REV-T and REV-A. Specific amplification products were detected in the 1st RT-PCR with these primers. By nested PCR with the env and the rel primer pairs, the products originating from REV-A and -T were identified. This system, using the env primer pairs, showed a specific amplification with several REV strains (REV-T, DE, CE, KI and 0202), but no amplified product was detected with MDV, NDV, IBV or ILTV. The 1st RT-PCR detected the virus in a concentration of 10 3 in 50% fluorescent antibody infectious dosse per ml (FAID 50/ml) and the nested PCR detected 10 1 FAID 50/ml virus. The sensitivity of the RT-PCR system wasfound to be higher than that of the FA assay. This system provides a rapid, sensitive and specific method for detection of contamination of MD vaccines with REV-RNA, and it may be applied for quality control of live vaccines.