Detection of contamination of vaccines with the reticuloendotheliosis virus by reverse transcriptase polymerase chain reaction (RT-PCR).
The reverse transcriptase polymerase chain reaction (RT-PCR) was applied to detect contamination of Marek's disease (MD) vaccine with reticuloendotheliosis 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 dose per ml (FAID50/ml) and the nested PCR detected 10(1) FAID50/ml virus. The sensitivity of the RT-PCR system was found 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.
Takagi
M
M
National Veterinary Assay Laboratory, Tokyo, Japan.
Ishikawa
K
K
Nagai
H
H
Sasaki
T
T
Gotoh
K
K
Koyama
H
H
D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D003001:Cloning, Molecular; D017931:DNA Primers; D004340:Drug Contamination; D015752:Genes, env; D008381:Herpesvirus 2, Gallid / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis; D012194:RNA-Directed DNA Polymerase; D017986:Reticuloendotheliosis Viruses, Avian / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014765:Viral Vaccines / Q000302:isolation & purification
D017931:DNA Primers; D012367:RNA, Viral; D014765:Viral Vaccines; D012194:RNA-Directed DNA Polymerase