Detection of specific UL49 sequences of Marek's disease virus CVI988/Rispens strain using loop-mediated isothermal amplification

Abstract

Marek's disease (MD) is a tumoral disease of chickens that can be controlled by vaccines based on non-pathogenic strains of turkey herpesvirus (HVT), SB-1 strain belonging to serotype 2, or the attenuated CVI988/Rispens strain belonging to serotype 1 of Marek's disease virus (MDV). Currently, the ‘gold standard’ in MD prophylaxis is the Rispens strain-based vaccine which protects against very virulent MDV and disease onset. Previous studies have shown that loop-mediated isothermal amplification (LAMP) is a rapid alternative to polymerase chain reaction (PCR) for detection and differentiation of HVT, SB-1 and virulent MDV strains. The aim of this study was to develop and evaluate a novel LAMP assay for the detection of the UL49 Rispens-specific region. This assay was validated using material from infected chicken embryo fibroblasts (CEFs) and tissue samples from vaccinated chickens. The analytical sensitivity of the assay was 10-times higher than PCR and reliably amplified 0.1 log10 TCID50/ml. The MDV Rispens was also detected at 18h after infection of CEFs. The results showed LAMP to be selective and a sensitive method to detect Rispens as early as 3 d.p.v. in all internal organs of chickens. Furthermore, the method was also capable to detect Rispens in 5 out of 26 chickens originating from different flocks. A mismatch amplification mutation assay (MAMA-PCR) confirmed the presence of Rispens strain in all LAMP-positive chickens. This is the first report of the specific visual detection of Rispens in vitro and in vivo using LAMP. The method may be useful for monitoring of successful chicken vaccination as well as in vitro studies in infected cell cultures.