Viral nucleocapsid gene based real-time polymerase chain reaction assay for estimation of Peste des petits ruminants virus titre in infected cells Bisht D., Saxena S.*, Khandare R., Kumar P., Shah P., Shrivastava S. Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute (Deemed University), Izatnagar-243122, Uttar Pradesh, India *Corresponding author E-mail id: sonalvet@gmail.com
Online published on 29 August, 2023. Abstract Peste des Petits Ruminants (PPR) is a highly contagious viral disease of small ruminants caused by the PPR virus, posing a significant threat to the livelihoods of small-scale farmers in developing countries. Quantification of viral infectious units is essential for monitoring the effectiveness of vaccination programs, evaluating the severity of outbreaks, and identifying the source of infections. Traditional methods based on measuring the endpoint titer of a virus (TCID50) are laborious, time -consuming, and subject to ambiguity. In the present study, a procedure was developed using a two-step RT-PCR and the SYBR Green detection method, targeting the immunodominant regions of the nucleocapsid gene of PPR virus, enabling rapid and efficient calculation of titre equivalents when working with cell culture propagated PPRVs. RT-qPCR was performed to measure the viral load of different dilutions of PPR vaccine virus (Sungri/96) with known tissue culture infectivity titre. Cycle threshold values against log TCID50 values were used to generate the comparable titre equivalents. The correlation value of r = -0.9931 between the two quantification methods implied that Ct measurements can serve as a reliable surrogate for estimating the tissue culture infectivity titres of PPRV within the specified titre range commonly used in laboratory research. Top Keywords Peste des petits ruminants, Nucleocapsid gene, Endpoint titration, Tissue culture infectious dose, Real-time RT-PCR. Top |