Application of real-time RT-PCR assay for detection and typing of foot-and-mouth disease virus in Tanzania

Masters ThesisSpeed is paramount in the diagnosis of highly infectious Foot-and-mouth disease (FMD). Recent developments in molecular biology have enabled specific detection of FMD virus by real-time RT-qPCR and sequencing in endemic settings in Africa. In this study, a laboratory-based experimental design was used to standardize real-time RT-qPCR assay for detection and typing of FMDV in selected regions of Tanzania. The optimized conditions for both pan-serotypic and serotype-specific real-time RT-qPCR assays were: reverse transcription (at 60°C for 30minutes), denaturation of reverse transcriptase and activation of DNA polymerase (at 95°C for 10minutes followed by 95°C for 15 seconds), annealing and elongation temperature (at 60°C for 1 minute) for 52 cycles. FMD virus were detected in 100% (n = 14) archived samples that were collected between 2010 and 2013 from confirmed FMD-cases. The standardized real-time RT-qPCR assays revealed 100% sensitivity and 100% specificity for detection and typing of FMDV, respectively.The frequency of FMDV detection among FMD-suspected cases of cattle collected from 2008 to 2014 in selected regions of Tanzania was 92% (n = 23). Of the 23 positive samples, 56.5% (n = 13), 8.7% (n = 2), 21.7% (n = 5), and 8.7% (n = 2) were typed into serotypes O, A, SAT 1 and SAT 2 respectively. One sample (4.3%) was positive for both serotype A and SAT 1. These findings indicate that the standardized pan-serotypic and serotype-specific real-time RT-qPCR assays have a potential use in detection and typing of FMDV field strains in endemic settings of Tanzania and Africa at large. Application of standardized real-time RT-qPCR assays could hasten diagnosis of FMDV that guide selection of effective FMD-control measures in the region. In-depth studies, including sequencing of one sample that revealed positive for two serotypes, are required to elucidate the possibility of mixed infection among FMD cases.Southern African Centre for Infectious Disease Surveillance (SACIDS