DEVELOPMENT OF A REAL-TIME PCR ASSAY FOR THE RAPID DETECTION OF netB GENE IN CLOSTRIDIUM PERFRINGENS

Necrotic enteritis (NE) has a huge economic impact in the poultry industry. Numerous studies have shown that necrotic enteritis toxin B (netB) is a key virulence factor in Clostridium perfringens strains that cause NE in chickens. Early detection of netB in C. perfringens is important to circumvent the spread of NE. In this study, we developed a novel combinatorial approach involving (i) a commercial 2-step DNA extraction kit and (ii) a real-time polymerase chain reaction (qPCR) for the detection of netB-positive C. perfringens in fecal samples. Melt curve analysis and specificity test demonstrated 100% specificity without any cross-reactivity in other bacterial species with a limit of detection of 102 cfu/g. Field validation was subsequently conducted on nine fecal composites collected from different layer houses at two commercial farms, leading to successful detection of four netB positive samples. The study presented a rapid diagnostic qPCR assay involving a 2-step DNA extraction protocol to screen for C. perfringens carrying netB gene in chicken fecal samples.