Study of Outer Membrane Proteins OMPs of Salmonella spp and development of OMP and virulence based rapid diagnostics for food and clinical samples

Abstract

Infections due to Salmonella spp. are of great risk to human health. It is known that genes involved in pathogenesis of Salmonella spp. are clustered within Salmonella pathogenicity islands. In this study, we have developed three multiplex PCRs to detect potentially pathogenic Salmonella directly from clinical and seafood samples. The multiplex PCR involving the hns gene could detect Salmonella up to a level of 102 cells/ml in 10 times diluted clinical samples. This study also aimed to detect the prevalence of 12 different virulence associated genes coded by 4 pathogenicity Islands namely, SPI-1, SPI-2, SPI-3 and SPI-5, in 58 Salmonella enterica spp. isolated from both food and clinical samples. Two sets of Hexaplex PCR were also developed. Results showed that the genes hilA, invA, ssaB, ssaP, ssaQ, ssaT, sseD and mgtC were 100% prevalent in all Salmonella isolates tested in this study, followed by rhuM (96.77%), sseC (93.5%), sopB (90.34%) and pipC (90.34%). The detection limit of the optimized multiplex PCR was tested with lowest detectable concentration of 0.005 ng and#956;l-1. The assay using the SPI virulence genes was reproducible and sensitive in detecting Salmonella. While in this study, the amplification of hns could point to the presence of Salmonella, the presence of virulence genes could determine its pathogenicity. Finding the presence or absence of virulence genes could explain the reason behind the stringent host adaptation of seafood associated serovars of Salmonella. In addition, this study also aimed in developing polyclonal and monoclonal antibodies targeting Salmonella outer membrane proteins (OMP?s) which are highly immunogenic and considered as diagnostic antigens. Primers designed in this study targeting the ompV and ompF genes were specific in detecting Salmonella. The OMPs genes were cloned and expressed in E. coli host system and the purified OmpF protein was used to raise monoclonal antibodies.