A icsA-targeted PCR for rapid identification of Shigella spp.

Abstract

Introduction: The genus Shigella contains four species (serogroups): S. dysenteria (serogroup A), S. flexneri (serogroup B), S. boydii (serogroup C), and S. sonnei (serogroup D). Shigella are highly infectious and a dose of 10–100 cells can cause infection. Virulent Shigella organisms cause the human illness known as bacillary dysentery. Bacillary dysentery (shigellosis) causes mild diarrhea, fever, abdominal cramps, and severe fluid loss. Shigella colonize and invade mucosal epithelial cells. Shigella produce icsA(intracellular spread)/VirG protein,a 116-kD surface-exposed outer membrane protein, which mediates actin polymerization to aid in bacterial movement inside the cell. The icsA protein is encoded by the virulence plasmid. IcsA consists of three major domains: an N-terminal signal sequence (amino acids [aa] 1 to 52), to direct Sec dependent protein transport across the inner-membrane; and a C-terminal translocation domain (aa 759 to 1102) that enables export of the remaining N-terminal region of icsA, the passenger domain (aa 53 to 758), across the OM. The purpose of this study was to develop speciesspecific PCR primers for detection of Shigella spp. Methods: A primer was designed for α-domain of icsA gene for the detection of Shigella. The icsA gene was amplified by using PCR and confirmed by sequencing. Results: Specificity data showed that PCR primers produce amplicons from all the Shigella spp., but not from the other species tested. These results suggested that these two PCR primers are quite sensitive in the detection of Shigella spp. in molecular epidemiological studies of shigellosis. Conclusion: This research proved that icsA gene specialized in Shigella spp. therefore icsA could be used for the detection of Shigella in clinical samples.