Saltatory Rolling Circle Amplification (SRCA): a Novel Nucleic Acid Isothermal Amplification Technique Applied for Rapid Detection of Shigella Spp. in Vegetable Salad

Shigella spp. are enteric pathogens that pose a serious threat to public health worldwide. A novel saltatory rolling circle amplification (SRCA) assay was developed to detect Shigella spp. in food targeting the ipaH gene. SRCA as an isothermal amplification method requires no expensive thermocycle instrument and could avoid electrophoresis as visualization results was successfully applied for SRCA. In order to confirm the specificity of this assay, 34 strains including 11 strains belonging to different Shigella species and 23 non-Shigella bacteria were detected with pure cultures. The sensitivity of Shigella flexneri by SRCA was evaluated using agarose gel electrophoresis, which was 7.3 x 10(1) fg/mu L. In addition, the amplification results were also determined by adding the fluorochrome, SYBR Green I (1 mu L of 1000x), allowing naked eye visualization of results, and the sensitivity was 7.3 x 10(0) fg/mu L. Moreover, the sensitivity of PCR was 7.3 x 10(2) fg/mu L, showing that the sensitivity of SRCA by electrophoresis and SYBR Green I fluorescence were 10- and 100-fold higher than that of PCR, respectively. The detection limit of SRCA was also evaluated with artificially inoculated vegetable salad without enrichment, and it was 4.7 x 10(2) and 4.7 x 10(1) CFU/g by electrophoresis and fluorescence, respectively. The detection limit by PCR was 4.7 x 10(3) CFU/g, which was 10- and 100-fold higher than that of SRCA. Therefore, SRCA is a potentially reliable tool for rapid and specific detection of Shigella in food and could be useful in underdeveloped countries with limited resources.