Saltatory Rolling Circle Amplification (SRCA): a Novel Nucleic Acid Isothermal Amplification Technique Applied for Rapid
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Saltatory Rolling Circle Amplification (SRCA): a Novel Nucleic Acid Isothermal Amplification Technique Applied for Rapid Detection of Shigella Spp. in Vegetable Salad Zhiyan Wang 1 & Qian Yang 2 & Yunzhe Zhang 1 & Zhaoxiang Meng 1 & Xiaoyan Ma 1 & Wei Zhang 1,2
Received: 14 April 2017 / Accepted: 13 August 2017 / Published online: 25 August 2017 # Springer Science+Business Media, LLC 2017
Abstract 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 × 101 fg/μL. In addition, the amplification results were also determined by adding the fluorochrome, SYBR Green I (1 μL of 1000×), allowing naked eye visualization of results, and the sensitivity was 7.3 × 100 fg/μL. Moreover, the sensitivity of PCR was 7.3 × 102 fg/μL, showing that the sensitivity of SRCA by electrophoresis and SYBR Green I fluorescence were 10and 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 × 102 and 4.7 × 101 CFU/g by electrophoresis and fluorescence, r e s p e c t i v e l y. T h e d e t e c t i o n l i m i t b y P C R w a s 4.7 × 103 CFU/g, which was 10- and 100-fold higher than that of SRCA. Therefore, SRCA is a potentially reliable tool for Zhiyan Wang and Qian Yang contributed equally to this work. * Wei Zhang [email protected] 1
College of Food Science and Technology, Hebei Agricultural University, Baoding 071001, China
2
College of Science and Technology, Hebei Agricultural University, Cangzhou 061100, China
rapid and specific detection of Shigella in food and could be useful in underdeveloped countries with limited resources. Keywords Saltatory rolling circle amplification (SRCA) . Shigella . Visualization . Detection
Introduction Shigella is a facultative gram-negative genus that is classified into four species: Shigella flexneri (S. flexneri), Shigella sonnei (S. sonnei), Shigella boydii (S. boydii), and Shigella dysenteriae (S. dysenteriae), based on the O-antigen structures of the membrane-associated lipopolysaccharide (Ranallo et al. 2014). They are recognized as significant enteric pathogens causing watery diarrhea and bacillary dysentery (shigellosis) (Nickerson et al. 2016; Thompson et al. 2016). Shigella spp. have a low infectious dose (Kentner et al. 2014) and could lead to a serious inflammatory reaction by penetration and destruction of colonic epithelia (Fritah
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