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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

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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 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 × 102 and 4.7 × 101 CFU/g by electrophoresis and fluorescence, respectively. The detection limit by PCR was 4.7 × 103 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.

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Funding

This work was supported by the National Natural Science Foundation of China (31371772), the Natural Science Foundation of Hebei Province of China (C2017204027), the Scientific Research Program of Hebei Education Department (ZD2017237), and the Science and Engineering Foundation of Hebei Agricultural University (ZD201620).

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Author notes

  1. Zhiyan Wang and Qian Yang contributed equally to this work.

Authors and Affiliations

  1. College of Food Science and Technology, Hebei Agricultural University, Baoding, 071001, China

    Zhiyan Wang, Yunzhe Zhang, Zhaoxiang Meng, Xiaoyan Ma & Wei Zhang

  2. College of Science and Technology, Hebei Agricultural University, Cangzhou, 061100, China

    Qian Yang & Wei Zhang

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  1. Zhiyan Wang
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  2. Qian Yang
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  3. Yunzhe Zhang
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  6. Wei Zhang
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Correspondence to Wei Zhang.

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Zhiyan Wang declares that she has no conflict of interest. Qian Yang declares that she has no conflict of interest. Yunzhe Zhang declares that she has no conflict of interest. Zhaoxiang Meng declares that he has no conflict of interest. Xiaoyan Ma declares that she has no conflict of interest. Wei Zhang declares that he has no conflict of interest.

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Wang, Z., Yang, Q., Zhang, Y. et al. Saltatory Rolling Circle Amplification (SRCA): a Novel Nucleic Acid Isothermal Amplification Technique Applied for Rapid Detection of Shigella Spp. in Vegetable Salad. Food Anal. Methods 11, 504–513 (2018). https://doi.org/10.1007/s12161-017-1021-0

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