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Rapid and visual detection of viable Staphylococcus aureus in pork and pork products by PMA and saltatory rolling circle amplification

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Abstract

The aim of this study was to establish a rapid and visual nucleic acid isothermal amplification method for the detection of viable Staphylococcus aureus (S. aureus) in pork and pork products. The method was based on saltatory rolling circle amplification (SRCA) combined with propidium monoazide (PMA). Eighteen S. aureus strains and 29 non-S. aureus strains were used for the specificity evaluation. The sensitivity and detection limit of PMA–SRCA assay through fluorescence visualization were 2.6 × 101 CFU/mL and 6.6 × 101 CFU/mL, respectively. Compared with PMA–PCR assay, the PMA–SRCA method by fluorescence visualization exhibited a 1000-fold higher sensitivity. A total of 123 pork samples were tested for S. aureus contamination, and the results indicated that the relative sensitivity, specificity and accuracy of PMA–SRCA method were 100, 99.12 and 99.19%, respectively, compared with ISO method. In conclusion, the PMA–SRCA assay can reduce the false-positive detection rate of S. aureus in pork and pork products.

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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 (C2019204342), Postdoctoral Foundation of Hebei Province (B2021005007), and the Key Research and Development Project of Hebei Province of China (18275501D).

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Author notes

  1. Wei Guo, Qian Yang, and Yunzhe Zhang have contributed equally to this work.

Authors and Affiliations

  1. College of Food Science and Technology, Hebei Agricultural University, Baoding, 071000, China

    Wei Guo, Yunzhe Zhang, Chenchen Wu, Jianxin Tan & Wei Zhang

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

    Qian Yang & Xin Lu

  3. College of Life Sciences, Hebei Agricultural University, Baoding, 071000, China

    Wei Zhang

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  1. Wei Guo
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  2. Qian Yang
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  4. Xin Lu
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Correspondence to Wei Zhang.

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Supplementary file1 (DOCX 19 KB)

217_2022_3990_MOESM2_ESM.tif

Supplementary file2 (TIF 3866 KB) Supplementary Fig 1 The schematic diagram of SRCA assay The lines in black indicate the target double strands DNA. The line in red and orange indicates the nucleotides added at the intersection and their complementary sequence. The forward primer (FWP) initiates an SRCA reaction. And the DNA strands is cyclized and the nucleotides are added at the junction of non-closed circular linear DNA under the action of Bst DNA polymerase. When the synthesized sequence crossed the gap, the newborn DNA strand is displaced. Subsequently, the reverse primer (RVP) binds to each tandem repeat (including target sequence and the added bases) generated by the rolling circle. To track the order of added bases, note that the addition sequence is composed of a few bases from either FWP or the complementary sequence of RVP, or both. With the elongation of original SRCA strand triggered by FWP, multiple priming events are initiated by RVP and the new generated DNA strands are displaced, thus generating new priming sites for FWP. A new branch begins to be displaced by RVP at the nth repeat, as RVP binds to the (n+1) th repeat. Subsequently, FWP bind to the replaced single strand DNA, generating double strand DNA with different repeats, and so on.

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Guo, W., Yang, Q., Zhang, Y. et al. Rapid and visual detection of viable Staphylococcus aureus in pork and pork products by PMA and saltatory rolling circle amplification. Eur Food Res Technol 248, 1625–1634 (2022). https://doi.org/10.1007/s00217-022-03990-4

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  • DOI: https://doi.org/10.1007/s00217-022-03990-4

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