Detection and Evaluation of Viable but Non-culturable Escherichia coli O157:H7 Induced by Low Temperature with a BCAC-EMA-Rti-LAMP Assay in Chicken Without Enrichment
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In this study, a rapid and sensitive method that combines ethidium bromide monoazide (EMA) staining with real-time loop-mediated isothermal amplification (Rti-LAMP) assay was tested in detecting and evaluating viable but non-culturable state (VBNC) Escherichia coli (E. coli) O157:H7 induced by low temperature. When 1.7 × 104 CFU/ml of E. coli O157:H7 was 3 cycles of freeze-thaw, the cells were all dead. However, E. coli O157:H7 in 1.7 × 106 CFU/ml and 1.7 × 108 CFU/ml gradually transferred into VBNC state reaching 6.8 × 102 CFU/ml (0.04%) and 4.1 × 105 CFU/ml (0.24%) after 6 cycles of freeze-thaw, respectively. Keeping E. coli O157:H7 (1.7 × 108 CFU/ml) at 4 °C and − 20 °C, the culturable cells persistently decreased in plate counting. Meanwhile, the VBNC cells increased from 0 to 1.1 × 106 CFU/ml and 5.5 × 106 CFU/ml detected by both EMA-Rti-LAMP and direct epifluorescence method (DEM) up to 258-day storage at 4 °C and − 20 °C, respectively. The EMA-Rti-LAMP had similar accuracy with DEM in detecting viable including VBNC cells, the former had specificity but not the later. Furthermore, the EMA-Rti-LAMP could detect as low as 25 CFU/g of VBNC E. coli O157:H7 derived from contaminated chicken combined with bentonite-coated activated carbon (BCAC) treatment to remove DNA amplification inhibitors, and the entire assay could be completed in 5 h. In addition, one same sample for E. coli O157:H7 positive was detected by both BCAC-EMA-Rti-LAMP and plate count method from 24 retail chicken samples, but four positive samples by BCAC-Rti-LAMP assay. The results obviously suggested that the BCAC-EMA-Rti-LAMP assay might be a rapid and sensitive method for detection of viable including VBNC E. coli O157:H7 cells in food without enrichment.
KeywordsEscherichia coli O157:H7 (E. coli O157:H7) Viable but non-culturable state (VBNC) Chicken Rti-LAMP Ethidium bromide monoazide (EMA) Bentonite-coated activated carbon (BCAC)
This study was funded by the National Natural Science Foundation of China (No. 31560480, 31760483), Jiangxi Natural Science Foundation of China (No. 20171ACB20013), and the Special Funds for Collaborative Innovation of Modern Agricultural Science and Research in Jiangxi, China (No. JXXTCX201703).
Compliance with Ethical Standards
Conflict of Interest
Hu Chen declares that he has no conflict of interest. Yuan Yang Zhao declares that he has no conflict of interest. Mei Su declares that she has no conflict of interest. Tian Tian Zhang declares that she has no conflict of interest. Yan Bi declares that she has no conflict of interest. Yan Yan Gao declares that she has no conflict of interest. Guo Ping Wu declares that he has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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