Abstract
In this study, a rapid and sensitive real-time loop-mediated isothermal amplification (Rti-LAMP) assay was developed for quantitative and evaluation of viable but non-culturable (VBNC) Salmonella. Four micrograms per milliliter of ethidium bromide monoazide (EMA) could significantly inhibit DNA amplification derived from dead cells in Rti-LAMP assays. The EMA-Rti-LAMP was used to monitor the culturable and VBNC Salmonella cells induced by 4 °C and − 20 °C, as direct fluorescence method (DEM) and plate counting method as controls. When 1.3 × 104 CFU/mL Salmonella was 5 cycles of freeze-thaw, the cells were all dead. However, Salmonella in 1.3 × 106 CFU/mL gradually transferred into VBNC state reaching 6.0 × 102 CFU/mL (0.05%) after 5 cycles of freeze-thaw. Keeping Salmonella 1.3 × 104 CFU/mL and 1.3 × 106 CFU/mL in 0.85% NaCl at 4 °C, the culturable cells persistently decreased in plate counting. Meanwhile, the VBNC cells generated gradually from 0 to 4.2 × 103 CFU/mL and 1.3 × 105 CFU/mL detected by both EMA-Rti-LAMP and DEM up to 110-day storage, respectively. While in − 20 °C, 1.3 × 104 CFU/mL Salmonella sharply inactivated during 20 days, but 1.3 × 106 CFU/mL increasingly transferred into VBNC state reaching 3.5 × 104 CFU/mL detected by both EMA-Rti-LAMP and DEM up to 110-day storage. The results indicated that the EMA-Rti-LAMP had similar accuracy with DEM in rapidly detecting viable including VBNC cells, and the former had specificity but the latter did not. The EMA-Rti-LAMP combined with bentonite-coated activated carbon (BCAC) treatment could detect as low as 35 CFU/g VBNC Salmonella derived from contaminated chicken, and the entire assay completed in 5 h. Furthermore, four identical samples were Salmonella positive from 24 retail frozen chicken samples detected by plate culture (GB4789.4-2016), BCAC-Rti-LAMP, and BCAC-EMA-Rti-LAMP. The BCAC-EMA-Rti-LAMP had one more sample for Salmonella positive than that of plate culture, but less two samples than that of BCAC-Rti-LAMP. Noticeably, the BCAC-EMA-Rti-LAMP had much more accuracy as plate counting than that of BCAC-Rti-LAMP in detection of viable Salmonella derived from chicken. These results strongly suggested that the BCAC-EMA-Rti-LAMP assay could be a rapid and sensitive method for detection of viable Salmonella including VBNC cells in chicken without enrichment.
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Funding
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).
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Hu Chen declares that he has no conflict of interest. Chan Zhong declares that she has no conflict of interest. Tian Tian Zhang declares that she has no conflict of interest. Mei Su declares that she has no conflict of interest. Li Ping Lin declares that she has no conflict of interest. Qiu Shui Luo declares that he has no conflict of interest. Guo Ping Wu declares that he has no conflict of interest.
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Chen, H., Zhong, C., Zhang, T. et al. Rapid and Sensitive Detection of Viable but Non-culturable Salmonella Induced by Low Temperature from Chicken Using EMA-Rti-LAMP Combined with BCAC. Food Anal. Methods 13, 313–324 (2020). https://doi.org/10.1007/s12161-019-01655-9
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DOI: https://doi.org/10.1007/s12161-019-01655-9