Rapid and Sensitive Detection of Viable but Non-culturable Salmonella Induced by Low Temperature from Chicken Using EMA-Rti-LAMP Combined with BCAC

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.