rapid and sensitive detection of viable but non-culturable salmonella induced by low temperature from chicken using ema-rti-lamp combined with bcac

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.