Rapid and Sensitive Detection of Viable but Non-culturable Salmonella Induced by Low Temperature from Chicken Using EMA-Rti-LAMP Combined with BCAC
Rapid and Sensitive Detection of Viable but Non-culturable Salmonella Induced by Low Temperature from Chicken Using EMA-Rti-LAMP Combined with BCAC
复制标题
使用 EMA-Rti-LAMP 结合 BCAC 快速、灵敏地检测鸡肉中低温诱导的活但不可培养的沙门氏菌
DOI：
10.1007/s12161-019-01655-9
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发表时间：
2019-10-23
影响因子：
2.9
通讯作者：
Wu, GuoPing
中科院分区：
文献类型：
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作者：
Chen, Hu;Zhong, Chan;Wu, GuoPing
在本研究中，开发了一种快速、灵敏的实时环介导等温扩增 (Rti-LAMP) 测定法，用于定量和评估活但不可培养 (VBNC) 沙门氏菌。在 Rti-LAMP 检测中，每毫升 4 微克的溴化乙锭单叠氮化物 (EMA) 可以显着抑制来自死细胞的 DNA 扩增。使用EMA-Rti-LAMP监测4℃和-20℃诱导的可培养和VBNC沙门氏菌细胞，作为直接荧光法（DEM）和平板计数法作为对照。 1.3×104 CFU/mL沙门氏菌冻融5个循环时，细胞全部死亡。然而，经过5次冻融循环后，1.3×106 CFU/mL的沙门氏菌逐渐转变为VBNC状态，达到6.0×102 CFU/mL（0.05%）。将沙门氏菌在 0.85% NaCl 中于 4 °C 保持为 1.3 × 104 CFU/mL 和 1.3 × 106 CFU/mL，平板计数中的可培养细胞持续减少。同时，通过 EMA-Rti-LAMP 和 DEM 检测，VBNC 细胞的生成量分别从 0 逐渐增加到 4.2 × 103 CFU/mL 和 1.3 × 105 CFU/mL，直至储存 110 天。在− 20 °C 下，1.3 × 104 CFU/mL 沙门氏菌在 20 天内急剧失活，但 1.3 × 106 CFU/mL 逐渐转变为 VBNC 状态，EMA-Rti-LAMP 和 DEM 检测到储存长达 110 天时达到 3.5 × 104 CFU/mL。结果表明，EMA-Rti-LAMP在快速检测包括VBNC在内的活细胞方面与DEM具有相似的准确性，并且前者具有特异性，而后者则没有。 EMA-Rti-LAMP 结合膨润土涂层活性炭 (BCAC) 处理可以检测到来自受污染鸡肉的低至 35 CFU/g VBNC 沙门氏菌，整个检测在 5 小时内完成。此外，通过平板培养 (GB4789.4-2016)、BCAC-Rti-LAMP 和 BCAC-EMA-Rti-LAMP 检测，24 个零售冷冻鸡肉样品中有 4 个相同样品呈沙门氏菌阳性。 BCAC-EMA-Rti-LAMP 比平板培养多 1 个沙门氏菌阳性样本，但比 BCAC-Rti-LAMP 少 2 个样本。值得注意的是，在检测来自鸡肉的活沙门氏菌时，BCAC-EMA-Rti-LAMP 的平板计数精度比 BCAC-Rti-LAMP 高得多。这些结果强烈表明，BCAC-EMA-Rti-LAMP 测定可能是一种快速、灵敏的方法，可用于检测鸡中活沙门氏菌（包括 VBNC 细胞），无需富集。
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.