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
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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使用 BCAC-EMA-Rti-LAMP 检测对未富集鸡中低温诱导的活但不可培养的大肠杆菌 O157:H7 进行检测和评估
DOI：
10.1007/s12161-018-1377-9
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发表时间：
2019
影响因子：
2.9
通讯作者：
Guo Ping Wu
中科院分区：
文献类型：
--
作者：
Chen Hu;Zhao Yuan Yang;Shu Mei;Zhang Tian Tian;Bi Yan;Gao Yan Yan;Guo Ping Wu
在这项研究中，测试了一种快速、灵敏的方法，该方法将溴化乙锭单叠氮（EMA）染色与实时环介导等温扩增（Rti-LAMP）检测相结合，用于检测和评价活的但不可培养状态（VBNC）大肠杆菌（E. coli）O157：H7的低温诱导。当E. colio157：H7冻融3次，细胞全部死亡。然而，E. 1.7 × 106CFU/ml和1.7 × 108CFU/ml的大肠杆菌H7经6次冻融后逐渐转化为VBNC状态，分别达到6.8 × 102CFU/ml（0.04%）和4.1 × 105CFU/ml（0.24%）。保持E。colio157：H7（1.7 × 108CFU/ml）培养时，平板计数持续下降。同时，在4 ° C和− 20 ° C下储存258天，通过EMA-Rti-LAMP和直接荧光法（DEM）检测到的VBNC细胞分别从0增加到1.1 × 106CFU/ml和5.5 × 106CFU/ml。EMA-Rti-LAMP与DEM检测活细胞（包括VBNC细胞）的准确性相似，前者具有特异性，后者则无特异性。此外，EMA-Rti-LAMP可以检测低至25 CFU/g的VBNCE。大肠杆菌O157：H7的DNA扩增抑制剂与蒙脱石包被活性炭（BCAC）结合，可在5 h内完成整个检测过程。另外，还对E. 24份零售鸡肉样品中，BCAC-EMA-Rti-LAMP法和平板计数法均检出colio157：H7阳性，而BCAC-Rti-LAMP法仅检出4份阳性。结果表明，BCAC-EMA-Rti-LAMP法是一种快速、灵敏的检测包括VBNCE在内的活菌的方法。大肠杆菌157：H7细胞在食品中没有富集。
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 × 104CFU/ml ofE. coliO157:H7 was 3 cycles of freeze-thaw, the cells were all dead. However,E. coliO157:H7 in 1.7 × 106CFU/ml and 1.7 × 108CFU/ml gradually transferred into VBNC state reaching 6.8 × 102CFU/ml (0.04%) and 4.1 × 105CFU/ml (0.24%) after 6 cycles of freeze-thaw, respectively. KeepingE. coliO157:H7 (1.7 × 108CFU/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 × 106CFU/ml and 5.5 × 106CFU/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 VBNCE. coliO157: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 forE. coliO157: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 VBNCE. coliO157:H7 cells in food without enrichment.