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.

中文翻译：

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EMA-Rti-LAMP结合BCAC可以快速灵敏地检测出低温诱导的活的沙门氏菌和不可培养的沙门氏菌

摘要

在这项研究中，开发了一种快速灵敏的实时环介导的等温扩增（Rti-LAMP）分析法，用于定量和评估可行但不可培养的沙门氏菌（VBNC）。在Rti-LAMP分析中，每毫升溴化乙叠氮化单乙胺（EMA）4微克可以显着抑制源自死细胞的DNA扩增。EMA-Rti-LAMP作为直接荧光法（DEM）和平板计数法，用于监测4°C和-20°C诱导的可培养和VBNC沙门氏菌细胞。当1.3×10 ⁴ CFU / mL沙门氏菌进行5次冻融循环时，细胞全部死亡。但是，沙门氏菌在1.3×10 ⁶经过5次冻融后，CFU / mL逐渐转变为VBNC状态，达到6.0×10 ² CFU / mL（0.05％）。在4°C下于0.85％NaCl中保持沙门氏菌1.3×10 ⁴ CFU / mL和1.3×10 ⁶ CFU / mL，可培养细胞在平板计数中持续减少。同时，通过EMA-Rti-LAMP和DEM分别检测到的VBNC细胞逐渐从0生成至4.2×10 ³ CFU / mL和1.3×10 ⁵ CFU / mL，最多可存储110天。在− 20°C时，在20天之内沙门氏菌急剧失活1.3×10 ⁴ CFU / mL ，但1.3×10 ⁶ CFU / mL逐渐转移到VBNC状态，达到3.5×10 ⁴EMA-Rti-LAMP和DEM最多可检测110天的储存CFU / mL。结果表明，EMA-Rti-LAMP在快速检测包括VBNC细胞在内的存活物中具有与DEM相似的准确性，并且前者具有特异性，而后者则没有。EMA-Rti-LAMP结合膨润土包被的活性炭（BCAC）处理可以检测到低至35 CFU / g受污染鸡源的VBNC沙门氏菌，整个检测过程在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检测到的24个零售冷冻鸡样品中，有四个相同的样品为沙门氏菌阳性。BCAC-EMA-Rti-LAMP还有一份沙门氏菌样品阳性比平板培养阳性，但比BCAC-Rti-LAMP少两个样品。值得注意的是，在检测源自鸡的活沙门氏菌时，BCAC-EMA-Rti-LAMP作为板计数的准确性比BCAC-Rti-LAMP高。这些结果强烈表明，BCAC-EMA-Rti-LAMP测定法可能是一种快速且灵敏的方法，用于检测鸡中包括VBNC细胞在内的活沙门氏菌而无需富集。