Rapid and accurate detection of viable Escherichia coli O157:H7 in milk using a combined IMS, sodium deoxycholate, PMA and real-time quantitative PCR process
Rapid and accurate detection of viable Escherichia coli O157:H7 in milk using a combined IMS, sodium deoxycholate, PMA and real-time quantitative PCR process
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使用 IMS、脱氧胆酸钠、PMA 和实时定量 PCR 组合工艺快速准确地检测牛奶中的活大肠杆菌 O157:H7
DOI：
10.1016/j.foodcont.2013.08.011
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发表时间：
2014-02
期刊：
影响因子：
6
通讯作者：
Wei, Hua
中科院分区：
文献类型：
--
作者：
Xu, Feng;Lai, Weihua;Xiong, Yonghua;Wei, Hua
为了提高大肠杆菌157:H7食源性疾病暴发检测方法的准确性，采用单叠氮丙啶（PMA）和脱氧胆酸钠（SD）两种试剂消除死亡和损伤细胞对qPCR的干扰，并结合免疫磁分离（IMS）进行IMS-SD-PMA-qPCR富集实验。最佳SD浓度为0.1%，最佳SD孵育时间为20ámin。将细胞悬液在63<e:1>温度下热处理或在<e:1>−20<e:1>温度下冷冻保存后，采用平板计数、qPCR、PMA-qPCR和SD-PMA-qPCR方法比较存活细菌的数量。经PMA处理或未经PMA处理的细胞悬浮液和经SD处理的细胞悬浮液的细菌存活数量明显低于未经SD处理的细胞悬浮液，这表明SD消除了死亡细胞的DNA。更重要的是，SD-PMA-qPCR分析的细菌存活数量与平板计数法分析的细菌存活数量直接相关。此外，为了提高检测限（LOD）和缩短检测时间，采用免疫磁分离（IMS）对目标菌进行捕获和富集。以加标牛奶为基质，IMS-SD-PMA-qPCR检测限为102áCFU/mL。值得注意的是，即使在106áCFU/mL的非目标细菌存在的情况下，SD-PMA-qPCR的LOD与IMS分离的结果一致。ácoliO157：加标乳基质中H7浓度为102áCFU/mL。这种联合试验有望检测foodborneE.ácoliO157:H7。
To improve the accuracy of current methods for the detection ofEscherichia coliO157:H7 foodborne disease outbreaks, two reagents, propidium monoazide (PMA) and sodium deoxycholate (SD), were utilized to eliminate the interference of dead and injured cells for qPCR that was combined with immunomagnetic separation (IMS) for cell enrichment in an IMS-SD-PMA-qPCR assay. The optimal SD concentration and the optimal incubation time with SD were recorded at 0.1% and 20ámin, respectively. The number of bacteria survivors was compared using plate counts, qPCR, PMA-qPCR, and SD-PMA-qPCR assays after the cell suspensions were heat treated at 63á░C or freeze stored atá−20á░C. Cell suspensions treated or not treated with PMA and treated with SD resulted in significantly lower number of bacteria survivors than those analyzed without SD treatment which indicated that dead cell DNA was eliminated with SD. More importantly, the number of bacteria survivors from those analyzed with SD-PMA-qPCR showed a direct correlation with those analyzed using the plate counts methods. In addition, in order to improve the limit of detection (LOD) and shorten the detection time, immunomagnetic separation (IMS) was adopted to capture and enrich the target bacteria. Using spiked milk as a matrix, the IMS-SD-PMA-qPCR showed a detection limit of 102áCFU/mL. Significantly, even in the presence of 106áCFU/mL of non-target bacteria, the LOD for the SD-PMA-qPCR with IMS separation forE.ácoliO157:H7 in spiked milk matrix was recorded at 102áCFU/mL. This combination assay holds promise for the detection of foodborneE.ácoliO157:H7.
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影响因子：
3.1
作者：
Lijun Wang;L. Ping;Youjun Yang;Hong Xu;Zoraida P. Aguilar;Hengyi Xu;Lin Yang;Feng Xu;W. Lai;Yonghua Xiong;Hua Wei
通讯作者：
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影响因子：
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影响因子：
2
作者：
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影响因子：
36.8
作者：
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通讯作者：
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影响因子：
5.4
作者：
Malorny, B;Tassios, PT;Hoorfar, J
通讯作者：
Hoorfar, J