A new application of a sodium deoxycholate-propidium monoazide-quantitative PCR assay for rapid and sensitive detection of viable Cronobacter sakazakii in powdered infant formula.
A new application of a sodium deoxycholate-propidium monoazide-quantitative PCR assay for rapid and sensitive detection of viable Cronobacter sakazakii in powdered infant formula.
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DOI：
10.3168/jds.2016-11538
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发表时间：
2016-12
影响因子：
3.5
通讯作者：
Baoqing Zhou;Bolu Chen;Xin Wu;Fan Li;Pei Yu;Zoraida P. Aguilar;Hua Wei;Hengyi Xu
中科院分区：
文献类型：
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作者：
Baoqing Zhou;Bolu Chen;Xin Wu;Fan Li;Pei Yu;Zoraida P. Aguilar;Hua Wei;Hengyi Xu
阪崎慢杆菌是一种可能导致严重新生儿疾病的常见食源性病原菌，现已建立了一种快速、可靠、敏感的检测方法。本研究建立了一种结合脱氧胆酸钠(SD)和单叠氮丙烷(PMA)的快速实时定量聚合酶链式反应(QPCR)检测婴儿配方奶粉(PIF)中阪崎肠杆菌污染的方法。这种方法可以消除死亡或受伤细菌的干扰。优化研究表明，SD和PMA的最佳用量分别为0.08%(wt/ol)和5µg/mL。此外，采用qPCR、PMA-qPCR、SD-PMA-qPCR和平板计数法计算在55℃水浴不同时间下细胞悬液中的活细菌数量。结果表明，PMA-qPCR检测的活菌数显著高于SD-PMA-qPCR法和平板计数法检测的活菌数。SD-PMA-qPCR的活菌数与传统的平板计数结果一致，说明SD处理可以消除死亡或损伤细胞的干扰。在优化的条件下，SD-PMA-qPCR方法在纯培养和添加PIF条件下的检测限分别为3.3×102cfu/m L和4.4×102cfu/g。金黄色葡萄球菌的检出限为5.6×102cfu/g(107cfu/ml)。SD-PMA-qPCR联合检测方法可用于PIF中阪崎肠杆菌的快速检测。
A rapid, reliable, and sensitive method for the detection of Cronobacter sakazakii, a common foodborne pathogen that may cause serious neonatal disease, has been developed. In this study, a rapid real-time quantitative PCR (qPCR) assay combined with sodium deoxycholate (SD) and propidium monoazide (PMA) was developed to detect C. sakazakii contamination in powdered infant formula (PIF). This method could eliminate the interference from dead or injured bacteria. Optimization studies indicated that SD and PMA at 0.08% (wt/vol) and 5µg/mL, respectively, were the most appropriate. In addition, qPCR, PMA-qPCR, SD-PMA-qPCR, and plate count assays were used to account for the number of viable bacteria in cell suspensions that were exposed to a 55°C water bath at different length of time. As a result, the viable number by PMA-qPCR showed significantly higher than of the number from SD-PMA-qPCR or plate counts. The number of viable bacteria was consistent between SD-PMA-qPCR and traditional plate counts, which indicated that SD treatment could eliminate the interference from dead or injured cells. Using the optimized parameters, the limit of detection with the SD-PMA-qPCR assay was 3.3×102 cfu/mL and 4.4×102 cfu/g in pure culture and in spiked PIF, respectively. A similar detection limit of 5.6×102 cfu/g was obtained in the presence of the Staphylococcus aureus (107 cfu/mL). The combined SD-PMA-qPCR assay holds promise for the rapid detection of viable C. sakazakii in PIF.