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Rapid Detection of Viable Salmonellae in Produce by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification

单叠氮丙二钠 环介导等温扩增 生物 联轴节(管道) 等温过程 沙门氏菌 微生物学 细菌 物理 遗传学 热力学 材料科学 聚合酶链反应 基因 DNA 冶金
作者
Siyi Chen,Fei Wang,John C. Beaulieu,Rebecca E. Stein,Beilei Ge
出处
期刊:Applied and Environmental Microbiology [American Society for Microbiology]
卷期号:77 (12): 4008-4016 被引量:165
标识
DOI:10.1128/aem.00354-11
摘要

Recent outbreaks linked to Salmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targeting Salmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killed Salmonella cells with concentrations up to 10(8) CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viable Salmonella cells in pure culture and 6.1 × 10(3) to 6.1 × 10(4) CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (T(T)) values and viable Salmonella cell numbers was high (R(2) = 0.949 to 0.993), with a quantification range (10(2) to 10(5) CFU/reaction in pure culture and 10(4) to 10(7) CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viable Salmonella cells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce.
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