ABSTRACTRecent outbreaks linked toSalmonella-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 targetingSalmonella. 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-killedSalmonellacells with concentrations up to 108CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viableSalmonellacells in pure culture and 6.1 × 103to 6.1 × 104CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (TT) values and viableSalmonellacell numbers was high (R2= 0.949 to 0.993), with a quantification range (102to 105CFU/reaction in pure culture and 104to 107CFU/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 viableSalmonellacells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce.
Visual Detection of Enterohemorrhagic Escherichia coli O157:H7 Using Loop-Mediated Isothermal Amplification
