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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVING THE SENSORY QUALITY AND SHELF LIFE OF FRESH-CUT FRUIT PRODUCTS Title: Rapid detection of viable salmonella in produce by coupling propidium monoazide with loop-mediated isothermal amplification (PMA-LAMP).

Authors
item Chen, Siyi -
item Wang, Fei -
item Ge, Beilei -
item Beaulieu, John
item Stein, Rebecca

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 10, 2011
Publication Date: June 1, 2011
Citation: Chen, S., Wang, F., Ge, B., Beaulieu, J.C., Stein, R.E. 2011. Rapid detection of viable salmonella in produce by coupling propidium monoazide with loop-mediated isothermal amplification (PMA-LAMP). Applied and Environmental Microbiology. 4008-4016.

Interpretive Summary: Recent outbreaks linked to Salmonella-contaminated fresh produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining viability of live versus dead bacteria cells. In this study, we examined a novel strategy for the rapid detection and quantification of viable Salmonella in produce by coupling a simple propidium monoazide (PMA) sample treatment with loop-mediated isothermal amplification (LAMP). The method was compared against the most accurate rapid methods currently available (quantitative polymerase chain reaction, qPCR, and PMA-PCR) for foodborne pathogen detection. First, a LAMP assay targeting Salmonella was designed and optimized. Second, the performance of PMA-LAMP for detecting and quantifying viable Salmonella 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 in produce. The detection limits for viable, live Salmonella were 3.4-34 cells in pure culture and 6.1 × 103 - 6.1 × 104 CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive in detecting viable Salmonella. The complete PMA-LAMP assay took about 3 h to complete when testing produce samples which, is significantly faster and more cost-effective than PMA-qPCR. In conclusion, this rapid, accurate, and cost-effective detection and quantification method for viable Salmonella in fresh produce may present a reliable tool for the produce industry to better control potential microbial hazards in produce. This is the first report examining the novel combination of PMA and LAMP in detecting viable bacterial cells. Dead Salmonella cells, up to 3.8 × 108 CFU/ml (4.2 × 108 CFU/g in spiked produce), were not detected by PMA-LAMP, illustrating excellent dead cell exclusivity.

Technical Abstract: 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 Salmonella 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 Salmonella 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 108 CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 - 34 viable Salmonella cells in pure culture and 6.1 × 103 - 6.1 × 104 CFU/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 viable Salmonella cell numbers was high (R2 = 0.949 - 0.993), with a quantification range (102 - 105 CFU/reaction in pure culture and 104 - 107 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, which is faster and more cost-effective than PMA-qPCR. In conclusion, this rapid, accurate, and cost-effective method to detect and quantify viable Salmonella cells in produce may present a reliable tool for the produce industry to better control potential microbial hazards in produce.

Last Modified: 10/25/2014
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