Rapid detection of viable salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal amplification

Appl Environ Microbiol. 2011 Jun;77(12):4008-16. doi: 10.1128/AEM.00354-11. Epub 2011 Apr 15.

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 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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azides / metabolism*
  • Bacteriological Techniques / methods*
  • Fruit / microbiology*
  • Microbial Viability
  • Nucleic Acid Amplification Techniques / methods*
  • Propidium / analogs & derivatives*
  • Propidium / metabolism
  • Salmonella / genetics
  • Salmonella / isolation & purification*
  • Salmonella / physiology
  • Sensitivity and Specificity
  • Time Factors
  • Vegetables / microbiology*

Substances

  • Azides
  • propidium monoazide
  • Propidium