title: Rapid detection of viable salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal amplification.
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.
authors.affiliation: Department of Food Science, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803, USA.
authors.forename: Siyi
authors.initials: S
authors.lastname: Chen
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.forename: John C
authors.initials: JC
authors.lastname: Beaulieu
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: Stein
authors.forename: Beilei
authors.initials: B
authors.lastname: Ge
mesh_terms: D001386:Azides / Q000378:metabolism*; D001431:Bacteriological Techniques / Q000379:methods*; D005638:Fruit / Q000382:microbiology*; D050296:Microbial Viability; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives* / Q000378:metabolism; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D012680:Sensitivity and Specificity; D013997:Time Factors; D014675:Vegetables / Q000382:microbiology*
chemical_list: D001386:Azides; C533957:propidium monoazide; D011419:Propidium