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UBC Theses and Dissertations

Rapid determination of viable but non-culturable Campylobacter jejuni in agri-food products by loop-mediated isothermal amplification coupling propidium monoazide treatment Petersen, Marlen Wilhelmine

Abstract

Campylobacter is the leading cause of foodborne human diarrhea in Canada and worldwide. This microbe in the viable but non-culturable (VBNC) state can evade the detection by routinely used culture-based methods, remain viable for extended periods of time, resume their metabolic activity and virulence leading to infections and diseases, and subsequently pose a severe concern to public health, food safety and the economy. In this study, an assay combining loop-mediated isothermal amplification (LAMP) and propidium monoazide (PMA) treatment was developed to detect and quantify VBNC C. jejuni. PMA-qLAMP targeting the hipO gene showed 100% specificity to C. jejuni. The limit of detection was determined to be 8.77×10² CFU/mL in pure bacterial culture with good quantitative capacity ranging from 8.77×10² CFU/mL to 8.77×10⁷ CFU/mL. C. jejuni was induced into the VBNC state by incubation in 7% (w/v) NaCl over 48 h. VBNC C. jejuni cells were determined by PMA-qLAMP coupled with the plating assay and spiked into three agri-foods. The limits of detection of PMA-qLAMP were 1.58×10² CFU/mL, 3.78×10² CFU/g and 4.33×10² CFU/g in milk, chicken breast meat and romaine lettuce, respectively. PMA-qLAMP demonstrated a rapid (from 25 to 40 min), specific (100% inclusivity and 100% exclusivity), sensitive (1.58×10² - 8.77×10² CFU/mL) and quantitative (R² of 0.9937–0.9999) detection of VBNC C. jejuni in pure bacterial culture, chicken meat, milk, and lettuce. Considering the rising Campylobacter infections worldwide and serious concerns of VBNC bacteria, this detection method has extensive potential to be applied in the agri-food industry so as to reduce the burden of C. jejuni infections to the public health and economy.

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Attribution-NonCommercial-NoDerivatives 4.0 International

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