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

Campylobacter is the leading cause of foodborne human diarrhea worldwide. This microbe in the viable but nonculturable (VBNC) state can evade detection by routinely used culture-based methods and remain viable for extended periods of time. Bacteria in this dormancy state can resume their metabolic activity and virulence by resuscitation under favorable conditions, and subsequently cause infections. In this study, an assay combining loop-mediated isothermal amplification (LAMP) and propidium monoazide (PMA) treatment was developed for the detection and quantification of VBNC C. jejuni in agri-foods. PMA-qLAMP targeting the hipO gene demonstrated 100% high specificity to C. jejuni. A linear detection of C. jejuni was achieved between 8.77 x 102 and 8.77 x 07 CFU/mL with a coefficient of determination (R2) of 0.9956, indicating a good quantitative capacity. C. jejuni was effectively induced into the VBNC state by osmotic stress (i.e., 7% NaCl, w/v) over 48 h. VBNC C. jejuni cells were spiked into three representative food products and determined by PMA-qLAMP coupled with plating assay. The detection limits of PMA-qLAMP were 1.58 x 102 CFU/mL in milk, 3.78 x 102 CFU/g in chicken breast meat, and 4.33 x 102 CFU/g in romaine lettuce. PMA-qLAMP demonstrated rapid (25-40 min), specific (100% inclusivity and 100% exclusivity) and sensitive (-102 CFU/mL) determination of VBNC C. jejuni. This method can be applied in the agri-food industry to decrease the risks related to the consumption of contaminated agri-foods with pathogenic bacteria in the VBNC state and reduce the burden of C. jejuni infections to public health.