Digital polymerase chain reaction combined with propidium monoazide (PMA) without PMA enhancer detects viable but non-culturable Campylobacter jejuni cells

Campylobacter jejuni is the leading cause of foodborne illness in humans, typically after consuming contaminated chicken meat, and it holds significant public health importance. C. jejuni enters viable but non-culturable (VBNC) state in response to various environmental stressors, including low temperatures and nutrient starvation. VBNC cells are undetectable using conventional culture-based methods. Propidium monoazide (PMA) treatment allows the sole detection of viable cells. Detection of VBNC C. jejuni cells using digital PCR (dPCR) has not been reported previously. Therefore, we compared the quantitative PCR (qPCR) and dPCR methods to establish a novel condition for effectively detecting viable C. jejuni cells. Culturable, dead, and VBNC cells were prepared by spiking 25 g of chicken meat for two rounds of PMA treatment. Additionally, PMA enhancer, a substance that enhances the differentiation between viable and dead gram-negative bacteria, was used along with PMA treatment. VBNC cell was suppressed by PMA enhancer treatment in PMA-qPCR. Dead cell detection was successfully suppressed using PMA treatment alone; however, viable cells were undetected under 1 × 10⁴ colony-forming unit (CFU) per 25 g using PMA-qPCR. In contrast, dPCR successfully detected viable, including VBNC, in all concentrations tested, and dead cell was effectively suppressed under 1 × 10⁵ CFU/25 g. To the best of our knowledge, this is the first report on VBNC C. jejuni cell detection using PMA-dPCR.