Detection of Salmonella in Food Based on PMA-ddPCR

Abstract:

The research aimed to establish a fast method to detect of live Salmonella in food with high sensitity. Propidium monoazide（PMA） permeated in the injured cells，then PMA and DNA conducted covalent cross-linking reaction，which could inhibit PCR amplification. Finally，bacterial genome DNA was extracted to detect by ddPCR. The optimization of experimental parameters showed that a final PMA concentration of 40.0 μg/mL and exposure of 15 min could restrain DNA amplification of 105 CFU/mL dead cells ，and the maximum PMA against DNA amplification from live Salmonella cells was 50.0 μg/mL. Under the different live/dead bacteria proportion，only live Salmonella cells was detected by PMA-ddPCR even in the existence of dead Salmonella cells. The detection limit was 8.0 copy/20 μL. PMA-ddPCR could detect 102 CFU/mL Salmonella cells in the codfish polluted by manual work. Furthermore，PMA-ddPCR showed better accuracy and stability. In conclusion，PMA-ddPCR showed huge potential in foodborne pathogenic bacteria detection.