Quantitative detection of viable but nonculturable state Escherichia coli O157:H7 by ddPCR combined with propidium monoazide

Escherichia coli O157:H7, the causative agent of haemorrhagic colitis and haemolytic uremic syndrome in humans, has been implicated in large food-borne outbreaks all over the world. When confronted with harsh environmental stresses, it can enter into a viable but nonculturable (VBNC) state, which poses a great risk to food safety and public health since conventional methods are invalid to detect VBNC cells. Herein, a system for detecting VBNC state Escherichia coli O157:H7 was established by combining droplet digital PCR (ddPCR) with propidium monoazide (PMA). This system took advantage of an absolute quantification without external calibration of ddPCR and DNA binding, modification capacity of PMA after penetrating into compromised cells, providing a sensitive detection limit of 5 copies/mu L, good accuracy and specificity. The PMA-ddPCR platform could be applied not only on detection of VBNC cells induced by non-thermal pasteurization technique, high pressure CO2 (HPCD), but also quantification of viable E. coli O157:H7 in food samples including prawn, squid, lettuce and pear juice. Taken together, our research provides an effective method for detecting VBNC pathogenic bacteria during food pasteurization process or in food samples.