Rapid and accurate flow cytometric enumeration of viable Listeria monocytogenes in beef via propidium monoazide and fluorescent molecular probe

Listeria monocytogenes ( L. monocytogenes ) is a significant foodborne pathogen worldwide and is associated with the highest fatality rate. Cows serve as asymptomatic carriers of L. monocytogenes , with 45.6% of beef carcasses testing positive for L. monocytogenes . Therefore, a rapid and accurate method to quantify L. monocytogenes cells in beef is urgently needed. This study presents a flow cytometry (FCM)-based method to rapidly and accurately enumerate L. monocytogenes without the need for cultivation. Initially, propidium monoazide (PMA) was used to recognize viable cells. A fluorescent molecular probe (FMP) was subsequently utilized to selectively identify L. monocytogenes cells. Finally, viable L. monocytogenes cells were visually and accurately enumerated by FCM. Compared with the plate-based method, the PMA-FMP-FCM method could complete pretreatment of beef samples, dual staining of viable L. monocytogenes , and FCM analysis within 2.5 h, demonstrating superior efficiency. The recovery rate of L. monocytogenes was 96.25%, indicating good repeatability and a strong linear correlation with the plate-based method ( R 2 = 0.9994). This is the first study to combine FCM with PMA and FMP to target L. monocytogenes , which provided proof of concept for the application of this assay to other microorganisms. The established method has great potential for application in the quality monitoring of beef samples. • Flow cytometry was first combined with PMA and FMP for counting L. monocytogenes . • CM was innovatively used as a reaction promoter of FMP-FCM in bacterial assay. • PMA-FMP-FCM method holds the shortest TTR for enumerating viable L. monocytogenes . • FMP could specifically recognize common L. monocytogenes serotypes found in food.