Simultaneous Detection of spp. and Quantification of spp. in a Real-Time Duplex PCR: Myth or Reality?

In Europe, there is a process hygiene criterion for and on broiler carcasses after chilling. The criterion gives indicative contamination values above which corrective actions are required by food business operators. The reference methods for verifying compliance with the criterion for and are international standards EN ISO 6579-1 (2017) and EN ISO 10272-2 (2017), respectively. These methods are time-consuming and expensive for food business operators. Therefore, it would be advantageous to simultaneously detect spp. and quantify in the same analysis, using the same sample after the pre-enrichment step for recovery. A duplex PCR for detection and spp. enumeration was developed. Considering the method as a whole, the LOD and LOQ for enumeration were slightly over the limit of 3 log CFU/g set by the process hygiene criterion. A comparison of the duplex PCR method developed with the ISO method on artificially contaminated bacterial suspensions and on naturally contaminated samples demonstrated a good correlation of the results for enumeration when the duplex PCR was performed on samples taken before or after the pre-enrichment step, but revealed a slight bias with a large standard deviation resulting in widely spaced limits of agreement.