Comparison of Most Probable Number-PCR and Real-Time PCR Methods for Quantitative Detection of Listeria Monocyotgenes in Milk

Detection and enumeration of Listeria monocytogenes as a majorfood-borne pathogen are of increasing interests, particularly after recent occasional outbreaks. Conventional culture based techniques for detection of L. monocytogenes are laborious, time consuming and are not very sensitive. To evaluate other quantitative methods which give earlier results two methods, Most Probable Number-Polymerase Chain Reaction (MPN-PCR1) and real-time PCR, have been evaluated by using milk as matrix media.Materials and methods:From pure cultures of L. monocytogenesand and 8 other background bacteria approximately to 107 and 101 CFU/ml suspensions were prepared. The 3 test tubes MPN sets were prepared from 101 CFU/ml of bacterial mix suspension in listeria enrichment broth. MPN sets were prepared. After incubation turbid MPN tubes were subjected to DNA isolation and applied to PCR (MPN-PCR). Ten-fold dilutions were prepared from genomic DNA isolate from bacterial mix suspension and L. monocytogenesstock suspension (corresponding approximately to 107-10-2 genomic DNA copy/ml) for real-time PCR assay as testing samples and standard samples respectively. MPN-PCR and real-time PCR assays were conducted in triplicate using appropriate primers for the prfAgene.Results:The detection limit of MPN-PCR was estimated as 101 CFU/ml. These estimations were more precise than real-time PCR results which just could detect bacteria in concentrations of ≥104 bacteria per milliliter.Conclusion:According to the findings of this study MPN-PCR can be used both as a detection and also an enumeration method to assess the presence of L. monocytogenes in milk samples.

کلیدواژه ها:

L. monocytogenes ، MPN-PCR ، real-time PCR