Resumen de Development of a Multiplex PCR Assay for Detection of Pseudomonas fluorescenswith Biofilm Formation Ability
Yu Xu, Wanyi Chen, Chunping You, Zhenmin Liu
Under the cold storage and processing conditions of raw milk, the psychrotrophic Pseudomonas fluorescensis usually found as predominant bacteria causing its spoilage. In this study, a multiplex PCR assay was developed for rapid and selective detection of P. fluorescenswith biofilm formation ability. The target sequences were 2 genes (adnAand fliC) related to biofilm formation and flagella biosynthesis of P. fluorescens. The specificity of the mPCR assay was evaluated with 7 reference strains, isolated from raw milk, belonging to P. fluorescens, Pseudomonas fragi, Pseudomonas lundensis, Pseudomonas putida, Pseudomonas monteilii, and 2 unclassified Pseudomonasspecies (Pseudomonassp1 and Pseudomonassp8). The detection limit for the target strain was 102CFU/mL. Seventy‐three strains were evaluated by the mPCR assay. The adnAgene was detected in 23 strains while fliC gene was detected in only 3 strains. However, both target genes (adnAand fliC) were detected by amplification in 12 strains belonging to P. fluorescensspecies. The biofilm formation ability of P. fluorescensfollowing cultivation in 10% UHT milk at 30 °C or 4 °C were evaluated by the microtiter plate assay. The result showed that all the P. fluorescensstrains with the target gene (adnAor fliC, or both 2 genes) had the biofilm‐forming ability. The phylogenetic analysis showed that adnAgene tree had a higher resolution than rpoBtree, and the strains in adnAphylogenetic dendrogram could be divided into 4 different groups according with the matrix of their biofilm‐forming ability. The results indicated a promising use of adnAgene as a taxonomic marker for subdividing P. fluorescens. A mPCR assay targeting adnAand fliCgenes showed rapid and reliable detection of P. fluorescenswith biofilm formation ability, which could be useful to detect this contamination in milk samples.
