Development of a Multiplex PCR Assay for Detection of Pseudomonas fluorescenswith Biofilm Formation Ability

• Autores: Yu Xu, Wanyi Chen, Chunping You, Zhenmin Liu
• Localización: Journal of food science, ISSN 0022-1147, Vol. 82, Nº 10, 2017, págs. 2337-2342
• Idioma: inglés
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• Resumen

  • 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.