Xu Yu, Chen Wanyi, You Chunping, Liu Zhenmin
State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Synergetic Innovation Center for Food Safety and Nutrition, Dairy Research Inst., Bright Dairy & Food Co, Ltd, 1518 West of Jiangchang Road, Shanghai 200436, P. R. China.
J Food Sci. 2017 Oct;82(10):2337-2342. doi: 10.1111/1750-3841.13845. Epub 2017 Sep 26.
Under the cold storage and processing conditions of raw milk, the psychrotrophic Pseudomonas fluorescens is usually found as predominant bacteria causing its spoilage. In this study, a multiplex PCR assay was developed for rapid and selective detection of P. fluorescens with biofilm formation ability. The target sequences were 2 genes (adnA and 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 Pseudomonas species (Pseudomonas sp1 and Pseudomonas sp8). The detection limit for the target strain was 10 CFU/mL. Seventy-three strains were evaluated by the mPCR assay. The adnA gene was detected in 23 strains while fliC gene was detected in only 3 strains. However, both target genes (adnA and fliC) were detected by amplification in 12 strains belonging to P. fluorescens species. The biofilm formation ability of P. fluorescens following 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. fluorescens strains with the target gene (adnA or fliC, or both 2 genes) had the biofilm-forming ability. The phylogenetic analysis showed that adnA gene tree had a higher resolution than rpoB tree, and the strains in adnA phylogenetic dendrogram could be divided into 4 different groups according with the matrix of their biofilm-forming ability. The results indicated a promising use of adnA gene as a taxonomic marker for subdividing P. fluorescens.
A mPCR assay targeting adnA and fliC genes showed rapid and reliable detection of P. fluorescens with biofilm formation ability, which could be useful to detect this contamination in milk samples.
在生乳的冷藏和加工条件下,嗜冷的荧光假单胞菌通常是导致其变质的主要细菌。在本研究中,开发了一种多重PCR检测方法,用于快速、选择性地检测具有生物膜形成能力的荧光假单胞菌。靶序列是与荧光假单胞菌生物膜形成和鞭毛生物合成相关的2个基因(adnA和fliC)。使用从生乳中分离的7株参考菌株评估了多重PCR检测方法的特异性,这些菌株分别属于荧光假单胞菌、脆弱假单胞菌、伦氏假单胞菌、恶臭假单胞菌、蒙氏假单胞菌以及2种未分类的假单胞菌(假单胞菌sp1和假单胞菌sp8)。靶菌株的检测限为10 CFU/mL。通过多重PCR检测方法评估了73株菌株。在23株菌株中检测到了adnA基因,而仅在3株菌株中检测到了fliC基因。然而,通过扩增在12株属于荧光假单胞菌属的菌株中检测到了两个靶基因(adnA和fliC)。通过微量滴定板试验评估了荧光假单胞菌在30℃或4℃的10%超高温灭菌乳中培养后的生物膜形成能力。结果表明,所有具有靶基因(adnA或fliC,或两个基因)的荧光假单胞菌菌株都具有生物膜形成能力。系统发育分析表明,adnA基因树的分辨率高于rpoB树,并且根据其生物膜形成能力的矩阵,adnA系统发育树状图中的菌株可分为4个不同的组。结果表明,adnA基因有望作为细分荧光假单胞菌的分类标记。
针对adnA和fliC基因的多重PCR检测方法显示,能够快速、可靠地检测具有生物膜形成能力的荧光假单胞菌,这对于检测牛奶样品中的这种污染可能是有用的。