Quantitative detection of Corynebacterium casei in cheese by real-time PCR
Quantitative detection of Corynebacterium casei in cheese by real-time PCR
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DOI：
10.1128/aem.01303-06
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发表时间：
2006-11-01
影响因子：
4.4
通讯作者：
Irlinger, Francoise
中科院分区：
文献类型：
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作者：
Monnet, Christophe;Correia, Karine;Irlinger, Francoise
涂抹成熟干酪表面的菌群由许多种类的细菌和酵母菌组成，它们有助于产生所需的感官特性。由于缺乏选择性培养基，很难对干酪表面细菌进行定量，因此，干酪表面微生物区系的生态学还没有得到广泛的研究。我们开发了一种SYBR green I实时荧光聚合酶链式反应方法来定量检测干酪棒状杆菌，干酪棒状杆菌是一种主要的涂片成熟干酪品种，使用针对16S rRNA基因的引物设计。从干酪基质中回收干酪假单胞菌基因组DNA的产率与从液体培养物中通过离心法回收干酪乳杆菌基因组DNA的得率几乎相同。定量在每克奶酪10(5)到10(10)cfu的范围内是线性的。用干酪假单胞菌相关物种的DNA以及奶酪菌群中的其他细菌和酵母菌的DNA证明了该方法的特异性。对9种商品干酪进行了实时荧光定量PCR分析，其中6种干酪的菌落总数超过105个菌落当量，其中2种干酪的菌落总数占菌群总数的比例接近40%。通过单链构象多态分析和平板计数和16S rRNA基因测序相结合的方法进一步证实了这些样品中C-酪蛋白的存在。结论：SYBR green I实时荧光定量聚合酶链式反应可作为一种可靠的定量检测奶酪表面细菌的方法。
The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 10(5) to 10(10) CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 105 CFU equivalents of C casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses.