Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR
Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR
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DOI：
10.1128/aem.63.4.1268-1273.1997
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发表时间：
1997-04-01
影响因子：
4.4
通讯作者：
Meile, L
中科院分区：
文献类型：
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作者：
Kaufmann, P;Pfefferkorn, A;Meile, L
在16S rRNA的V9变量区域中的双歧杆菌特异性靶序列已详细阐述，并用于开发杂交探针，该探针的特异性，称为LM3（5'-CGG GTGCTI*CCCATTTCATTCATG-3'），WAS，WAS，用于识别所有已知类型菌株并将其与其他细菌区分开，所有30种类型的菌株在德国培养物收藏中可用的双歧杆菌Deutsche Sammlung von Mikroegranismen和Zellkulturen，6种市售的生产菌株，以及34个密切相关的相关菌株（负面对照）（作为负面对照）均已测试，所有测试的双杆菌均均显示出不同的双杆菌，均通过群体造成的阳性信号，而群体均为所有群体，所有测试的阳性信号控制措施除了阴道Gardnerella之外没有明显的点DSM4944和弗洛德氏菌杆菌Shermanii DSM4902，它发出了轻微的信号，此外，我们还建立了一种方法，我们通过使用PCR分析了PCR分析，而无需隔离DNA，但使用这种蛋白酶K，通过这种方法隔离细胞，从而从食物中隔离和鉴定食物双歧杆菌，但使用这种方法。所有双歧杆菌菌株导致预期的DNA产物大小，我们还建立了一个快速测定，以通过稀释镀层和菌落杂交定量地测量食物和粪便中的双歧杆菌计数，我们能够证明2.1 x 10（6）至2.3 x 10（7）含有含有酸奶的牛奶与特异性核苷酸探针杂交的双歧杆菌与这两种方法：特异性属菌落杂交和现在可以轻松，准确地检测出食物和粪便样品中的任何双歧杆菌，并区分它们和其他属成员之间的任何双歧杆菌。
A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe, The specificity of this probe, named lm3 (5'-CGG GTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria, All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested, All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp, shermanii DSM4902, which gave slight signals, Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K, By this method, all Bifidobacterium strains lead to a DNA product of the expected size, We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization, We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe, With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera.