Specific oligonucleotide probes for in situ detection of a major group of Gram-positive bacteria with low DNA G+C content
Specific oligonucleotide probes for in situ detection of a major group of Gram-positive bacteria with low DNA G+C content
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DOI：
10.1016/s0723-2020(99)80065-4
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发表时间：
1999-05-01
影响因子：
3.4
通讯作者：
Schleifer, KH
中科院分区：
文献类型：
--
作者：
Meier, H;Amann, R;Schleifer, KH
利用ARE软件对公共数据库中近千株DNA G+C含量较低的革兰氏阳性菌的16S rRNA序列进行了分析。G+C含量低的革兰氏阳性菌的芽孢杆菌分支在第354-371位(大肠杆菌编号)之间发现了一个标志区，前者是芽孢杆菌目和乳杆菌目。设计了三种寡核苷酸探针，即LGC354A、LGC354B和LGC354C，以此为靶点。它们的荧光衍生物适合于荧光原位杂交(FISH)全细胞检测。调整杂交条件，以区分目标和相关的非目标参考物种。当将FISH应用于公共污水处理厂活性污泥样本中的整个细菌细胞时，在用多聚甲醛固定的样本中检测到0.01%的细胞，在用乙醇固定并用溶菌酶处理的相同样本中检测到超过8%的细胞。讨论了生物膜絮体中DNA G+C含量低的革兰氏阳性菌原位定量分析中存在的问题，并提出了建议。在来自不同污水处理厂的活性污泥样本中检测到不同数量的芽孢杆菌亚支成员。
Almost one thousand 16S rRNA sequences of Gram-positive bacteria with a low DNA G+C content from public databases were analyzed using the ARE software package. A signature region was identified between positions 354 and 371 (E. coli numbering) for the Bacillus sub-branch of the Gram-positive bacteria with a low DNA G+C content, the former orders Bacillales and Lactobacillales. Three oligonucleotide probes, namely LGC354A, LGC354B, and LGC354C, were designed to target this diagnostic site. Their fluorescent derivatives were suitable for whole cell detection by fluorescence in situ hybridization (FISH). Hybridization conditions were adjusted for differentiation of target and related non-target reference species. When applying FISH to whole bacterial cells in a sample of activated sludge from a communal wastewater treatment plant, members of the Bacillus sub-branch were detected at levels from 0.01% of cells in samples fixed with paraformaldehyde to over 8 percent in the same samples fixed with ethanol and treated with lysozyme. The problems of quantitative in situ analysis of Gram-positive bacteria with a low DNA G+C content in biofilm flocs are discussed and recommendations made. Members of the Bacillus sub-branch were detected in different abundances in activated sludge samples from different wastewater plants.