USE OF A SIMPLIFIED CELL BLOT TECHNIQUE AND 16S RIBOSOMAL-RNA-DIRECTED PROBES FOR IDENTIFICATION OF COMMON ENVIRONMENTAL ISOLATES
USE OF A SIMPLIFIED CELL BLOT TECHNIQUE AND 16S RIBOSOMAL-RNA-DIRECTED PROBES FOR IDENTIFICATION OF COMMON ENVIRONMENTAL ISOLATES
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DOI：
10.1128/aem.59.10.3219-3224.1993
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发表时间：
1993-10-01
影响因子：
4.4
通讯作者：
NIERZWICKIBAUER, SA
中科院分区：
文献类型：
--
作者：
BRAUNHOWLAND, EB;VESCIO, PA;NIERZWICKIBAUER, SA
一种简单的技术，其中rRNA靶向的寡脱氧核苷酸探针用于识别固定在膜上的细菌。通过使用菌落升降机，细菌直接从平板转移到未处理的硝酸纤维素膜上。或者，可以通过在高盐条件下使用真空歧管将从集落重悬的细胞施加到膜上。通过使用标准方案在严格条件下烘烤和杂交印迹细胞。用十二烷基硫酸钠、尿素、甲醛或蛋白酶处理印迹细胞对杂交信号没有明显影响。与冷藏储存6天的细胞的rRNA杂交很容易检测到;然而，需要5倍多的细胞（约10（7）个细胞）才能获得与未储存细胞产生的杂交信号相当的杂交信号。提供了洋葱假单胞菌、睾丸酮丛毛单胞菌和醋酸钙不动杆菌特异性寡核苷酸探针的序列，以及鉴定铜绿假单胞菌、门多萨假单胞菌、荧光假单胞菌、食酸丛毛单胞菌和“黄杆菌”的组探针。结合殖民地举升杂交法，对从河水中分离的细菌进行了鉴定。
A simple technique in which rRNA-targeted oligodeoxynucleotide probes are used to identify bacteria immobilized on membranes is described. By using colony lifts, bacteria are directly transferred from plates to untreated nitrocellulose membranes. Alternatively, cells resuspended from colonies can be applied to membranes by using a vacuum manifold under high-salt conditions. Blotted cells are baked and hybridized under stringent conditions by using standard protocols. Treatment of blotted cells with sodium dodecyl sulfate, urea, formaldehyde, or protease had no apparent effect on hybridization signals. Hybridization to rRNA from cells that had been stored refrigerated for 6 days was readily detected; however, fivefold more cells (approximately 10(7) cells) were required to obtain hybridization signals comparable to those generated by cells not subjected to storage. The sequences of oligonucleotide probes specific for Pseudomonas cepacia, Comamonas testosteroni, and Acinetobacter calcoaceticus and a group probe identifying Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas fluorescens, Comamonas acidovorans, and ''Flavobacterium'' lutescens are presented. In conjunction with the colony lift hybridization procedure, bacteria isolated from river water were identified by using these probes.