Trebesius K, Harmsen D, Rakin A, Schmelz J, Heesemann J
Max-von-Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig Maximilians Universität München, D-80336 Munich, Germany.
J Clin Microbiol. 1998 Sep;36(9):2557-64. doi: 10.1128/JCM.36.9.2557-2564.1998.
In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiate Yersinia species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three different Yersinia species and partial 23S rDNA sequences for three Y. pestis and three Y. pseudotuberculosis strains were determined. While 16S rDNA sequences of Y. pestis and Y. pseudotuberculosis were found to be identical, one base difference was identified within a highly variable region of 23S rDNA. The rDNA sequences were used to develop primers and fluorescently tagged oligonucleotide probes suitable for differential detection of Yersinia species by PCR and in situ hybridization, respectively. As few as 10(2) Yersinia cells per ml could be detected by PCR with a seminested approach. Amplification with a subgenus-specific primer pair followed by a second PCR allowed differentiation of Y. enterocolitica biogroup 1B from biogroups 2 to 5 or from other pathogenic Yersinia species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenic Yersinia species (in particular Y. pestis and Y. pseudotuberculosis) was developed. The applicability of this technique was demonstrated by detection of Y. pestis and Y. pseudotuberculosis in spiked throat and stool samples, respectively. These probes were also capable of identifying Y. enterocolitica within cryosections of experimentally infected mouse tissue by the use of confocal laser scanning microscopy.
在本报告中，我们介绍了两种基于16S和23S rRNA序列数据的快速分子检测技术的详细信息，用于从临床和环境样本中鉴定和区分耶尔森菌属菌种。测定了三种不同耶尔森菌属菌种的近乎全长的16S rRNA基因（rDNA）序列以及三株鼠疫耶尔森菌和三株假结核耶尔森菌的部分23S rDNA序列。虽然发现鼠疫耶尔森菌和假结核耶尔森菌的16S rDNA序列相同，但在23S rDNA的一个高度可变区域内鉴定出一个碱基差异。这些rDNA序列被用于分别开发适用于通过PCR和原位杂交对耶尔森菌属菌种进行差异检测的引物和荧光标记寡核苷酸探针。采用半巢式方法通过PCR每毫升可检测到低至10²个耶尔森菌细胞。用亚属特异性引物对进行扩增，随后进行第二次PCR，可将小肠结肠炎耶尔森菌生物群1B与生物群2至5或其他致病性耶尔森菌属菌种区分开来。此外，还开发了一组适用于快速（3小时）原位检测和区分三种致病性耶尔森菌属菌种（特别是鼠疫耶尔森菌和假结核耶尔森菌）的寡核苷酸探针。通过分别在加标的咽喉和粪便样本中检测鼠疫耶尔森菌和假结核耶尔森菌，证明了该技术的适用性。通过使用共聚焦激光扫描显微镜，这些探针还能够在实验感染小鼠组织的冰冻切片中鉴定出小肠结肠炎耶尔森菌。
