Kenny John H, Zhou Yan, Schriefer Martin E, Bearden Scott W
Bacterial Diseases Branch, Division of Vector-Borne Infectious Diseases, National Center for Zoonotic, Vector-Borne and Enteric Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521, United States.
J Microbiol Methods. 2008 Oct;75(2):293-301. doi: 10.1016/j.mimet.2008.06.021. Epub 2008 Jul 3.
A successful method has been developed for the detection of live Yersinia pestis, the plague bacillus, which incorporates nascent RNA synthesis. A fluorescent in situ hybridization (FISH) assay using peptide nucleic acid (PNA) probes was developed specifically to differentiate Y. pestis strains from closely related bacteria. PNA probes were chosen to target high copy mRNA of the Y. pestis caf1 gene, encoding the Fraction 1 (F1) antigen, and 16S ribosomal RNA. Among Yersinia strains tested, PNA probes Yp-16S-426 and Yp-F1-55 exhibited binding specificities of 100% and 98%, respectively. Y. pestis grown in the presence of competing bacteria, as might be encountered when recovering Y. pestis from environmental surfaces in a post-release bioterrorism event, was recognized by PNA probes and neither hybridization nor fluorescence was inhibited by competing bacterial strains which exhibited faster growth rates. Using fluorescence microscopy, individual Y. pestis bacteria were clearly differentiated from competing bacteria with an average detection sensitivity of 7.9x10(3) cells by fluorescence microscopy. In the current system, this would require an average of 2.56x10(5) viable Y. pestis organisms be recovered from a post-release environmental sample in order to achieve the minimum threshold for detection. The PNA-FISH assays described in this study allow for the sensitive and specific detection of viable Y. pestis bacteria in a timely manner.
已开发出一种成功的方法来检测鼠疫杆菌（即鼠疫耶尔森菌）的活菌，该方法涉及新生RNA合成。专门开发了一种使用肽核酸（PNA）探针的荧光原位杂交（FISH）检测方法，以区分鼠疫耶尔森菌菌株与密切相关的细菌。选择PNA探针靶向鼠疫耶尔森菌caf1基因的高拷贝mRNA，该基因编码第1组分（F1）抗原以及16S核糖体RNA。在所测试的耶尔森菌菌株中，PNA探针Yp - 16S - 426和Yp - F1 - 55的结合特异性分别为100%和98%。在存在竞争细菌的情况下生长的鼠疫耶尔森菌（例如在生物恐怖袭击事件后从环境表面回收鼠疫耶尔森菌时可能遇到的情况），能被PNA探针识别，且杂交和荧光均不受生长速度更快的竞争菌株的抑制。使用荧光显微镜，单个鼠疫耶尔森菌能与竞争细菌清晰区分，荧光显微镜的平均检测灵敏度为7.9×10³个细胞。在当前系统中，这要求从生物恐怖袭击事件后的环境样本中平均回收2.56×10⁵个活的鼠疫耶尔森菌生物体，以达到最低检测阈值。本研究中描述的PNA - FISH检测方法能够及时、灵敏且特异检测活的鼠疫耶尔森菌。
