利用原位分子杂交结合酪酰胺放大检验军团菌性肺炎组织嗜肺军团菌的研究  

A research on detecting Legionella pneumophila in guinea pig model of L. pneumophila pneumonia by in situ molecular hybridization with tyramide signal amplification method

作　　者：吴守芝[1] 李恒新[1] 黄河清[1] 张金[1] 张锋[1] 

机构地区：[1]西安市疾病预防控制中心,陕西西安710054

出　　处：《现代预防医学》2013年第10期1910-1913,共4页Modern Preventive Medicine

基　　金：陕西省自然科学基金资助项目(2006C223);西安市支撑计划资助项目(YF07154)

摘　　要：目的利用针对嗜肺军团菌种特异性16SrRNA和军团菌致病基因—巨噬感染增强因子(mip)序列探针原位分子杂交结合酪酰胺放大技术(Tyramide signal amplification method,TSA)建立军团菌性肺炎组织中嗜肺军团菌的快速、特异性检验方法。方法设计特异性针对嗜肺军团菌种特异性16SrRNA、mip序列探针,分别采用荧光、地高辛标记探针对嗜肺军团菌性豚鼠肺炎模型支气管肺泡灌洗液、肺组织进行原位杂交结合TSA检验嗜肺军团菌。结果荧光原位杂交显示支气管肺泡灌洗液细胞中存在16SrRNA、mip序列双杂交阳性杆状细菌;地高辛标记原位杂交结合TSA显示肺组织中也存在杂交阳性杆状细菌,分布于肺泡壁与细胞内,整个检验周期小于24h。结论结果表明采用荧光、地高辛标记探针结合TSA的原位杂交能迅速准确灵敏检验出肺组织、支气管肺泡灌洗液中的嗜肺军团菌,是一种快速、特异性检验方法。OBJECTIVE To develop a rapid and sensitive method for identifying Legionella pneumophila （L. pneumophila） in clinical samples. METHODS Based on comparative sequence analysis, we designed oligonucleotide probes for a region of 16S rRNA or macrophage infection potentiator （mip） gene sequences of L. pneumophila respectively, which allowed the differentia- tion of L. pneumophila from other impathogenic L.species without cultivation, labeled these probes by fluorescein and digoxin respectively, detected L. pneumophila in bronchoalveolar lavage （BAL）, lung tissue by in situ hybridization with tyramide sig- nal amplification method. RESULTS Using fluorescent in situ hybridization with tyramide signal amplification method, we found that the strong red fluorescent signal specific for 16S rRNA probe could be detected to be contained in the macrophage of BAL and showed as bacillus in guinea pig model of L. pneumophila pneumonia. Almost all red fluorescent signal was double labeled with green fluorescent signal of the probe targeted mip of L. pneumophila. By the probe labeled with digoxin, we found that the signals of hybridization with the probe for mip were distributed in the cells of the lungs and showed as bacillus, the signals of hybridization with the probe for 16S rRNA were also distributed in the cells or alveoli of the lungs of guinea pigs inoculated with L. pneumophila. This assay allowed us to quantify the L. pneumophila after Legionella infection within 20h. CONCLUSION These results show this new approach appears fast and reliable and may be useful for the rapid detection for the L. pneumophila of Legionella pneumonia.

关 键 词：嗜肺军团菌 肺炎 支气管肺泡灌洗液 原位杂交 酪酰胺放大技术 

分 类 号：R115[医药卫生—公共卫生与预防医学]