利用荧光素原位分子杂交检验水样中嗜肺军团菌的研究  

Detection of Legionella pneumophila in water samples by fluorescent in situ hybridization for 16S rRNA and mip gene

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

机构地区：[1]西安市疾病预防控制中心,西安710054

出　　处：《中国卫生检验杂志》2007年第4期609-611,625,共4页Chinese Journal of Health Laboratory Technology

基　　金：陕西省自然科学基金资助项目(2006C223)

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-巨噬细胞感染增强因子（mip）序列探针原位分子杂交建立嗜肺军团菌的快速、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和mip探针，采用荧光素标记探针对采自西安市8家宾馆中央空调冷凝水、贮水池水各8份进行原位分子杂交，并且结合细菌分离、生化鉴定技术检验水样中嗜肺军团菌。结果：细菌分离鉴定表明2份冷凝水、5份贮水池水分离到可疑菌落，抗体凝集实验证实嗜肺军团菌血清Ⅰ型3份、Ⅴ、Ⅵ型各1份，2份阴性。实验周期约10～12d；原位分子杂交显示16SrRNA、mip的探针杂交信号成堆分布于原虫细胞内，形如杆菌状，16SrRNA与mip的杂交信号存在完全双标记，但有少数mip的杂交信号不与16SrRNA共存，5株鉴定为嗜肺军团菌Ⅰ、Ⅴ、Ⅵ型的水样原虫中，经16SrRNA、mip探针杂交均为阳性，对2株非嗜肺军团菌水样原虫进行16SrRNA、mip的杂交结果均为阴性。实验周期约20h。结论：结果表明该方法适用于对存在于原虫内致病性嗜肺军团菌进行检验，是种快速、特异性的致病性嗜肺军团菌检验方法。Objective： To develop a rapid identification and sensitive detection method for Legionella pneumophila （L pneumophila） in water samples. Methods ：Based on comparative sequence analysis, we designed oligonucleotide probes complementary to a region of 16S rRNA or macrophage infection potentiator （mip） gene sequences of L. pneumophila respectively, which allows the differentiation of L pneumophila from other impathogenic L species without cultivation, detected L pneumophila in 16 water samples（8 instore whirlpool spa water samples, 8 cooling tower water samples from 8 hostels in Xi＇an）using fluorescent in situ hybridization （FISH） with bacteria isolation and cultivation, biochemical identification methods. All probes used in FISH were synthesized commercially and 5＇ - end - labelled with fluorescein texas - red, providing a red signal or with fluorescein isothiocyanate （ FITC）, providing a green signal. Results： By bacteria cultivation, L. pneumophila was found in 2 cooling tower water samples and 3 in - store whirlpool spa water samples and verified as serogroup Ⅰ（3）, Ⅴ（ 1 ）, Ⅵ（ 1 ） respectively by a direct antibody agglutination test. By FISH, the strong red fluorescent signal of L. pneumophila specific 16S rRNA probe could be detected to be contained in protozoan and showed as bacillus. Almost all red fluorescent signal was double labeled with green fluorescent signal of the probe targeted mip of L. pneumophila. In 5 water samples contaminated by L. pneumophila serogroup Ⅰ, Ⅴ, Ⅵ respectively, Lpneumophila could be detected by FISH. This assay allowed us to quantify the Lpneumophila contamination in water samples within 20 h. Conclusion： This new method appears fast and reliable and may be useful for the rapid detection for L. pneumophila in water samples without cultivation.

关 键 词：嗜肺军团菌 原位分子杂交 16SrRNA 巨噬细胞感染增强因子 鉴定 

分 类 号：R517.9[医药卫生—内科学]