长滩军团菌荧光原位杂交探针的设计研究  

Development of novel FISH probe for detection of Legionella longbeachae

作　　者：顾全[1] 胡朝晖[1] 赵利伟[1] 范晓姗 严慧[1] 朱庆义[1] 

机构地区：[1]广州金域医学检验中心有限公司科研开发部,广东广州510330

出　　处：《环境与健康杂志》2014年第1期47-50,共4页Journal of Environment and Health

基　　金：国家自然科学基金面上项目(31270049);国家自然科学基金青年基金(31300004)

摘　　要：目的设计一条特异性的长滩军团菌荧光原位杂交探针,并验证其特异性与敏感度,初步建立长滩军团菌的荧光原位杂交检测法。方法以长滩军团菌16S rRNA特异性位点设计杂交探针。在数据库中对探针序列进行特异性检验,优化反应条件,以军团菌标准菌株和环境分离菌株验证探针的特异性,并以模拟水样验证探针灵敏度。结果生物信息学分析表明,设计的探针LEG long序列与长滩军团菌16S rRNA序列完全配对,与其他军团菌种均存在1个碱基以上的错配。菌株验证实验证明LEG long探针可与长滩军团菌的标准菌株和环境分离株特异性杂交,与其他军团菌种和非军团菌属菌株无杂交信号。探针检测灵敏度可达1.49×103 CFU/ml。结论本研究设计的探针LEG long可用于长滩军团菌的高特异性和高灵敏度检测。Objective To design a fluorescence in situ hybridization （FISH） probe, named LEG long, targeted on 16S rRNA, to differentiate L.longbeachea from Legionella.spp. Methods The FISH probe, which targeted the particular loci of L.longbeachea 16S rRNA, was submitted to RDP database and ProbeCheck database to test the probe specificity. Then, the sensitivity and specificity of the probe was tested with several type strains of Legionella and non-Legionella and some wild strains isolated by our laboratory. Results The bioinformatics analysis indicated the probe LEG long completely matched the 16S rRNA sequences of L.longbeachea, and mismatched the other Legionella specie at last one base.The experimental results were consistent with the conclusion of bioinformatics analysis. All type strains and wild strains of L.longbeachea could hybridize with the probe and emit fluorescence. Hybridization signal was not detected when probe hybridized with non-L.longbeachea and non-Legionellca The lowest detection limit of LEG long probe was 1.49×10^3 CFU/ml. Conclusions The FISH probe designed in the present study is applicable to the identification of L.longbeachea specifically.

关 键 词：长滩军团菌 荧光原位杂交 探针 

分 类 号：R117[医药卫生—公共卫生与预防医学]