摘要
目的：设计一种弹回引物荧光PCR方法鉴定军团菌属和嗜肺军团菌。方法通过分析军团菌16S rRNA基因序列，采用生物信息学方法设计引物，优化实验条件，对方法的特异性、灵敏度进行评估，并对186株环境军团菌分离株与15份环境水样进行鉴定。结果经弹回引物检测，军团菌属菌株在85℃～86℃处有扩增子熔解峰，嗜肺军团菌在71℃处有探针结合区熔解峰，非军团菌未检测到熔解峰。弹回引物对标准菌株DNA与模拟水样灵敏度分别为1 ng／μl与（1×10^3～1×10^4）／ml。弹回引物对环境分离株验证实验中，成功鉴定了186株军团菌和44株嗜肺军团菌；对15份环境水样直接检测，检出12份军团菌属阳性水样与4份嗜肺军团菌阳性水样。结论弹回引物荧光PCR方法可用于军团菌属和嗜肺军团菌的鉴定，具有较高的特异性与灵敏度。
Objective To test a snapback primer for the identification of Legionella and Legionella pneumophila （ L.pneumophila） strains in a one-step real-time PCR assay.Methods A novel primer was designed with a pair of genus-specific primers of Legionella strains.The species-specific probe sequences of L.pneumophila strains were linked at the 5′end of the reverse primer.The sensitivity and specificity of the novel PCR assay were tested with 43 types of Legionella and non-Legionella strains.The established PCR as-say was used to identify 186 wild Legionella strains isolated from 11 provinces of China and 15 environmental water samples.Results The amplicon melting peak of Legionella strains was detected at 85-86℃.The snapback melting peak of L.pneumophila was detected at 71℃.No melting peak of non-Legionella strains was detected.The sensitivity of the standard strains and simulated water samples were 1 ng/μl DNA tem-plates and （1×10^3-1×10^4 ）/ml, respectively.186 Legionella strains and 44 L.pneumophila strains isolated from environmental water samples were successfully identified with the snapback primer.Twealve Legionella strains and 4 L.pneumophila strains were identified from 15 environmental water samples with the snapback primer as compared with 8 Legionella strains identified with the culture method.Conclusion The snapback primer mediated one-step PCR assay could be used for the identification of Legionella and L.pneumophila strains with the advantages of high specificity and sensitivity.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2015年第2期121-126,共6页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目（31300004）
关键词
军团菌
嗜肺军团菌
弹回引物
荧光PCR
Legionella
Legionella pneumophila
Snapback primer
Real-time PCR
