摘要
目的建立一种特异、快速、敏感,同时能区分嗜肺和非嗜肺军团菌的荧光定量PCR方法,用于检测临床和环境样品。方法利用军团菌属特异性23S rRNA基因和嗜肺军团菌种mip基因的保守序列,设计引物和TaqMan探针。提取嗜肺军团菌标准菌株(LP1)DNA,分别构建2个含有目的基因的标准质粒作为阳性模板。优化荧光PCR反应条件和反应体系,对方法的特异性、敏感性、重复性进行评价。并对环境水样及80份临床痰样进行检测。结果该方法检测灵敏度达10标准质粒拷贝数/反应,具有高度特异性、稳定性。整个过程约需2 h。对35份环境水样进行检测,检出3株嗜肺军团菌和3株非嗜肺军团菌,所有水样经传统分离培养法和普通PCR法检测均显示为阴性。对临床80份随机痰液标本进行检测,2份嗜肺军团菌阳性。结论 TaqMan探针荧光定量PCR双管检测是一种可同时区分嗜肺与非嗜肺军团菌的特异、敏感、稳定的方法,可用于基层医院对环境标本及临床痰样的检测。
Objectives To develop a specific,rapid and sensitive real-time fluorescence quantitative PCR assay for the detection of legionella pneumophila( LP) and for the differentiation of it from other legionella spp. in clinical and environmental samples. Methods The primers and TaqMan probes were designed in conservative sequence of LP mip gene and all legionella spp. 23S rRNA gene. Fragments were amplified from the standard strain of LP serotype I by PCR to construct 2 plasmid standards. The PCR reaction conditions and system were optimized. The specificity,sensitivity and reproducibility of the assay were evaluated. Some environmental samples and 80 clinical sputum samples were detected. Results The sensitivity of this real-time PCR assay was 10 copies / reaction. This assay proved to be specific and reproducible. The whole process could be done in about 2 hours. Of the 35 environmental samples for testing legionella,3 were found to be positive for LP and 3 were found to be positive for other legionella spp. All environmental samples tested by both ordinary PCR and culture method were negative. Of 80 clinical sputum samples detected by this assay,2 were found to be positive for LP,and all others were negative. Conclusions The study has developed a highly sensitive and specific TaqMan real-time PCR assay,which can detect both legionella at the genus level and LP. This assay could be used for detecting clinical and environmental samples in small hospitals.
出处
《环境卫生学杂志》
北大核心
2014年第1期76-80,共5页
JOURNAL OF ENVIRONMENTAL HYGIENE
