应用TaqMan荧光定量PCR检测土拉弗朗西斯菌

目的：利用Roche LightCycler实时定量PCR系统建立一种快速、灵敏、特异的检测土拉弗朗西斯菌的方法。方法：基于TaqMan荧光探针实时定量PCR技术,选择土拉弗朗西斯菌染色体上的特异序列醇醛酮还原酶（AKR）和外膜蛋白FopA基因]作为检测靶序列,建立土拉弗朗西斯菌实时定量PCR检测方法;评价该检测方法的特异性和灵敏性;采用克隆菌株污染环境土壤来模拟实际样品,评价该检测方法在快速检测与现场检测等实际应用中的表现。结果：优化筛选基因组中的FT-AKR和FT-fopA片段作为检测靶序列,所建立的土拉弗朗西斯菌实时定量PCR检测方法检测克隆菌株质粒的灵敏度均为10个拷贝/每个反应体系;以其他非土拉弗朗西斯菌为模板未出现非特异扩增;模拟环境土壤样品检测灵敏度2个引物对分别为440和960CFU/g土壤;盲测实验结果显示对于灵敏度范围内的阳性样本均能正确识别,并能正确检测出不同浓度的阳性样本。以FT-fopA片段为靶序列的扩增效率不及基于FT-AKR引物对的扩增。结论：基于FT-AKR片段的引物对扩增效率高,检测土拉弗朗西斯菌具有特异、灵敏的特点,对临床诊断、环境污染监测、防治生物突发事件等具有重要意义。

Establishment of Real-Time PCR for Detection of Francisella tularensis

Objective： To develop TaqMan-based real-time PCR systems for rapid and sensitive detection of Francisella tularensis using the Roche LightCycler. Methods： Based on the Roche LightCyeler real-time PCR technology, we designed TaqMan primers and fluorescent probes against specific sequences genes encoding aldo/keto reductase （AKR） and outer membrance associated protein FopA] within F.tularensis chromosome for detecting this pathogen. The specificity and sensi- tivity of this assay were investigated, respectively. The sensitivity was evaluated by serial dilution of the recombinant plas- mids; and the specificity by amplifying DNAs from F.tularensis or non-Ftularensis. The spiked soil samples with different concentrations of E.coli containing the corresponding recombinant plasmid were used to evaluate the PCR＇s feasibility in quantitative detection of target genes in soil samples. Results： The TaqMan real-time PCR assays were developed based on AKR and fopA genes, which can detect as few as 10 copies of cloned bacterial plasmid per reaction, whereas non- specific amplifications were not detected for non-F.tularensis templates. The two assays based on AKR and fopA genes could detect 440 and 960 CFU bacteria per gram spiked soil. A blind detection of the target genes in the spiked soil samples demonstrated the correct quantitative detection within the range of sensitivity. The amplification efficiency of fopA- based primers was higher than that of AKR-based primers. Conclusion： The TaqMan real-time PCR assay based on AKR primers could specifically and sensitively detect F.tularensis from soil samples with high efficiency. This assay would help greatly in cases of clinical diagnosis, environmental pollution monitoring and biohazard control.