实时荧光PCR快速检测嗜肺军团菌的研究

Research on detection of Legionella pneumophila by real-time fluorescence PCR

目的建立TaqMan-MGB探针实时荧光PCR快速检测嗜肺军团菌技术，为临床和环境样品检测嗜肺军团菌提供可实用工具。方法在对嗜肺军团菌mip序列进行分析、比较基础上，设计一对特异性引物和TaqMan—MGB探针，通过实时荧光PCR反应条件和反应体系的优化，实现对嗜肺军团菌的快速检测；用克隆到pMD-19T载体上的嗜肺军团菌mip基因阳参片段和不同菌株验证方法的敏感性和特异性。结果当用热裂解法提取DNA，25μl的反应体系中包括上、下游引物（20μmoL／L）各0．6μl，探针（20μmol／L）0．4μl，模板DNA6．0μl，反应条件为预变95℃20s，变性95℃10s，退火50℃40s，40个循环时，TaqMan—MGB探针实时荧光PCR技术对嗜肺军团菌mip基因阳参片段最低检测浓度为0．71拷贝μl，其循环阈值（Ct值）与模板浓度具有极好的对应关系（r=0．999）；1株嗜肺军团菌标准株、12株嗜肺军团菌分离株的Ct值在13．23—16．04之间，而包括金黄葡萄球菌、鼠伤寒沙门菌、副溶血性弧菌、大肠埃希菌、铜绿假单胞菌、痢疾志贺菌共计76株其他菌PCR Ct值均大于30；整个检测过程仅需1．5h。结论TaqMan-MGB探针的嗜肺军团菌实时荧光PCR检测方法具有特异性和敏感性、易操作、结果准确可靠等优点，可用于嗜肺军团菌检测。

Objective To develop a real-time fluorescence quantitative PCR method for detection of Legionella pneumophila as a tool for environmental and clinical examination. Methods A pair of degenerated primers and one TaqMan-MGB probe were designed to test the conserved region at the macrophage infective potentiation （mip） gene of Legionella pneumophila. TaqMan MGB real-time quantitative PCR assay was developed with pMD-19T plasmid including mip gene of Legionella pneumophila as standard sample. The sensitivity and specificity of the real-time quantitative PCR was evaluated using the standard sample and different strains. Results The detection limit of 0.71 copy/μl was obtained for the standard sample in a reaction system of 0.6 μl of sense and antisense primers （20 μmoL/L）, respectively, 0.4 μl of probe （20 μmol/L） and 6.0 μl of DNA temple. Conditions for the PCR reactions were as follows. After an initial denaturation at 95℃ for 20 s, 40 amplification cycles were performed. Each cycle consisted of denaturation at 95℃ for 10 s, primer annealing at 50℃ for 45 s. The PCR Ct value of a standard strain and 12 isolates was in a scale of 13.23 and 16.04. However, the Ct values of the strains of Staphylococcus aureus, Salmonella typhimurium, Vibrio parahaemolyticus, Escherichia coli, Pseudomonas aeruginosa and Shigella sonnei were greater than 30. Conclusion The real-time quantitative PCR method has good sensitivity and specificity and the result has potentiality of applying for detecting Legionella pneumophila.