Research on detection of Legionella pneumophila by real-time fluorescence PCR / 中华微生物学和免疫学杂志

ABSTRACT

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 degen-erated primers and one TaqMan-MGB probe were designed to test the conserved region at the macrophage in-fective 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 dif-ferent strains. Results The detection limit of 0.71 copy/μl was obtained for the standard sample in a reac-tion system of 0.6μl of sense and antisense primers (20μmol/L), respectively, 0.4μl of probe (20μmol/L) and 6.0μl of DNA temple. Conditions for the PCR reactions were as follows. After an initial de-naturation at 95℃ for 20s, 40 amplification cycles were performed. Each cycle consisted of denaturation at 95℃ for 10 s, primer annealing at 50℃ for45s. 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.