实时荧光PCR熔解曲线法快速鉴定嗜肺军团菌

Rapid identification of Legionella pneumophila using real-time PCR melt curve analysis

目的建立特异、快速、敏感的实时荧光PCR熔解曲线基因分型方法,并探讨该法在鉴定嗜肺军团菌中的应用价值。方法根据嗜肺军团菌16S rRNA基因保守序列设计引物和猝灭FRET探针建立实时荧光PCR熔解曲线方法,优化引物与探针浓度,用15株嗜肺军团菌、30株非嗜肺军团菌及7株其他病原菌标准菌株验证该方法的特异性、敏感性和重复性,用该法检测117例临床痰标本并进行基因测序。结果 15株不同血清型的嗜肺军团菌Tm值均为57℃;7株非嗜肺军团菌Tm值为43℃,1株Tm值为45℃;其余22株非嗜肺军团菌和7株其他病原菌均无明显Tm值。该方法最低检出限可达0.1 pg/μL。检测117例痰标本发现3株嗜肺军团菌,与PCR基因测序法检测结果一致。结论实时荧光PCR熔解曲线法可特异、快速和敏感地检测嗜肺军团菌,适用于临床痰标本的嗜肺军团菌检测。

Objective To develop a new method for the detection of Legionella pneumophila（L.Legionella） using melt curve based on real-time PCR.Methods The primers and fluorescence hybridisation probes were designed according to sequences of 16S rRNA gene of L.Legionella.The system and conditions of reaction were optimized and the specificity,sensitivity and repeatability of the method were verified by detecting 15 strains L.pneumophila,30 strains non-L.pneumophila and 7 strains other bacteria.117 sputum specimens were detected by melt curve and DNA sequencing.Results The melt curve showed that the Tm of 15 strains L.pneumophila were 57 ℃,the Tm of 7 strains non-L.pneumophila were 43 ℃,and the Tm of 1 strain non-L.pneumophila was 45 ℃.No significant Tm was found in 22 strains of non-L.pneumophila and 7 strains of other bacteria.The assay detection limit was 0.1 pg/μL.L.pneumophila was detectable in 3 of 117 sputum specimens from the patients with pneumonia which were consistent with those of DNA sequencing.Conclusion The method of melt curve based on real-time PCR method can be used for detection of L.pneumophila.The assay was fast,sensitive and specific,so it should be suitable for clinical investigation and the rapid detection of L.pneumophila infection.