实时荧光聚合酶链反应检测肺部感染患者痰标本中军团菌

Real-time fluorescence polymerase chain reaction for detecting Legionella in sputum specimens of patients with pulmonary infection

目的建立实时荧光定量聚合酶链反应(PCR),检测肺部感染患者痰液中军团菌属特异性16SrRNA基因。方法利用军团菌属特异性16SrRNA基因保守序列设计引物和探针,优化反应条件和反应体系,对嗜肺军团菌、非嗜肺军团菌及其他病原菌标准菌株进行检测,验证该方法的特异性、敏感性、重复性;并对557例肺部感染患者痰液标本进行检测,同时采用PCR酶切法作比较,阳性者对基因扩增产物测序做验证试验。结果该方法检测军团菌属所有标准菌株均出现阳性信号,其他非军团菌属检测结果均为阴性,灵敏度为102 CFU/mL;577例肺部感染患者的痰液标本,实时荧光定量PCR军团菌检出阳性率为23.1%,PCR酶切法检出阳性率为19.9%,经16SrRNA基因测序验证军团菌阳性率为17.2%,3种方法检出阳性率差异无统计学意义(P>0.05)。结论实时荧光定量PCR检测患者痰液标本中军团菌,具有快速、简便的特点,可作为临床军团菌感染患者的一种辅助诊断试验。

Objective To establish a real‐time fluorescent polymerase chain reaction and detect 16S rRNA gene of Legionella strains isolated from sputum specimens of patients with pulmonary infection by using this method .Methods 16s rRNA gene of Le‐gionella was used to design primers and probes .The reaction system and reaction conditions were optimized and the specificity ,sen‐sitivity and repeatability of this method were verified by detecting Legionella pneumophila ,non‐Legionella pneumophila and other bacteria .A total of 557 sputum specimens of patients with pulmonary infection were detected ,and PCR‐digestion identification method was carried out as control .Otherwise ,sequences of 16S rRNA were verified in patients with positive detection results .Re‐sults The results showed that all reference strains of Legionella were positive ,while all of other bacteria were negative ,and the sensitivity was 102 CFU/mL .Among sputum specimens collected from 577 cases of patients with pulmonary infection ,the positive rate of Legionella detected by using real‐time fluorescent PCR and PCR‐digestion identification method was 23 .1% and 19 .9% re‐spectively ,while the positive rate was 17 .2% by verifying the sequences of 16s rRNA .There were no statistically significant differ‐ences of positive rate among the three methods（P〉0 .05） .Conclusion The real‐time fluorescent PCR is fast and convenient in de‐tection of Legionella strains isolated from sputum specimens of patients ,which could be an assisted method for clinically diagnosing Legionella infection .