实时荧光定量PCR检测痰标本中军团菌属

Real-time fluorescence quantitative PCR detecting legionella in sputum specimens

目的建立荧光定量PCR方法,用于检测军团菌属特异性16S rRNA基因,并探讨广州地区军团菌属感染状况。方法利用军团菌属特异性16S rRNA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细菌进行检测,验证该方法的特异性、敏感性、重复性,对广东省中医院采集的532例肺部感染患者痰液标本进行检测。结果该方法检测军团菌属所有标准菌株均出现阳性信号,其他非军团菌属细菌检测结果均为阴性;检测灵敏度为102CFU/ml,临床检测532例肺部感染患者的痰液标本,荧光法检出军团菌阳性43例,阳性率为8.08%,16S rRNA PCR法加基因测序验证阳性率为7.71%,两者检测结果差异无统计学意义,表明其具有较好的符合性和等效性。结论 16S rRNA基因荧光定量PCR法检测患者痰液标本中军团菌属,具有快速、敏感、特异等特点,适用于临床军团菌属感染调查及快速检测。

OBJECTIVE To establish a fluorescent quantitative PCR method of Legionella-specific 16S rRNA gene, and to investigate the patients with pneumonia of Legionella infection in Guangzhou. METHODS 16S rRNA gene of Legionella spp. was used to design primers and probes. The reaction system and reaction conditions were optimized and the specificity, sensitivity and repeatability of this method were verified by detecting Legionella pneumophila, non-Legionella pneumophila and other bacteria. Totally 532 sputum specimens of patients with pneumonia in Guangdong Provincial Hospital of Chinese Medicine were detected. RESULTS The results showed that all reference strains of Legionella were positive, while all of other bacteria were negative, and the sensitivity was 10 CFU/ml. The positive rate of fluorescent quantitative PCR was 8. 08% sputum specimens, while the 16S rRNA PCR and gene sequencing was 7.71%（41/532） positive. This results showed that no significant difference between the two groups, which had good compliance and equivalence. CONCLUSION 16S rRNA gene fluorescent quantitative PCR method can detect Legionella, it is fast, sensitive and specified and suitable for clinical investigation of legionella infection and the rapid detection.