基于16S rRNA的聚合酶链反应技术用于肺部感染患者痰标本中军团菌的检测

Polymerase chain reaction based on 16S rRNA in detection of Legionella in sputum specimens from patients with pulmonary Legionella infection

目的建立实时荧光定量聚合酶链反应(real-time PCR)方法,检测肺部感染患者痰液中军团菌属特异性16S rRNA基因。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病原菌标准菌株进行检测,验证该方法的特异性和敏感性。对150例肺部感染患者痰液标本进行检测,阳性者对基因扩增产物测序作验证试验。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10 cfu/ml。150例肺部感染患者的痰液标本,经荧光定量PCR法检出军团菌阳性27例,阳性率为18.0%(27/150),经16S rRNA基因测序验证,阳性率为15.0%(22/150),经χ2检验二者差异无统计学意义(χ2=3.2,P﹥0.05),表明其较好的符合性和等效性。结论 realtime PCR法检测患者痰液标本中军团菌,具有快速、敏感、特异等特点,适用于临床军团菌感染调查及快速检测。

Objective To establish a fluorescent quantitative polymerase chain reaction（ real-time PCR） assay to detect the specific16S rRNA gene of Legionella in the sputum specimens patients with pulmonary Legionella infection.Methods 16S rRNA gene of Legionella was used to design primers and probes. The reaction system and reaction conditions were optimized. The specificity and sensitivity of the assay were evaluated by using Legionella pneumophila,non-Legionella pneumophila and other bacteria. The assay was used to detect 150 sputum specimens from patients with pulmonary Legionella infection,the positive ones were confirmed by gene sequencing of PCR products. Results The results showed that the results of 10 reference Legionella strains were all positive,but the results of other bacteria were all negative,and the assay’s sensitivity was 10 cfu /ml. Among the 150 sputum specimens,27 were positive for Legionella by fluorescent quantitative PCR（ 18. 0%） and 22 were confirmed to be positive by gene sequencing（15.0%）. The difference had no statistical significance（χ2= 3. 2,P ﹥ 0. 05）,indicating their good consistency and equivalence. Conclusion Real-time fluorescent quantitative PCR can be used to detect Legionella,it is specific and sensitive,and is sui Tablefor clinical investigation of Legionella infection and rapid detection.