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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 real-time quantitative polymerase chain reaction (real-time PCR) method for detection of Legionella-specific 16S rRNA gene in sputum of patients with pulmonary infection. Methods Primers and probes were designed based on the conserved sequence of 16S rRNA gene of Legionella and the reaction conditions and reaction system were optimized. Legionella pneumophila, non-Legionella pneumophila and other standard strains of pathogenic bacteria were tested to verify the specificity And sensitivity. Sputum samples from 150 patients with pulmonary infection were tested, and the positive samples were verified by sequencing the gene amplification products. Results The method showed that all the standard strains of Legionella showed positive signals. The results of other non-Legionella strains were negative and the sensitivity was 10 cfu / ml. The sputum samples from 150 patients with pulmonary infection were positive for the detection of Legionella pneumophila by fluorescence quantitative PCR, the positive rate was 18.0% (27/150). The 16S rRNA gene sequencing confirmed that the positive rate was 15.0% (22 / 150). There was no significant difference between them (χ2 = 3.2, P> 0.05) by χ2 test, indicating good compliance and equivalence. Conclusion The detection of Legionella in sputum samples by realtime PCR is rapid, sensitive and specific. It is suitable for the investigation and rapid detection of clinical Legionella infection.