双重PCR法检测痰标本对早期诊断军团菌肺炎的意义

Evaluation of Duplex PCR Method for Sputum Samples in Early Diagnosis of Legionella Pneumonia

为探讨双重PCR方法 (DPCR)检测痰标本中军团菌DNA在早期诊断军团菌肺炎方面的意义 ,采用DPCR方法对军团菌肺炎组、临床疑似军团菌肺炎组、普通肺炎组和肺结核组 4组患者留取的痰标本进行检测。DPCR过程采用根据军团菌 1 6SrRNA基因和mip基因设计的 2对引物 ,同时扩增军团菌 3 86bp 1 6SrRNA基因片段和 2 0 6bpmip基因片段。结果 :军团菌肺炎组的DPCR阳性检出率为 1 0 0 % ,明显高于其他 3组 (分别为 1 7.2 4%、0、0 ,P均 <0 .0 1 )。模拟痰标本DPCR的最低检出限为 1× 1 0 3 cfu/mL。初步研究表明 ,DPCR方法检测痰标本中的军团菌DNA具有较好的敏感性、特异性和稳定性 。

To evaluate a Duplex PCR(DPCR) method in early diagnosis of legionella pneumonia by detecting legionella DNA in sputum samples, the sputum samples of patients from four groups, including legionella pneumonia group( n =15), clinical suspected legionella pneumonia group( n =29), ordinary pneumonia group( n =31) and pulmonary tuberculosis group( n =15) were detected with the DPCR method. Two different sets of oligonucleotide primer were stimultaneously used to amplify 386 bp 16SrRNA gene fragment and 206 bp mip gene fragment of legionella during the DPCR process. The DPCR positive rate of legionella pneumonia group(100%) was significantly higher than that of the other three groups(17.24%, 0, 0, respectively, P <0.01). The lowest detection level was 1×10 3 cfu/mL for the simulated sputum sample. Primary study showed the DPCR method adopted for detecting legionella DNA in sputum samples had satisfactory sensitivity, specificity and stability. The method has its value in early diagnosis of legionella pneumonia.