军团菌双重PCR检测方法的建立及其应用研究

DEVELOPMENT AND APPLICATION OF A DUPLEX PCR METHOD FOR DETECTING OF LEGIONELLA

目的 建立一种双重PCR方法 ,用于简便 ,快速检测军团菌。方法 根据军团菌 16SrRNA基因和mip基因序列设计合成引物 ,可以扩增 386bp 16SrRNA基因和 2 0 6bpmip基因片段。用该方法对军团菌标准参考菌株和 47份临床标本进行了检测。结果 通过一次PCR反应 ,不仅能够检测嗜肺军团菌 ,而且能够检测非嗜肺的军团菌 ,从而克服了单一引物应用时的不足。 2 1株对照菌株扩增阴性 ,最小检出限为 2× 10 1 cfu/ml。 47份临床标本 (包括 12份血 ,2 0份支气管肺泡灌洗液 ,7份胸水 ,8份痰 )PCR检测的阳性标本为 2 8份 (2 8/4 7) ,在临床上均支持为军团菌感染 ,高于血清抗体检测的阳性标本数 (2 4/4 7)。结论 该方法用于军团菌的检测简便、快捷 ,具有很高的特异性和敏感性 。

Aim\ To develop a duplex PCR method for detecting Legionella. Methods \ Two different sets of oligonucleotide primer were simultaneously used to amplify 386bp 16SrRNA gene fragment and 206bp mip gene fragment. These two primers were designed according to the sequences of 16SrRNA gene and mip gene of Legionella. We have applied the method to detect 47 clinical samples including 12 bloods, 20 bronchalveolar lavage fluids(BAL), 7 pleural fluids, 8 sputum samples from patients. Results\ Through a single procedure of PCR, L.pneumophila or non-pneumophila Legionella spp. could be distinguished. 21 control strains were negative by the duplex PCR. The lowest detection level was 2×10\+1cfu/ml. Among 47 clinical samples, 28 samples which were demonstrated Legionellosis were positive by the duplex PCR. The number of the positive samples detected by duplex PCR was higher than that of the positive samples of antibody assay(24/47). Conclusion \ The results indicate that the duplex PCR is a high specific and sensitive one. It is a rapid, reliable and simple approach and provides an effective method for the epidemiology investigation and early diagnosis of Legionellosis.