Cloud J L, Carroll K C, Pixton P, Erali M, Hillyard D R
Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA.
J Clin Microbiol. 2000 May;38(5):1709-12. doi: 10.1128/JCM.38.5.1709-1712.2000.
Legionella spp. are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomial pneumonia. A PCR method for the detection of legionellae in respiratory samples was evaluated and was compared to culture. The procedure can be performed in 6 to 8 h with a commercially available DNA extraction kit (Qiagen, Valencia, Calif.) and by PCR with gel detection. PCR is performed with primers previously determined to amplify a 386-bp product within the 16S rRNA gene of Legionella pneumophila. We can specifically detect the clinically significant Legionella species including L. pneumophila, L. micdadei, L. longbeachae, L. bozemanii, L. feeleii, and L. dumoffii. The assay detects 10 fg (approximately two organisms) of legionella DNA in each PCR. Of 212 clinical specimens examined by culture, 100% of the culture-positive samples (31 of 31) were positive by this assay. By gel detection of amplification products, 12 of 181 culture-negative samples were positive for Legionella species by PCR, resulting in 93% specificity. Four of the 12 samples with discrepant results (culture negative, PCR positive) were confirmed to be positive for Legionella species by sequencing of the amplicons. The legionella-specific PCR assay that is described demonstrates high sensitivity and high specificity for routine detection of legionellae in respiratory samples.
嗜肺军团菌属是社区获得性呼吸道感染的常见病因，也是医院获得性肺炎的偶发病因。我们评估了一种用于检测呼吸道样本中嗜肺军团菌属的聚合酶链反应（PCR）方法，并将其与培养法进行比较。该检测程序可使用市售DNA提取试剂盒（Qiagen公司，加利福尼亚州瓦伦西亚），并通过凝胶检测的PCR技术在6至8小时内完成。PCR使用先前确定的引物，以扩增嗜肺军团菌16S rRNA基因内一段长度为386bp的产物。我们能够特异性检测具有临床意义的嗜肺军团菌属菌种，包括嗜肺军团菌、米克戴德军团菌、长滩军团菌、博兹曼军团菌、菲尔军团菌和杜莫夫军团菌。该检测方法在每次PCR中可检测到10fg（约两个菌体）的嗜肺军团菌DNA。在通过培养法检测的212份临床标本中，所有培养阳性样本（31份中的31份）通过该检测方法均呈阳性。通过凝胶检测扩增产物，181份培养阴性样本中有12份通过PCR检测出嗜肺军团菌属菌种呈阳性，特异性为93%。12份结果不一致的样本（培养阴性，PCR阳性）中有4份通过扩增子测序被确认为嗜肺军团菌属菌种呈阳性。所描述的嗜肺军团菌属特异性PCR检测方法在呼吸道样本中嗜肺军团菌属的常规检测中显示出高灵敏度和高特异性。
