Weir S C, Fischer S H, Stock F, Gill V J
Microbiology Service, Clinical Pathology Department, National Institutes of Health, Bethesda, Maryland 20892, USA.
Am J Clin Pathol. 1998 Sep;110(3):295-300. doi: 10.1093/ajcp/110.3.295.
Using polymerase chain reaction (PCR) for the detection of pathogens that are difficult to grow, such as Legionella species, may reduce difficulties encountered with culture and immunofluorescent staining. We evaluated a commercial PCR and hybridization kit, designed for environmental samples, for the detection of Legionella in respiratory specimens. Sixteen Legionella species cultures tested positive with the Perkin Elmer Legionella EnviroAmp Amplification and Detection kits (Perkin Elmer, Foster City, Calif). The assay detected as few as 100 colony-forming units per milliliter of spiked bronchoalveolar lavage (BAL) fluid, and no false-negative results were obtained. PCR inhibition by blood in the specimens was removed by washing pelleted specimens in sterile distilled water. Of 126 specimens screened with the kit, 1 induced sputum and 3 BAL specimens were positive by PCR. All 4 were validated as true-positive results by culture or serologic testing. The entire PCR and hybridization assay can be completed in less than 6 hours, whereas isolation and identification by culture requires up to 12 days, and serologic conversion may not be demonstrated for weeks. Molecular techniques based on direct extraction and amplification of DNA from respiratory specimens nay be useful for the timely diagnosis of legionellosis.
使用聚合酶链反应（PCR）检测难以培养的病原体，如军团菌属，可能会减少培养和免疫荧光染色所遇到的困难。我们评估了一种专为环境样本设计的商用PCR和杂交试剂盒，用于检测呼吸道标本中的军团菌。16种军团菌属培养物经珀金埃尔默军团菌环境扩增检测试剂盒（珀金埃尔默公司，加利福尼亚州福斯特城）检测呈阳性。该检测方法能检测到每毫升加标的支气管肺泡灌洗（BAL）液中低至100个菌落形成单位，且未获得假阴性结果。通过用无菌蒸馏水洗涤沉淀的标本可消除标本中血液对PCR的抑制作用。用该试剂盒筛查的126份标本中，1份诱导痰标本和3份BAL标本经PCR检测呈阳性。所有4份标本经培养或血清学检测均被确认为真阳性结果。整个PCR和杂交检测可在不到6小时内完成，而通过培养进行分离和鉴定则需要长达12天，血清学转换可能数周都无法显示。基于直接从呼吸道标本中提取和扩增DNA的分子技术可能有助于及时诊断军团菌病。
