Clinical Application of a Multiplex Real-Time PCR Assay for Simultaneous Detection of Legionella Species, Legionella pneumophila, and Legionella pneumophila Serogroup 1 Alvaro J. Benitez, Jonas M. Winchell Respiratory Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA We developed a single-tube multiplex real-time PCR assay capable of simultaneously detecting and discriminating Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1 in primary specimens. Evaluation of 21 clinical speci- mens and 115 clinical isolates demonstrated this assay to be a rapid, high-throughput diagnostic test with 100% specificity that may aid during legionellosis outbreaks and epidemiologic investigations. L egionellae are facultative intracellular Gram-negative bacteria found in soil and water environments, where they parasitize and proliferate within free-living protozoa (1). Legionellae pos- sess the ability to replicate in environmental protozoa and within mammalian alveolar macrophages and epithelial cells (2–4). Le- gionellae are common contaminates of artificial water systems, including air-conditioning systems, cooling towers, and jacuzzis, where conditions may be optimal for growth and proliferation (5, 6). Once aerosolized, the bacteria can enter the human respiratory tract and cause disease manifesting as Legionnaires’ disease, a se- vere form of pneumonia, or Pontiac fever, a self-limiting flu-like illness (1). More than 50 species comprising 70 distinct serogroups have been identified to date (http://www.bacterio.cict.fr/l/legionella .html). Although more than 90% of isolates associated with Le- gionnaires’ disease are Legionella pneumophila, with 84% being L. pneumophila serogroup 1 (sg1), nearly one-half of Legionella spe- cies have been associated with human disease (1, 7, 8). Given this epidemiological background, an ideal diagnostic assay would rap- idly detect the presence of Legionella and simultaneously identify the species and, if applicable, the serogroup. Although bacterial culture remains the gold standard for the diagnosis of Legion- naires’ disease, it requires specialized media and expertise and is time-consuming, requiring several days to obtain definitive and reliable results. The urine antigen test is a common and conve- nient test used for diagnosis but detects only L. pneumophila sg1. Serology-based tests, although widely used, suffer from specificity limitations (1). Currently, molecular diagnosis is based largely on detection of the 16S rRNA gene for identification of the Legionella genus and the mip gene, used for identification to the species level, along with sequence based-typing (SBT) for L. pneumophila char- acterization (9–11). These tests require post-PCR analysis and/or sequencing to distinguish L. pneumophila sg1 from other sero- groups and/or non-pneumophila species of Legionella, thus mak- ing them less ideal (12, 13). The lack of available diagnostic tests for species other than L. pneumophila may lead to underreporting and/or unrecognized cases of legionellosis. Since the worldwide burden of disease caused by non-sg1 L. pneumophila and other species is largely unknown but is suspected to be significant (14, 15), an improved detection method would ideally integrate a more inclusive approach for identification and characterization. This article reports the development and evaluation of a single- tube multiplex real-time PCR assay that allows simultaneous de- tection and differentiation of Legionella spp., Legionella pneumo- phila, and Legionella pneumophila sg1 for clinical isolates and in primary specimens. The assay targets the ssrA (Legionella species) gene (16), the mip (L. pneumophila) gene, and the recently iden- tified wzm gene (17), specific for L. pneumophila sg1. Multiple TaqMan primer-probe sets were designed targeting the ssrA gene (GenBank accession no. AE017354 [bp 172917 to 173015]), the mip gene (GenBank accession no. AJ810179 [bp 76 to 190]), and the wzm gene (GenBank accession no. AM778127 [complement bp 6098 to 6167]) either manually (ssrA) or using the software program Primer Express 3.0 (Applied Biosystems, Foster City, CA). The RNase P assay designed to detect human DNA was described previously (18). Primers and probe sets were initially tested and optimized in singleplex format (data not shown). Primer and probe sequences, their final concentrations, and the distinct fluorophores for each probe used in the multiplex assay are listed in Table S1 in the supplemental material. Purified total nucleic acid from L. pneumophila sg1 strain Philadelphia was used as a positive control. The multiplex real-time PCR mixture was prepared in a total volume of 25 l containing the following, per reaction: 12.5 l of PerfeCTa Multiplex qPCR SuperMix (Quanta Biosciences, Gaithersburg, MD), the appropriate volume of each primer and probe (see Table S1), 5 l of template, and nuclease-free water (Promega), to achieve a 25-l final volume. The assay was carried out using the ABI 7500 real-time PCR sys- tem (Applied Biosystems) under the following conditions: 95°C for 5 min, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. Received 19 September 2012 Returned for modification 19 October 2012 Accepted 2 November 2012 Published ahead of print 7 November 2012 Address correspondence to Jonas M. Winchell, jwinchell@cdc.gov. Supplemental material for this article may be found at http://dx.doi.org /10.1128/JCM.02510-12. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.02510-12 348 jcm.asm.org Journal of Clinical Microbiology p. 348 –351 January 2013 Volume 51 Number 1 on January 23, 2018 by guest http://jcm.asm.org/ Downloaded from on January 23, 2018 by guest http://jcm.asm.org/ Downloaded from on January 23, 2018 by guest http://jcm.asm.org/ Downloaded from