Chang Bin, Sugiyama Kanji, Taguri Toshitsugu, Amemura-Maekawa Junko, Kura Fumiaki, Watanabe Haruo
Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan.
Appl Environ Microbiol. 2009 Jan;75(1):147-53. doi: 10.1128/AEM.00604-08. Epub 2008 Oct 31.
Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat- and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 10(4)- to 10(5)-fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.
嗜肺军团菌在人工水系统中普遍存在，并可导致人类感染军团病。评估了一种结合叠氮溴化乙锭（EMA）与PCR/实时PCR的活嗜肺军团菌细胞快速检测方法。EMA能够特异性地插入并切割经加热和氯处理的死亡嗜肺军团菌细胞的基因组DNA。EMA-PCR检测清楚地显示了来自活细胞的嗜肺军团菌DNA特异性扩增片段，但对于死细胞的DNA则无法扩增出该片段。通过实时PCR估计，经EMA处理的死亡嗜肺军团菌细胞数量与未经EMA处理的死亡嗜肺军团菌细胞数量相比，减少了10⁴至10⁵倍。相反，实时PCR检测未发现经EMA处理和未处理的活嗜肺军团菌细胞数量有显著差异。该联合检测方法也被证实可用于从温泉水样中特异性检测可培养的嗜肺军团菌细胞。因此，EMA与PCR/实时PCR的联合使用能够快速、特异性地检测活嗜肺军团菌细胞，可能在军团菌的环境监测中发挥作用。
