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首页> 外文期刊>Applied and Environmental Microbiology >Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples
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Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples

机译：生存力PCR，一种独立于培养的方法，用于快速选择性地定量环境水样品中的嗜肺军团菌细胞

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PCR-based methods have been developed to rapidly screen for Legionella pneumophila in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for L. pneumophila that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable L. pneumophila cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the L. pneumophila PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of L. pneumophila cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70°C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating L. pneumophila bacteria in water and, in comparison with conventional qPCR techniques used to monitor Legionella, has the advantage of selectively amplifying only viable cells.
机译：已开发出基于PCR的方法来快速筛选水中的嗜肺军团菌，以替代耗时的培养技术。但是，这些方法无法区分活细菌和死细菌。在这里，我们报告了一种结合了单叠氮化乙锭溴化物和定量实时PCR（qPCR）的嗜肺乳杆菌的活力测定（活力PCR [v-PCR]）。用透化剂，甲苯或异丙醇证实了v-PCR区分活的和非活的肺炎支原体细胞的能力。 v-PCR在无活力培养物中抑制了超过99.9％的嗜肺乳杆菌PCR信号，并且能够区分混合样品中的有活力细胞。通过v-PCR，常规qPCR和培养方法同时定量嗜肺乳杆菌细胞后，在64个生活热水样品中观察到了从可培养到死细胞的各种生理状态。 v-PCR计数等于或高于通过培养获得的计数，并且低于或等于常规qPCR计数。 v-PCR用于成功监测加热到70°C的消毒效果以及戊二醛和氯的治疗方法。 v-PCR方法似乎是一种用于计数水中嗜肺乳杆菌细菌的有前途且快速的技术，并且与用于监测军团菌的常规qPCR技术相比，它具有仅选择性扩增活细胞的优势。

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