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首页> 外文期刊>Applied and Environmental Microbiology >Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water.
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Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water.

机译：一种新型半巢式PCR方法检测军团菌的方法及其在医院冷却塔水中军团菌的监测中的应用。

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The presence of PCR inhibitors in water samples is well known and contributes to the fact that a practical PCR assay has not been developed for legionella surveillance. In this study, we devised a new seminested PCR assay for detection of Legionella spp. in water samples as a means of overriding the PCR inhibitors without loss of sensitivity. The seminested PCR assay utilized primers to amplify the 16S rRNA gene (LEG primers) of 39 Legionella spp. The assay was specific to legionellae, and the sensitivity was 1 fg of extracted Legionella DNA in laboratory examination. To evaluate the feasibility and sensitivity of the PCR assay in identifying the presence of legionellae, it was used to survey Legionella contamination in the water of 49 cooling towers of 32 hospitals. A commercially available EnviroAmp Legionella kit and a culture method were also used in the survey for comparison with the seminested PCR assay. The detection rates of legionellae in the samples were 91.8% (45 of 49) by the PCR assay and 79.5% (39 of 49) by the culture method. The EnviroAmp kit revealed that 30.6% of the water samples (15 of 49) contained inhibitors of the PCR amplification. However, the seminested PCR assay could produce the Legionella-specific DNA bands in 14 of the 15 samples. Although 8 of the 14 samples were positive in the first-step PCR, 6 of the 14 samples became positive in the second-step PCR. These results suggest that the effect of PCR inhibitors in samples, if any, can be reduced because of the dilution of the sample in the second-step PCR and that sensitivity of detection can be increased by the second-step PCR. Thus, the seminested PCR assay with LEG primers to amplify the 16S rRNA gene of 39 Legionella spp. was a practical and sensitive method to detect Legionella spp. in water samples.
机译：水样品中存在PCR抑制剂是众所周知的，这有助于为军团菌的监测开发一种实用的PCR检测方法。在这项研究中，我们设计了一种新的半巢式PCR检测试剂盒来检测军团菌。在水样品中作为覆盖PCR抑制剂而又不降低灵敏度的方法。半巢式PCR分析利用引物扩增39军团菌属的16S rRNA基因（LEG引物）。该测定法对军团菌具有特异性，在实验室检查中灵敏度为1 fg提取的军团菌DNA。为了评估PCR检测法鉴定军团菌存在的可行性和敏感性，它被用于调查32家医院49座冷却塔水中军团菌的污染。调查中还使用了市售的EnviroAmp Legionella试剂盒和培养方法，以与半巢式PCR检测法进行比较。通过PCR测定，样品中军团菌的检出率为91.8％（49例中的45例），通过培养方法检出率为79.5％（49例中的39例）。 EnviroAmp试剂盒显示30.6％的水样品（49个中的15个）含有PCR扩增抑制剂。但是，半巢式PCR检测可以在15个样本中的14个中产生军团菌特异性DNA条带。尽管14个样品中的8个在第一步PCR中呈阳性，但14个样品中的6个在第二步PCR中变为阳性。这些结果表明，由于在第二步PCR中稀释了样品，因此可以降低样品中PCR抑制剂的作用，并且通过第二步PCR可以提高检测的灵敏度。因此，用LEG引物进行的半巢式PCR测定法扩增了39个军团菌的16S rRNA基因。是检测军团菌的一种实用而灵敏的方法。在水样中。

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