Nayak A K, Rose J B
Michigan State University, Department of Fisheries and Wildlife, East Lansing, MI 48824, USA.
J Appl Microbiol. 2007 Nov;103(5):1931-41. doi: 10.1111/j.1365-2672.2007.03435.x.
To demonstrate the application of a new quantitative polymerase chain reaction (qPCR) technique for the determination of Helicobacter pylori concentrations in water, and to use this method to investigate the occurrence of the bacteria in sewage. The other aim was to study the survival capacity and detectability of the bacteria in artificially contaminated groundwater at different temperatures of 4 and 15 degrees C.
The detection of H. pylori in water was aided by PCR using specific primers designed for the amplification of a fragment within the major vacuolating cytotoxin gene. Conventional culture was compared with conventional PCR and the new real-time (RT) qPCR approach for the quantification of the bacterium. Helicobacter pylori remained culturable for 120 h at 4 degrees C as opposed to only 24 h at 15 degrees C. RT qPCR demonstrated a 100-fold greater sensitivity for the detection of H. pylori DNA in comparison with conventional PCR. Scanning electron microscopic (SEM) observation showed that the normal spiral form changed to a coccoid form after 24 and 72 h at 15 and 4 degrees C, respectively. Helicobacter pylori was found at 2-28 cells ml(-1) in sewage, of the 23 sewage samples - 84% were positive for H. pylori species-specific vacuolating cyctotoxin gene (vacA) by RT qPCR, but were negative by conventional PCR.
The RT qPCR assay provided a specific, sensitive and rapid method for the quantitative detection of H. pylori in sewage. This molecular method would be valuable in studying the prevalence of H. pylori as required by the United States Environmental Protection Agency Contaminant Candidate List, particularly in nondisinfected ground waters, in sewage as a source of contamination, and for addressing the possible presence of viable but nonculturable of H. pylori.
The quantitative detection of H. pylori by rapid and less-expensive methods than the TaqMan Assay using SYBR green could be an important tool to monitor infection in community by measuring the concentrations in sewage and to meet the new regulatory and risk-based frameworks for water supplies.
展示一种新的定量聚合酶链反应（qPCR）技术在测定水中幽门螺杆菌浓度方面的应用，并使用该方法调查污水中该细菌的存在情况。另一个目的是研究该细菌在4℃和15℃不同温度的人工污染地下水中的存活能力和可检测性。
利用针对主要空泡细胞毒素基因内一段片段扩增设计的特异性引物，通过PCR辅助检测水中的幽门螺杆菌。将传统培养法与传统PCR法以及用于该细菌定量的新型实时（RT）qPCR方法进行比较。幽门螺杆菌在4℃下可培养120小时，而在15℃下仅可培养24小时。与传统PCR相比，RT qPCR检测幽门螺杆菌DNA的灵敏度高100倍。扫描电子显微镜（SEM）观察表明，在15℃和4℃下分别培养24小时和72小时后，正常螺旋形态分别变为球菌形态。在污水中检测到幽门螺杆菌的浓度为2 - 28个细胞/毫升，在23个污水样本中，84%通过RT qPCR检测幽门螺杆菌特异性空泡细胞毒素基因（vacA）呈阳性，但传统PCR检测为阴性。
RT qPCR检测法为污水中幽门螺杆菌的定量检测提供了一种特异、灵敏且快速的方法。这种分子方法对于按照美国环境保护局污染物候选清单的要求研究幽门螺杆菌的流行情况具有重要价值，特别是在未消毒的地下水中、作为污染源的污水中，以及解决幽门螺杆菌可能存在的活的但不可培养的状态方面。
采用比使用SYBR绿的TaqMan检测法更快且成本更低的方法对幽门螺杆菌进行定量检测，可能是通过测量污水中的浓度来监测社区感染情况以及满足新的供水监管和基于风险的框架的重要工具。
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