School of Environmental Sciences, University of Guelph, Guelph, ON, Canada.
J Microbiol Methods. 2011 Dec;87(3):263-72. doi: 10.1016/j.mimet.2011.09.007. Epub 2011 Sep 22.
Sewage sludge is the solid, organic material remaining after wastewater is treated and discharged from a wastewater treatment plant. Sludge is treated to stabilize the organic matter and reduce the amount of human pathogens. Once government regulations are met, including material quality standards (e.g., E. coli levels and heavy metal content) sludge is termed "biosolids", which may be disposed of by land application according to regulations. Live-culture techniques have traditionally been used to enumerate select pathogens and/or indicator organisms to demonstrate compliance with regulatory requirements. However, these methods may result in underestimates of viable microorganisms due to several problems, including their inability to detect viable but non-culturable (VBNC) cells. Real-time quantitative polymerase chain reaction (qPCR) is currently under investigation as a fast, sensitive, and specific molecular tool for enumeration of pathogens in biosolids. Its main limitation is that it amplifies all target DNAs, including that from non-viable cells. This can be overcome by coupling qPCR with propidium monoazide (PMA), a microbial membrane-impermeant dye that binds to extracellular DNA and DNA in dead or membrane-compromised cells, inhibiting its amplification. PMA has successfully been used to monitor the presence of viable pathogens in several different matrices. In this review the use of PMA-qPCR is discussed as a suitable approach for viable microbial enumeration in biosolids. Recommendations for optimization of the method are made, with a focus on DNA extraction, dilution of sample turbidity, reagent concentration, and light exposure time.
污水污泥是废水经过处理并从污水处理厂排放后剩余的固体有机物质。污泥经过处理以稳定有机物并减少人类病原体的数量。一旦达到政府规定，包括材料质量标准（例如大肠杆菌水平和重金属含量），污泥就被称为“生物固体”，可以根据规定通过土地应用进行处置。传统上，一直使用活菌培养技术来计数选择的病原体和/或指示生物，以证明符合法规要求。然而，这些方法可能由于几个问题导致对活微生物的低估，包括它们无法检测到活但非可培养（VBNC）细胞。实时定量聚合酶链反应（qPCR）目前作为一种快速、敏感和特异的分子工具，用于生物固体中病原体的计数。它的主要限制是它扩增所有目标 DNA，包括来自非存活细胞的 DNA。通过将 qPCR 与吖啶橙（PMA）偶联，可以克服这一限制，吖啶橙是一种微生物膜不可渗透的染料，与细胞外 DNA 和死亡或膜受损细胞中的 DNA 结合，抑制其扩增。PMA 已成功用于监测几种不同基质中活病原体的存在。在这篇综述中，讨论了 PMA-qPCR 作为生物固体中活微生物计数的合适方法。对该方法进行了优化的建议，重点是 DNA 提取、样品浊度稀释、试剂浓度和光照时间。
