Opening Pandora's box Unmasking the Need for a Continuous Sample Processing Control for PCR Based Fecal Marker Analysis


Linke R., ZEKİ S., Mayer R., Keiblinger K., Mach R., Sommer R., ...More

19th International Symposium on Health-Related Water Microbiology, North Carolina, United States Of America, 15 - 19 May 2017, pp.59

  • Publication Type: Conference Paper / Full Text
  • City: North Carolina
  • Country: United States Of America
  • Page Numbers: pp.59

Abstract

PCR based techniques for the detection and tracking of faecal pollution have gained considerable popularity. Molecular techniques are opening the "black box" of faecal pollution in order to guide a more problem oriented quality management of water resources. However, applying molecular diagnostics to environmental matrices can be challenging, e.g. the PCR reaction can be inhibited and thus strongly biased by compounds such as humic acids or heavy metals. The aim of this study was a detailed process evaluation of the cell filtration, DNAextraction and qPCR-performance characteristics applied to water samples from one habitat with a very diverse and changing matrix composition. The aquatic habitat selected was Lake Neusiedl, located in the Eastern part of Austria, an important recreation area for the large urban area of Vienna. High and fluctuating levels of salt, humic acid, and suspended solids (TSS) characterize the water matrix of Lake Neusiedl. Therefore, this habitat represents a perfect "realistic world" model to study the basic performance characteristics of PCR-based diagnostics on water samples with changing matrix conditions. A filtration/extraction process control was added to each sample by spiking with a defined cell standard (DeTaCS). In addition, a strict evaluation of PCR-inhibition by using an internal amplification control and different dilutions for each sample was performed. DNA was extracted using a phenol-chloroform and CTAB based method. Results clearly demonstrated that inhibition of the PCR reaction played only a minor role in (some of) the samples investigated. In contrast, the presence of high amounts of (inorganic) TSS led to a complete loss of DNA during the process of DNA-extraction (i.e. recovery rates ≤1%). To test the hypothesis that the adsorption of DNA to surface-reactive matrix components during DNA-extraction was the causing mechanism, experiments with different amounts of sodium pyrophosphate and salmon sperm were conducted in the laboratory. Addition of these adsorption site competitors helped to recover DNA concentrations to near control levels. The results of the presented study impressively show the importance of using a stringent and continuous process control on a "sample-per-sample" basis. Using this approach, we were able to unmask the challenges of diverse matrices of water samples within a single habitat and could even adapt the DNA-extraction protocol in order to get comparable results. We postulate that the herein observed challenges are rather the rule than the exception. We think that this situation is conferrable to many surface waters, as high amounts of TSS are frequently observed (e.g. during event situation). We thus propose a continuous use of a stringent filtration/extraction process control as basis for a robust quality management of qPCR-based diagnostics for water quality monitoring. This is a joint investigation within the Interuniversity Cooperation Centre for Water and Health.