Identifying Inorganic Turbidity in Water Samples as Potential Loss Factor During Nucleic Acid Extraction: Implications for Molecular Fecal Pollution Diagnostics and Source Tracking

Abstract

Molecular diagnostic methods are increasingly applied for food and environmental analysis. Since several steps are involved in sample processing which can affect the outcome (e.g., adhesion of DNA to the sample matrix, inefficient precipitation of DNA, pipetting errors and (partial) loss of the DNA pellet during DNA isolation), quality control is essential at all processing levels. In soil microbiology, particular attention has been paid to the inorganic component of the sample matrix affecting DNA extractability. In water quality testing, however, this aspect has mostly been neglected so far, although it is conceivable that these mechanisms have a similar impact. The present study was therefore dedicated to investigate possible matrix effects on results of water quality analysis. Field testing in an aquatic environment with pronounced chemo-physical gradients [total suspended solids (TSS), inorganic turbidity, total organic carbon (TOC), and conductivity] indicated a negative association between DNA extractability (using a standard phenol/chloroform extraction procedure) and turbidity (spearman ρ = −0.72, p < 0.001, n = 21). Further detailed laboratory experiments on sediment suspensions confirmed the hypothesis of inorganic turbidity being the main driver for reduced DNA extractability. The observed effects, as known from soil samples, were also indicated to result from competitive effects for free charges on clay minerals, leading to adsorption of DNA to these inorganic particles. A protocol modification by supplementing the extraction buffer with salmon sperm DNA, to coat charged surfaces prior to cell lysis, was then applied on environmental water samples and compared to the standard protocol. At sites characterized by high inorganic turbidity, DNA extractability was significantly improved or made possible in the first place by applying the adapted protocol. This became apparent from intestinal enterococci and microbial source tracking (MST)-marker levels measured by quantitative polymerase chain reaction (qPCR) (100 to 10,000-fold median increase in target concentrations). The present study emphasizes the need to consider inorganic turbidity as a potential loss factor in DNA extraction from water-matrices. Negligence of these effects can lead to a massive bias, by up to several orders of magnitude, in the results of molecular MST and fecal pollution diagnostics.