Seems fishy: environmental DNA impacts on sketa22 quality control in salmonidae dominated waterbodies using qPCR and ddPCR

Abstract

Globally, water resources used for recreation and drinking water are threatened by fecal pollution. These pollutants can cause gastrointestinal illness and environmental degradation. Additionally, most sources of fecal pollution are non-point sources stemming from multiple species. Identifying these sources is vital to categorizing the exposure risk from contact and improving remediation efforts. A common technique to provide species-specific information for fecal source identification is microbial source tracking (MST). MST quantifies DNA of host or host-associated microorganisms through polymerase chain reaction (PCR) technologies such as quantitative PCR (qPCR) or droplet digital PCR (ddPCR). MST techniques have been implemented globally and are used for routine monitoring. In the United States (US), the US Environmental Protection Agency has provided several approved standard PCR methods for MST and other recreational water quality applications. These methods have specified quality controls including sample processing controls (SPC) and assessments for sample inhibition. A standard SPC used in EPA methods involves spiking samples with salmon testes DNA (nominally originating from Chum Salmon, Oncorhynchus keta and quantifying them using Sketa22, a genus specific TaqManTM assay). This quality control (QC) behaves similarly to the microbial species being monitored. MST testing in Fall 2022 indicated elevated Sketa22 recoveries and re-analysis of samples indicated the detection of external Salmonidae DNA on both qPCR and ddPCR platforms. Our research was designed to identify the cause of this interference. Results indicate that the primer probe set may react with wild Salmonidae DNA. Analyzing the Sketa22 sequence using BLAST indicated matches with many species of Salmonidae present in the sampled stream system. Consequently, further research is required to identify the effectiveness of Sketa22 as a QC when native and migratory Salmonidae are present. General recommendations are provided to account for excess ambient Salmonidae DNA.