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

Globally, water resources used for recreation and drinkingwater 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 fromcontact and improving remediation efforts. Acommontechnique to provide species-specific information for fecal source identification is microbial source tracking (MST). MSTquantifiesDNAof host or host-associated microorganisms through polymerase chain reaction (PCR) technologies such as quantitative PCR(qPCR) or droplet digitalPCR(ddPCR). MST techniques have been implemented globally and are used for routinemonitoring. In theUnited States (US), theUS Environmental ProtectionAgency has provided several approved standardPCRmethods forMST and other recreationalwater quality applications. Thesemethods have specified quality controls including sample processing controls (SPC) and assessments for sample inhibition. Astandard SPCused in EPA methods involves spiking sampleswithsalmon testesDNA(nominally originating fromChumSalmon, Oncorhynchus keta and quantifying them using Sketa22, a genus specificTaqMan(TM) assay). Thisquality control (QC) behaves similarly to themicrobial species being monitored. MSTtesting in Fall 2022 indicated elevated Sketa22 recoveries and re-analysis of samples indicated the detection of external SalmonidaeDNAon both qPCRandddPCR 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 matcheswithmany species of Salmonidae present in the sampled streamsystem. Consequently, further research is required to identify the effectiveness of Sketa22 as aQCwhen native andmigratory Salmonidae are present. General recommendations are provided to account for excess ambient SalmonidaeDNA.