Generic qPCR assays for quality control in environmental DNA research

Abstract Environmental DNA (eDNA) has been widely used for species surveillance. However, the lack of adequate quality control in many eDNA research projects and applications can lead to false‐negative results, greatly affecting biosecurity surveillance and conservation efforts. Exogenous DNA is routinely added to eDNA samples and used as a positive control, typically after DNA extraction. However, this type of positive control is only able to identify false negatives due to errors at the amplification stage. Therefore, errors in upstream processes, such as sample collection will not be identified by an exogenous control. We designed two independent sets of generic quality control qPCR assays (QCqPCR) targeting abundant endogenous DNA that is obtained during sample collection. Our QCqPCR assays target the chloroplast 16S and 23S ribosomal RNA sequences. In silico analyses indicated these regions were highly conserved among plants, algae and bacteria commonly found in freshwater, marine, or terrestrial environments. These QCqPCR assays were purposely mismatched against the human genome to avoid false positives resulting from human DNA contamination. Both assays remained highly efficient and sensitive under annealing temperatures between 58 and 62°C, allowing them to be multiplexed with most qPCR analyses. We validated our assays by multiplexing with a species‐specific Murray cod ( Maccullochella peelii ) assay on field‐collected environmental water samples. Potential false‐negative reactions can be identified by the failed or suppressed QCqPCR assay and the negative species‐specific assay. We recommend incorporating either one of the QCqPCR assays in qPCR‐based eDNA analysis to identify potential false negatives and improve the reliability of eDNA surveys.