Environmental DNA (eDNA)‐based methods are increasingly used by government agencies to detect pests and threatened species, and for broader biodiversity monitoring. Given rapid technological advances and a growing number of commercial service providers, there is a need to standardize methods for quality assurance and to maintain confidence in eDNA‐based results. Here, we introduce two documents to provide best‐practice guidelines for Australian and New Zealand eDNA researchers and end‐users (available from https://sednasociety.com/publications): the Environmental DNA protocol development guide for biomonitoring provides minimum standard considerations for eDNA and environmental RNA projects across the complete workflow, from ethical considerations and experimental design to interpreting and communicating results. The Environmental DNA test validation guidelines outline key steps to be used in assay development and validation for species‐specific testing and metabarcoding. Both guidelines were developed as an initiative of the Australian Government Department of Agriculture, Fisheries and Forestry and led by the Southern eDNA Society in a collaborative process including multiple consultation rounds with eDNA experts, end‐users, and stakeholders to adapt the guidelines to Australian and New Zealand needs. The aim of these guidelines is not to be prescriptive, but to set minimum standards to support a consistent and best‐practice approach to eDNA testing. We anticipate that the guidelines will be reviewed and regularly updated as required. Our aspiration is that these best‐practice guidelines will ensure environmental managers are provided with robust scientific evidence to support decision‐making.

Khapra beetle, Trogoderma granarium Everts, 1898, is a serious pest of stored grain products globally. Environmental DNA (eDNA)-based methods offer sensitive detection tools used to inform biosecurity officers on the presence of high-risk pests. This study tested laboratory and portable molecular technologies to detect khapra beetle environmental DNA extracted from dust samples collected during biosecurity responses (Tuggeranong and Fyshwick) to khapra beetle incursions in Australia. Airborne and floor dust samples were collected opportunistically using handheld vacuum cleaners and eDNA was extracted using either field or laboratory-based extraction methods and analyzed using laboratory benchtop real time PCR machines and portable machines with two TaqMan and one LAMP-based assay. We successfully collected, extracted, and amplified khapra beetle eDNA from dust samples by qPCR, but failed to amplify T. granarium eDNA using LAMP. The Laboratory qPCR machine showed significantly higher mean Ct values (p < 0.001) and significantly higher positive detections for both assays (p < 0.001) compared to the portable thermocycler. DNA yield was significantly higher in samples extracted using laboratory-based kits compared to field kits (p < 0.001) for both vacuumed and airborne samples (Mean DNA ± S.D. = 5.52 ± 4.45 and 4.77 ± 1.68 ng/μL, respectively), compared to field kits, (1.75 ± 1.17 and 1.36± 1.29 ng/μL for vacuumed and airborne samples, respectively). There were no significant differences in DNA yield between collection methods or differences in amplification associated to extraction or collection methods in either platform tested in this study. Portable technologies tested in this study (Franklin™ Real Time Thermocycler and Genie III) accurately amplified all tissue derived DNA during assay optimisation and field testing, highlighting the capacity of these technologies to complement biosecurity in confirming specimen ID. There was a high incidence of positive detections in field negative controls (Tuggeranong = 12.3 % and Fyshwick = 50 %), mostly attributed to the use of contaminated vacuum cleaners. We discuss suitable methods to minimize sample cross-contamination, the potential of portable molecular technologies as tools for biosecurity applications, and the suitability of eDNA-based molecular detection methods to complement global trade biosecurity for one of the most invasive and important grain pests worldwide.

Effective border control relies on stringent biosecurity protocols to detect and prevent introductions of exotic pests and diseases. Detection of pathogens and parasites in the live ornamental fish trade using environmental DNA (eDNA) techniques has the potential to improve current biosecurity practices. We examined water samples from 11 target consignments (cyprinids susceptible to Dactylogyrus spp. infections) and seven non-target fish consignments (non-cyprinids, not susceptible to Dactylogyrus spp. infections) imported from Southeast Asia to Australia for the presence of eDNA from five Dactylogyrus species (Monogenea: Dactylogyridae). A four-step predictive framework was used to predict putative positive and putative negative detections from quantitative PCR assays. Both target and non-target consignments were positive for Dactylogyrus spp. eDNA as confirmed by Sanger sequencing. Positive detections for Dactylogyrus spp. eDNA in non-target fish consignments demonstrates the possibility of source water contamination, limiting the applicability of eDNA screening methods at border control. This study suggests that screening for parasite eDNA within ornamental fish consignments should be tested during pre-export quarantine periods to avoid false positive detections at border control. Lastly, the proposed predictive framework has a broad utility for minimizing false positive and false negative eDNA detections of aquatic organisms.