DOI: 10.5061/dryad.jwstqjq96
title: Detection of Khapra Beetle environmental DNA using portable technologies in Australian biosecurity

description: 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 &lt; 0.001) and significantly higher positive detections
 for both assays (p &lt; 0.001) compared to the portable
 thermocycler. DNA yield was significantly higher in samples extracted
 using laboratory-based kits compared to field kits (p &lt; 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.
description: This dataset contains amplification data for all environmental
 DNA samples collected during Australian biosecurity responses for Khapra
 beetle. All Samples were run in triplicate (including negative extraction
 blanks, field and negative controls) using TaqMan assays designed in
 previous studies. We considered samples as positive if amplification
 crossed a common threshold determined individually within each qPCR run
 and if detections occured within each assay's cutoff values. If no
 positive amplification was observed in any qPCR replicates then samples
 were deemed negative. Quantitative PCR reactions were done using the ViiA™
 7 Real-Time PCR System (Applied Biosystems, Vic., Australia) and in a
 Franklin™ portable Realtime Thermocycler (Biomeme, USA). All data
 associated to exporting/importing companies have been removed to ensure
 confidentiality.