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RESEARCH ARTICLE (Open Access)
Contents Vol 51(1)

Developing Loop Mediated Isothermal Amplification (LAMP) assays for rapid, presumptive DNA detection of an invasive reptile (Boa constrictor)

Nathan Deliveyne https://orcid.org/0000-0002-9128-4285 A B * , Jeremy J. Austin https://orcid.org/0000-0003-4244-2942 B and Phillip Cassey https://orcid.org/0000-0002-2626-0172 A B
+ Author Affiliations
- Author Affiliations

A Invasion Science & Wildlife Ecology Lab, The University of Adelaide, Adelaide, SA 5000, Australia.

B School of Biological Sciences and The Environment Institute, The University of Adelaide, Adelaide, SA 5005, Australia.

* Correspondence to: nathan.deliveyne@adelaide.edu.au

Handling Editor: Adam Stow

Wildlife Research 51, WR23053 https://doi.org/10.1071/WR23053
Submitted: 11 May 2023  Accepted: 2 October 2023  Published: 25 October 2023

© 2024 The Author(s) (or their employer(s)). Published by CSIRO Publishing. This is an open access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND)

Abstract

Context

Wildlife trade is a prominent pathway for invasive species introductions into novel environments. Deliberate or accidental release of exotic pets can result in the establishment of alien populations, with damaging impacts for native species and environmental assets. This process is well documented for reptiles globally and is of considerable biosecurity concern in Australia. Boa constrictor is one species at high risk of establishment in Australia, and has insufficient biosecurity detection and post-border control capacity.

Aims

We aimed to develop rapid DNA-based presumptive testing capacity for detecting B. constrictor, with appropriate sensitivity and specificity to operate in a trace DNA biosecurity context.

Methods

Loop Mediated Isothermal Amplification (LAMP) is an emerging biosecurity tool that provides highly specific, sensitive, low-resource methods for detection of trace DNA in the absence of physical evidence. We developed colourimetric and fluorescent LAMP assays targeting the mitochondrial DNA control region of B. constrictor. We tested and validated these assays against synthetic DNA fragments, as well as DNA extracted from: (1) vouchered museum B. constrictor tissue; (2) shed B. constrictor skin samples; (3) a range of non-target species to test specificity; and (4) trace DNA recovered from glass tanks post B. constrictor presence.

Key results

We successfully detected synthetic target DNA down to 1 fg and genomic B. constrictor DNA from tissue and shed skins down to <10 pg in under 30 minutes with our fluorescence-based LAMP assay. Additionally, we were able to detect B. constrictor trace DNA following 24 h of presence utilising a traditional laboratory-based DNA extraction method (approximately 180 min) and a rapid lysis step (approximately 8 min).

Conclusions

Both colourimetric and fluorescent assays show promise for the specific detection of B. constrictor in biosecurity contexts, including post-border enforcement and compliance checks in the domestic illicit wildlife trade.

Implications

Our findings greatly strengthen the ongoing development of biosecurity tools for trace DNA detection of commonly traded and trafficked species (i.e. reptiles) in wildlife enforcement contexts, advancing both preparedness and surveillance.

Keywords: Biosecurity, Boa constrictor, DNA detection, invasive species, Loop Mediated Isothermal Amplification (LAMP), reptiles, wildlife forensics, wildlife trade.

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