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

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 checksin 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.