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 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.

The Utilization of the SaLux19 -Based LAMP Assay for the Rapid and Sensitive Identification of Minute Amounts of Biological Material

“…Current methods based on DNA analysis, which determine the presence of species-specific biological materials, are fast and have excellent sensitivity [6]. This study aimed to develop an inhouse method that exploits a novel amplification technique called loop-mediated amplification (LAMP), particularly using the device SaLux19 (Max-Planck Innovation prototype, Germany), allowing fluorescent real-time control of amplification and focusing on DNA identification from Sus scrofa biological fluid (venous blood) from forensically sized samples (~2 μl or less, diluted 100x, 1000x, at their appropriate concentration units within the range from ng to pg of DNA [7]).…”

Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens

“…In the last 4–5 years, this situation has changed, and now this type of formulation has been reaching the markets. Due to its novelty, independent studies comparing these reagents are very limited, such as the one performed by Domesle et al [ 1 , 13 ] or Deliveyne et al [ 14 ]. Even though this may seem trivial, the success of any given method relying on this technique is highly dependent on the appropriate selection of reagents.…”