A new molecular tool for detection of the highly invasive gecko, Hemidactylus frenatus.

The Common House Gecko, Hemidactylus frenatus, also known as the Asian House Gecko (AHG), is the most significant invasive gecko globally. Detecting this species can be challenging because it closely resembles other geckos, and is often not directly observed, being cryptic and nocturnal. Traces such as scats, however, are more readily observed than the animal itself. Here, we developed and tested a new diagnostic mitochondrial ND2 LAMP (Loop-mediated isothermal amplification) assay to detect and distinguish AHG from other geckos. Testing DNA from twenty-five non-target gecko and skink species present in Western Australia demonstrated the species-specificity of the assay. This new molecular assay showed amplification in under 15 minutes from AHG DNA. Intraspecific variation did not adversely affect the LAMP assay, with all AHG tissue samples successfully amplifying. This included samples from the Cocos (Keeling) Islands territory of Australia which were >6% genetically divergent from mainland Australian samples, representing a genetic group that was previously unknown, referred to here as AHG "clade E". The assay was found to be highly sensitive, capable of amplifying AHG DNA at very low levels, down to 0.0001 ng/µL of AHG DNA, within 25 minutes. The new LAMP assay has been fully optimised for in-field use, including development and testing of a non-destructive DNA extraction method for in-field extractions from both AHG tissue and scat samples, as well as a gBlock gene fragment for use as a synthetic positive control. The in-field protocols were tested on 100 field collected scats, from multiple lizard species in Western Australia, demonstrating the AHG specificity of the assay, with amplification successful on 79% of AHG scats tested in-field. While the assay was highly effective, scat DNA degradation and inhibitors limited detection in 21% of AHG samples, highlighting preservation challenges. This new assay has already been applied operationally in the field, providing early detection of AHG, and preventing potential introduction of this species into new areas.