Rapid and Portable Presumptive Loop-Mediated Isothermal Amplification Assays for the Detection of the Invasive Corn Snake (Pantherophis guttatus)

Abstract

The exotic pet trade is a major pathway for the introduction, establishment, and spread of novel invasive alien species. Reptiles are common in the exotic pet trade and are prominent invasive alien vertebrate species that have dire impacts if allowed to establish. The North American corn snake (Pantherophis guttatus) is particularly common in the international pet trade and has been identified as a vertebrate pest priority species in Australia due to widespread climate suitability and prevalence in pre- and post-border seizure records. Consequently, rapid, and presumptive post-border biosecurity detection is essential to prevent its establishment and spread. Loop-mediated isothermal amplification (LAMP) is an emerging biosecurity tool that has shown promise for rapid detection of several high-risk species. We developed two LAMP assays for the detection of P. guttatus, validated against: synthetic DNA; DNA extracted from snap-frozen tissue, and shed skins; and then compared their performance for the detection of trace DNA collected from swabs of glass tanks post reptile presence. Our results include laboratory optimization and assessment of two mobile devices for in-field integration (Franklin Real-Time PCR Thermocycler, Biomeme, USA, and Genie III, Optigene, UK). The results indicate that LAMP is a viable biosecurity tool, with DNA detection possible for a range of sample types in a total of c.30 min, when including a rapid extraction step (8 min). Herein, we provide tools for rapid, presumptive detection of the North American corn snake from trace DNA samples in Australian biosecurity and wildlife compliance settings.