Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 nanograms of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.

Assessment of Fungal Succession in Decomposing Swine Carcasses (Sus scrofa L.) Using DNA Metabarcoding.

The decomposition of animal bodies is a process defined by specific stages, described by the state of the body and participation of certain guilds of invertebrates and microorganisms. While the participation of invertebrates in decomposing is well-studied and actively used in crime scene investigations, information on bacteria and fungi from the scene is rarely collected or used in the identification of important factors such as estimated time of death. Modern molecular techniques such as DNA metabarcoding allow the identification and quantification of the composition of microbial communities. In this study, we used DNA metabarcoding to monitor fungal succession during the decomposition of juvenile pigs in grasslands of New Jersey, USA. Our findings show that decomposition stages differ in a diversity of fungal communities. In particular, we noted increased fungal species richness in the more advanced stages of decomposition (e.g., bloat and decay stages), with unique fungal taxa becoming active with the progression of decay. Overall, our findings improve knowledge of how fungi contribute to forensically relevant decomposition and could help with the assessment of crime scenes.