Differentiation of Leishmania in the Sand fly Vector

Multiplex assays will be developed for identifying Leishmania from sand flies and for determining the species of the parasites. A multiplex PCR will be developed to amplify the gene loci needed to distinguish the closely related species of the pathogenic species. Analysis of the PCR products will be performed on an electronic microarray, a method that is well suited for the rapid analysis of complex gene products. Electronic addressing of the target and hybridization occurs within 1 2 minutes. This method allows high levels of multiplex analysis and is excellent at detecting point mutations as well deletions/insertions. The assay will be adapted to a small, inexpensive point-ofcare instrument that will integrate the nucleic acid sample preparation with amplification and the electronic microarray detection. This approach offers a rapid method of detection coupled with high levels of multiplexing which will facilitate the identification of multiple species of pathogenic Lieshmania and allow them to be distinguished from the non-pathogenic species with which they coexist in the environment. The combination of automated sample preparation with rapid PCR and rapid detection method will allow a time to result of less than 4 hours making this assay suitable for use in small labs or remote locations.