A Real Time PCR Assay for Quantification of Parasite Burden in Murine Models of Leishmaniasis

Abstract

Eukaryotic parasites in the genus Leishmania place approximately 350 million people per year at risk of disease. In addition to their global health significance, Leishmania spp. have served as an important model for delineating basic concepts in immunology such as T-helper cell polarization. There have been many qPCR based assays reported for measuring parasite burden in humans and animals. However, these are largely optimized for use in clinical diagnosis and not specifically for animal models. This has led several of these assays to have suboptimal characteristics for use in animal models. For example, multi-copy number genes have been frequently used to increase sensitivity, but are subject to greater plasticity within the genome and thus may confound effects of experimental manipulations in animal models. In this study, we develop a sybr-green based quantitative touchdown PCR assay for a highly conserved and single copy, putative RNA binding protein, DRBD3. With primers nearly perfectly conserved across all Leishmania spp., this assay rivals the sensitivity of previously reported qPCR based methods of parasite quantitation and successfully detected L. major from mouse infection. Use of this protocol in the future will lead to improved accuracy in animal based models and help to tease apart differences in biology of host-parasite interactions.