Development of a real-time polymerase chain reaction assay for the quantification of Leishmania species and the monitoring of systemic distribution of the pathogen

Abstract We have developed a highly accurate and sensitive real-time polymerase chain reaction (PCR) assay to detect and quantify Leishmania parasites. The assay targets GP63 , a highly conserved gene in Leishmania . We demonstrate that, with a single assay, we are able to detect and quantify several species of Leishmania . Our assay system detects Leishmania donovani and Leishmania major down to 0.1 parasite. The dynamic range of the assay extends over 6 log cycles of target, with an average correlation coefficient >0.988. In addition, we utilize a simple approach to distinguish between Leishmania species causing diverse spectra of disease. We have also used this assay to follow the course of cutaneous disease in CBA/CaJ mice, known to be resistant to L. major . The assay is sensitive enough to quantify parasite load in the absence of overt lesions and reveals a systemic distribution of Leishmania , which has implications for our understanding of the disease.