Validation of quantitative real-time PCR for the in vitro assessment of antileishmanial drug activity

Abstract

In vitro assays play an important role in the discovery and development of new antileishmanial drugs. The classic macrophage-amastigote models using murine peritoneal macrophages or human-monocyte derived macrophages as host cells are useful for drug screening. A major limitation of these models is the dependence on microscopic counting, a time-consuming and subjective method of analysis. The present study describes a detailed protocol for applying quantitative real-time PCR (qPCR) as an accurate and sensitive tool to assess parasite load in an amastigote-macrophage model. This assay can be performed in a standardized medium-to-high throughput procedure, replacing traditional readout of number of amastigote per macrophages by DNA load measurement.