title: 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.
authors.affiliation: Laboratory of Clinical Research, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz-Fiocruz, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Luciana I
authors.initials: LI
authors.lastname: Gomes
authors.forename: Felipe M
authors.initials: FM
authors.lastname: Gonzaga
authors.forename: Eliane
authors.initials: E
authors.lastname: de Morais-Teixeira
authors.forename: Bruna S
authors.initials: BS
authors.lastname: de Souza-Lima
authors.forename: Verônica V
authors.initials: VV
authors.lastname: Freire
authors.forename: Ana
authors.initials: A
authors.lastname: Rabello
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D016054:DNA, Protozoan / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D020128:Inhibitory Concentration 50; D018314:Leishmania infantum / Q000187:drug effects* / Q000235:genetics; D017737:Macrophages, Peritoneal / Q000469:parasitology*; D051379:Mice; D008807:Mice, Inbred BALB C; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*
chemical_list: D000981:Antiprotozoal Agents; D016054:DNA, Protozoan; D000666:Amphotericin B