Validation of quantitative real-time PCR for the in vitro assessment of antileishmanial drug activity
- 作者：Luciana I. Gomes ; Felipe M. Gonzaga ; Eliane de Morais-Teixeira ; Bruna S. de Souza-Lima ; Verô ; nica V. Freire ; Ana Rabello ; ana@cpqrr.fiocruz.br
- 关键词：Leishmania ; Drug screening ; In vitro assays ; Quantitative real-time PCR
- 刊名：Experimental Parasitology
- 出版年：2012
- 期刊代码：99_00144894
- 类别：imm
- 出版时间：June, 2012
- 卷：131
- 期：2
- 页码：175-179
- 文件大小：213 K
摘要
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.