Ramírez Juan Carlos, da Cruz Moreira Otacilio
Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres" (INGEBI), CONICET, Buenos Aires, Argentina.
Laboratório de Biología Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, Brazil.
Methods Mol Biol. 2019;1955:215-225. doi: 10.1007/978-1-4939-9148-8_16.
The development of accurate diagnostic tools and surrogate markers of parasitological response to treatment are priorities in Chagas disease (CD) research. For years, the detection of Trypanosoma cruzi DNA by PCR has proved to be useful in some clinical scenarios like acute CD, including cases of congenital transmission, CD reactivation in immunosuppressed patients, and posttreatment follow-up. In that sense, the implementation of quantitative real-time PCR (qPCR) assays was an important step in the development of more reliable tools for CD molecular diagnostics and treatment follow-up. In the last decade, two multicenter PCR studies allowed the harmonization and validation of standard operating procedures for PCR-based detection and quantification of T. cruzi DNA in blood samples. Herein we describe the two most used protocols to quantify parasitic load in human blood samples by multiplex qPCR assays and discuss some aspects to consider during planning and executing these procedures.
开发精确的诊断工具以及寄生虫学治疗反应替代标志物是恰加斯病（CD）研究的重点。多年来，通过聚合酶链反应（PCR）检测克氏锥虫DNA已被证明在某些临床情况下有用，如急性CD，包括先天性传播病例、免疫抑制患者的CD再激活以及治疗后随访。从这个意义上说，定量实时PCR（qPCR）检测的实施是开发更可靠的CD分子诊断和治疗随访工具的重要一步。在过去十年中，两项多中心PCR研究实现了基于PCR的血液样本中克氏锥虫DNA检测和定量标准操作程序的协调与验证。在此，我们描述了通过多重qPCR检测定量人类血液样本中寄生虫载量的两种最常用方案，并讨论了在规划和执行这些程序时需要考虑的一些方面。
