Secció de Parasitologia, Departament de Biologia, Sanitat i Medi Ambient, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, Barcelona, Spain.
ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic-Universitat de Barcelona, Barcelona, Spain.
PLoS One. 2018 Apr 17;13(4):e0195738. doi: 10.1371/journal.pone.0195738. eCollection 2018.
Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process.
METHODOLOGY/PRINCIPAL FINDINGS: We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately.
CONCLUSIONS/SIGNIFICANCE: This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.
聚合酶链反应（PCR）已成为诊断克氏锥虫感染的有用工具。自动 DNA 提取方法和 PCR 系统的开发是常规诊断中标准化方案的重要步骤。迄今为止，只有两种市售的实时 PCR 检测方法可用于常规实验室检测临床样本中的 T. cruzi DNA：TCRUZIDNA.CE（Diagnostic Bioprobes Srl）和 RealCycler CHAG（Progenie Molecular）。我们的目的是评估 RealCycler CHAG 检测方法，同时考虑整个过程。
方法/主要发现：我们评估了一种基于磁性颗粒的自动化 DNA 提取系统（EZ1 Virus Mini Kit v2.0，Qiagen）与市售针对 T. cruzi 卫星 DNA（SatDNA）的实时 PCR 检测方法（RealCycler CHAG）相结合的实用性，这种方法用于我们医院的常规诊断。它与一种众所周知的策略进行了比较，该策略结合了一种基于硅基柱的商业 DNA 分离试剂盒（High Pure PCR Template Preparation Kit，Roche Diagnostics）和一种针对 SatDNA 的内部实时 PCR。两种方法的结果几乎完全一致，表明它们可以互换使用。然而，当应用协议因素的变化（样品处理、提取方法和实时 PCR）时，结果就不那么令人信服了。对整个过程进行全面调整是获得成功结果的关键。由于抑制作用，胍 EDTA 血（GEB）样本不适合基于磁性颗粒的 DNA 提取，至少在样本未立即处理时是这样。
结论/意义：这是第一项考虑整个过程（包括三个变量：样品处理、提取方法和实时 PCR）评估 RealCycler CHAG 检测方法的研究。我们的研究结果可能有助于实验室之间方案的协调，并有助于更广泛地将实时 PCR 应用于与保健中心相关的分子诊断实验室。
