Abstract：
The molecular diagnosis of toxoplasmosis lacks standardisation due to the use of numerous methods with variable performance. This diversity of methods also impairs robust performance comparisons between laboratories. The harmonisation of practices by diffusion of technical guidelines is a useful way to improve these performances. The knowledge of methods and practices used for this molecular diagnosis is an essential step to provide guidelines for Toxoplasma-PCR. In the present study, we aimed (i) to describe the methods and practices of Toxoplasma-PCR used by clinical microbiology laboratories in France and (ii) to propose technical guidelines to improve molecular diagnosis of toxoplasmosis. To do so, a yearly self-administered questionnaire-based survey was undertaken in proficient French laboratories from 2008 to 2015, and guidelines were proposed based on the results of those as well as previously published work. This period saw the progressive abandonment of conventional PCR methods, of Toxoplasma-PCR targeting the B1 gene and of the use of two concomitant molecular methods for this diagnosis. The diversity of practices persisted during the study, in spite of the increasing use of commercial kits such as PCR kits, DNA extraction controls and PCR inhibition controls. We also observed a tendency towards the automation of DNA extraction. The evolution of practices did not always go together with an improvement in those, as reported notably by the declining use of Uracil-DNA Glycosylase to avoid carry-over contamination. We here propose technical recommendations which correspond to items explored during the survey, with respect to DNA extraction, Toxoplasma-PCR and good PCR practices.
摘要：
由于使用了许多具有可变性能的方法，弓形虫病的分子诊断缺乏标准化。这种方法的多样性也削弱了实验室之间稳健的性能比较。通过传播技术准则来协调实践是改善这些性能的有用方法。用于这种分子诊断的方法和实践的知识是提供弓形虫PCR指南的重要步骤。在本研究中,我们的目的是(i)描述法国临床微生物学实验室使用的弓形虫-PCR方法和实践;(ii)提出技术指南,以改善弓形虫病的分子诊断.要做到这一点，从2008年至2015年,我们在熟练的法国实验室进行了一项基于问卷调查的年度自我管理式调查,并根据这些调查和以前发表的研究结果提出了指南.这一时期逐渐放弃了传统的PCR方法，针对B1基因的弓形虫PCR以及使用两种伴随的分子方法进行诊断。在研究期间，实践的多样性仍然存在，尽管越来越多地使用商业试剂盒，如PCR试剂盒，DNA提取对照和PCR抑制对照。我们还观察到DNA提取自动化的趋势。实践的演变并不总是伴随着这些方面的改进，据报道，尿嘧啶-DNA糖基化酶的使用正在减少，以避免残留污染。我们在这里提出与调查期间探讨的项目相对应的技术建议，关于DNA提取，弓形虫PCR和良好的PCR实践。
