Abstract

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77–100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

中文翻译：

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Mucorales PCR 检测血清标本的实验室间评估：真菌 PCR 倡议和 Modimucor 研究组的一项研究

摘要

开发了毛霉菌 qPCR 检测的实验室间评估，以评估当前使用的方法的重现性和性能。参与者包括来自法国大学医院的 12 个实验室（其中 9 个参与 Modimucor 研究）和 11 个参与真菌 PCR 计划的实验室。对于图 1，三个血清分别加入来自三个不同物种（Rhizomucor pusillus、Lichtheimia corymbifera、Rhizopus oryzae）的DNA 。对于第 2 组，含三种浓度的R. pusillus和L. corymbifera 的六种血清(1、10 和 100 个基因组/毫升)。每个面板包括盲阴性对照血清。每个小组分发了一张表格，用于收集结果和所需的技术信息，包括 DNA 提取方法、使用的样本量、DNA 洗脱量、qPCR 方法、qPCR 模板输入量、qPCR 总反应量、qPCR 平台和使用的 qPCR 试剂。对于面板 1，评估 18 种不同的协议，定性结果（阳性或阴性）在 97% 的案例中是正确的 (70/72)。观察到 Cq 值（SD = 1.89 个循环）的实验室间变异性非常低。对于评估 26 种不同方案的第 2 组，5/6 加标血清的检出率很高 (77–100%)。qPCR 平台与性能之间存在显着关联。然而，某些技术步骤和因素的最佳组合也可能影响性能。本研究中证明的良好重现性和性能支持使用毛霉菌 qPCR 作为毛霉菌病诊断策略的一部分。