Animal Production and Health Laboratory, Joint FAO/IAEA Centre for Nuclear Applications in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Friedenstrasse 1, A-2444, Seibersdorf, Austria.
Animal Production and Health Laboratory, Joint FAO/IAEA Centre for Nuclear Applications in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Friedenstrasse 1, A-2444, Seibersdorf, Austria.
J Virol Methods. 2021 Sep;295:114200. doi: 10.1016/j.jviromet.2021.114200. Epub 2021 Jun 1.
Transmission mitigation of SARS-CoV-2 requires the availability of accurate and sensitive detection methods. There are several commercial ad hoc molecular diagnostic kits currently on the market, many of which have been evaluated by different groups. However, in low resource settings the availability and cost of these commercial kits can be a limiting factor for many diagnostic laboratories. In such cases alternatives need to be identified. With this in mind, eight commercial reverse transcription quantitative real-time PCR (RT-qPCR) master mixes from Applied Biosystems (Thermo Fisher Scientific), Bio-Rad, Biotech Rabbit, Promega, Qiagen, QuantaBio, Invitrogen (Thermo Fisher Scientific) and Takara using the same commercial primer and probe mix [LightMix® Modular SARS and Wuhan CoV E-gene mix (TIB MolBiol, Germany)] were evaluated. Three ad hoc molecular diagnostic kits [GeneFinder™ COVID-19 Plus RealAmp kit (Osang Healthcare); genesig® Real-Time PCR Coronavirus COVID-19 (Primerdesign); and ViroReal® Kit SARS-CoV-2 & SARS-CoV (Ingenetix)] were also included in the study. The limit of detection was calculated for each assay using serial dilutions of a defined clinical sample. The performances of the assays were compared using a panel of 178 clinical samples and their analytical specificity assessed against a panel of human betacoronaviruses. Inter assay agreement was assessed using statistical tests (Bland-Altman, Fleiss-Kappa and Cohen's Kappa) and was shown to be excellent to good in all cases. We conclude that all of the assays evaluated in this study can be used for the routine detection of SARS-CoV-2 and that the RT-qPCR master mixes are a valid alternative to ad hoc molecular diagnostic kits.
SARS-CoV-2 的传播抑制需要准确和敏感的检测方法。目前市场上有几种商业专用分子诊断试剂盒，其中许多已被不同的小组进行了评估。然而，在资源有限的情况下，这些商业试剂盒的可用性和成本可能是许多诊断实验室的一个限制因素。在这种情况下，需要确定替代方案。考虑到这一点，我们评估了来自 Applied Biosystems（Thermo Fisher Scientific）、Bio-Rad、Biotech Rabbit、Promega、Qiagen、QuantaBio、Invitrogen（Thermo Fisher Scientific）和 Takara 的八种商业逆转录定量实时 PCR（RT-qPCR）主混合物，这些主混合物使用相同的商业引物和探针混合物[LightMix® Modular SARS 和 Wuhan CoV E-gene mix（TIB MolBiol，德国）]。该研究还包括三种专用分子诊断试剂盒[GeneFinder™ COVID-19 Plus RealAmp 试剂盒（Osang Healthcare）；genesig® Real-Time PCR Coronavirus COVID-19（Primerdesign）；和 ViroReal® Kit SARS-CoV-2 & SARS-CoV（Ingenetix）]。使用定义明确的临床样本的系列稀释液计算每个检测的检测限。使用 178 个临床样本的面板比较检测的性能，并针对人类贝塔冠状病毒面板评估其分析特异性。使用统计检验（Bland-Altman、Fleiss-Kappa 和 Cohen's Kappa）评估了试验间的一致性，结果表明在所有情况下均为优秀至良好。我们得出结论，本研究评估的所有检测均可用于 SARS-CoV-2 的常规检测，RT-qPCR 主混合物是专用分子诊断试剂盒的有效替代方案。
