SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B–F) to evaluate their sensitivity, specificity, predictive values and accuracy. Fifty-two clinical samples were used including throat swabs (n = 30), nasal swabs (n = 7), nasopharyngeal swabs (n = 7) and sputum specimens (n = 8), comprising confirmed (n = 26) and negative cases (n = 26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits’ evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients. For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa ≥ 0.75); Kits D and E were less congruent (0.4 ≤ Kappa < 0.75). Differences between all kits were statistically significant (P < 0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. This is the first comparative study to evaluate CPA and RT-qPCR kits’ specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic.

中文翻译：

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六种用于SARS-CoV-2 RNA检测的核酸扩增试剂盒的比较评价


SARS-CoV-2 是一种新出现的冠状病毒，导致 2019 年 12 月爆发了 2019 年冠状病毒病（COVID-19）。由于 COVID-19 的药物和疫苗仍在开发中，准确的病毒检测在当前公众中发挥着至关重要的作用健康危机。自 COVID-19 爆发以来，定量实时逆转录酶聚合酶链反应 (RT-qPCR) 试剂盒已可靠地用于检测 SARS-CoV-2 RNA，而基于等温核酸扩增的点检测护理自动化套件也被认为是一种更简单、更快速的替代方案。然而，由于这些试剂盒的开发和临床应用时间很短，迄今为止其临床性能尚未得到充分评估。我们描述了新开发的交叉引发等温扩增 (CPA) 试剂盒（试剂盒 A）和五种 RT-qPCR 试剂盒（试剂盒 B–F）之间的比较研究，以评估其敏感性、特异性、预测值和准确性。使用了 52 份临床样本，包括咽拭子（n = 30）、鼻拭子（n = 7）、鼻咽拭子（n = 7）和痰标本（n = 8），其中包括确诊病例（n = 26）和阴性病例（n = 26）。使用六个核酸扩增试剂盒同时对每个样本进行 SARS-CoV-2 检测。使用临床表现和分子诊断作为参考标准评估每个试剂盒的敏感性、特异性、阳性/阴性预测值（PPV/NPV）和准确性。三位不同的操作员使用 SARS-CoV-2 RNA 阳性样本对 RT-qPCR 试剂盒的重现性进行了三次评估。 在六种试剂盒评估结果的基础上，将CPA试剂盒（试剂盒A）和两种RT-qPCR试剂盒（试剂盒B和F）应用于COVID-19患者密切接触者中的SARS-CoV-2检测。对于试剂盒 A，灵敏度、特异性、PPV/NPV 和准确度均为 100%。 5种RT-qPCR试剂盒中，试剂盒B、C、F与临床诊断报告吻合较好（Kappa≥0.75）；套件 D 和 E 一致性较差 (0.4 ≤ Kappa < 0.75)。所有试剂盒之间的差异具有统计学显着性（P< 0.001）。 RT-qPCR 试剂盒的重现性由 0.95% 至 2.57% 之间的变异系数 (CV) 确定，表明重现性良好。这是第一项评估 CPA 和 RT-qPCR 试剂盒检测 SARS-CoV-2 的特异性和敏感性的比较研究，可以作为临床实验室的参考，从而为快速发展的 COVID-19 大流行中的检测方案提供信息。