Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Department of Pathology, Hospital Sungai Buloh, Selangor, Malaysia.
PLoS One. 2021 Jan 6;16(1):e0245164. doi: 10.1371/journal.pone.0245164. eCollection 2021.
Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.
快速诊断是管理严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 爆发的重要干预措施。实时逆转录聚合酶链反应 (RT-qPCR) 仍然是诊断新病毒株的主要手段,但它既耗时又昂贵。重组酶聚合酶扩增 (RPA) 是一种不需要 PCR 机器的等温扩增检测方法。它是一种经济实惠、快速且简单的检测方法。在这项研究中,我们使用 SYBR Green I 和/或侧流 (LF) 条带开发并优化了一种用于快速检测 SARS-CoV-2 的灵敏 RT-RPA 检测方法。通过使用 10 倍系列稀释的合成 RNA 和类似病毒的基因组 RNA 分别测试了 RT-RPA 检测方法的分析灵敏度和特异性。使用 78 个阳性和 35 个阴性鼻咽样本进行了 RT-RPA 检测方法的临床灵敏度和特异性检测。RPA 和 RT-qPCR 检测方法的检测限分别为 7.659 和 5 拷贝/μL RNA,与其他病毒无交叉反应。RT-RPA 的临床灵敏度和特异性分别为 98%和 100%。我们的研究表明,RT-RPA 是 RT-qPCR 检测 SARS-CoV-2 的一种可行替代方法,尤其是在基础设施有限的地区。
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