El-Kafrawy Sherif A, El-Daly Mai M, Hassan Ahmed M, Harakeh Steve M, Alandijany Thamir A, Azhar Esam I
Special Infectious Agents Unit-BSL3, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
Diagnostics (Basel). 2022 Mar 28;12(4):828. doi: 10.3390/diagnostics12040828.
The global pandemic coronavirus SARS-CoV-2 has a healthcare, social and economic burden. To limit the spread of the virus, the World Health Organization (WHO) urgently called for extensive screening of suspected individuals; thus, a quick, simple, and sensitive diagnostic assay is always in need. We applied reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for the detection of SARS-CoV-2. The RT-LAMP method was optimized by evaluating two fluorescence amplification mixes and several reaction times, and results were compared to the standard real-time RT-PCR (rtRT-PCR). The assay was validated using 200 nasopharyngeal swabs collected in viral transport media (62 positive for SARS-CoV-2, and 138 negative for SARS-CoV-2 detected by the rtRT-PCR method). The samples were diluted 1:4 in diethylpyrocarbonate (DEPC)-treated water, utilized for RT-LAMP using different singleplex and multiplex sets of LAMP primers (N gene, S gene, and orf1ab gene), and incubated at 65 °C using real-time PCR 7500. Our direct detection with the RT-LAMP protocol showed 100% concordance (sensitivity and specificity) with the standard protocol used for the detection of SARS-CoV-2 nucleic acid. In this study, we set up a rapid, simple, and sensitive RT-LAMP assay for the detection of SARS-CoV-2 in clinical samples. The assay is suitable for point of care detection in public hospitals, medical centers in rural areas, and in transportation hubs.
全球大流行的冠状病毒SARS-CoV-2造成了医疗、社会和经济负担。为限制病毒传播，世界卫生组织（WHO）紧急呼吁对疑似个体进行广泛筛查；因此，一直需要一种快速、简便且灵敏的诊断检测方法。我们应用逆转录环介导等温扩增技术（RT-LAMP）检测SARS-CoV-2。通过评估两种荧光扩增混合物和几个反应时间对RT-LAMP方法进行了优化，并将结果与标准实时逆转录聚合酶链反应（rtRT-PCR）进行比较。使用在病毒运输培养基中收集的200份鼻咽拭子（rtRT-PCR方法检测出62份SARS-CoV-2阳性，138份SARS-CoV-2阴性）对该检测方法进行验证。将样本在经焦碳酸二乙酯（DEPC）处理的水中按1:4稀释，使用不同的单重和多重LAMP引物组（N基因、S基因和orf1ab基因）进行RT-LAMP检测，并使用实时荧光定量PCR仪7500在65℃下孵育。我们采用RT-LAMP方案的直接检测结果与用于检测SARS-CoV-2核酸的标准方案显示出100%的一致性（敏感性和特异性）。在本研究中，我们建立了一种快速、简便且灵敏的RT-LAMP检测方法，用于检测临床样本中的SARS-CoV-2。该检测方法适用于公立医院、农村地区医疗中心和交通枢纽的即时检测。
