Laboratory of Molecular Virology and Oncology, Department of Bioengineering and Technology, Gauhati University, Guwahati, Assam, 781014, India.
Department of Microbiology, GMCH, Guwahati, Assam, 781032, India.
Sci Rep. 2021 Sep 9;11(1):17878. doi: 10.1038/s41598-021-97502-1.
As the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.
随着 COVID-19 感染继续肆虐全球，高效且经济的现有基于 RT-PCR 的检测方法的出现，在这些检测时代基本上可以成为一种福音，并显著有助于大流行的管理。本研究展示了一种基于创新多重 PCR 的 COVID-19 测试的创新和快速验证。通过使用相同的引物进行快速常规和 HRM 耦合多重实时 PCR，对同时扩增 SARS-CoV-2 基因 E、S 和 RdRp 的最佳 PCR 条件进行了评估。通过扩增来自 COVID-19 疾病存档阳性样本的十倍稀释的已知靶序列，研究了所有具有实际价值的变量。带有新设计的 E、S 和 RdRp 引物的多重扩增显示出对 SARS-CoV-2 靶区域的有效扩增。在热循环仪中，在 37 分钟内观察到明显的扩增，而在 HRM 耦合实时检测中，使用 SYBR 绿在广泛的模板浓度范围内需要 96 分钟。我们的发现与其他市售检测试剂盒具有良好的一致性。这种快速 HRM 耦合多重实时 PCR 与 SYBR 绿方法除了具有用于常规多重 PCR 的引物兼容性的额外优势外，还提供了一种具有成本效益的快速和敏感的 SARS-CoV-2 检测方法。这种高度可重复的新方法除了为资源有限的环境提供缓解之外，还可以推动可持续商业产品的开发和扩展应用。
