Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka, 1000, Bangladesh; Department of Biochemistry & Molecular Biology, Tejgaon College, Tejgaon, Dhaka, 1215, Bangladesh.
Molecular Division, OMC Healthcare (Pvt.) Ltd, Rupnagar, Dhaka, 1216, Bangladesh.
J Virol Methods. 2021 Jul;293:114147. doi: 10.1016/j.jviromet.2021.114147. Epub 2021 Apr 1.
The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic is posing a great threat to global health and economy. Due to the lack of broad diagnostic setup, consistent reagent supply lines, and access to laboratory instruments and equipment, it is undoubtedly an enormous burden for developing countries to face the crisis.
To develop a cost-effective, reliable and sensitive multiplex assay for SARS-CoV-2 screening which would expand the testing capacities of a developing and low-income country like Bangladesh.
Initially a singleplex and then a multiplex real-time reverse-transcriptase PCR assays were developed targeting 2 nucleocapsid genes of SARS-CoV-2, and the human RNase P gene as an internal control using laboratory-made mastermixes. Three sets of primer- probes were designed for each of the target genes and one set was optimized for the final reaction set-up. Limit of detection, cross-reactivity and reproducibility were checked in order to assess the sensitivity and specificity of the assays, and validation was done using clinical specimens.
Clinical evaluation of the new assays using 240 nasopharyngeal swabs showed 100 % sensitivity, specificity, and accuracy in detecting SARS-CoV-2 infection in human. Equal efficiency and concordant results were observed between the singleplex and multiplex approaches. Notably, the kit was able to detect SARS-CoV-2 RNA at very low concentration upto 5 copies/reaction.
This is the first locally developed multiplex rRT-PCR kit in Bangladesh providing rapid and low-cost screening of COVID-19 which would be valuable for infection prevention and clinical management in the perspective of Bangladesh.
严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）的出现对全球健康和经济构成了巨大威胁。由于缺乏广泛的诊断设备、稳定的试剂供应线以及实验室仪器和设备的获取，发展中国家无疑面临着巨大的危机应对负担。
开发一种具有成本效益、可靠且敏感的 SARS-CoV-2 筛查多重检测方法，以扩大孟加拉国等发展中国家和低收入国家的检测能力。
最初开发了一种单重实时逆转录-聚合酶链反应（rRT-PCR）检测方法，然后开发了一种多重 rRT-PCR 检测方法，该方法针对 SARS-CoV-2 的 2 个核衣壳基因和人类 RNase P 基因作为内参，使用实验室自制的主混合物。为每个靶基因设计了 3 组引物-探针，其中一组针对最终反应方案进行了优化。为了评估检测方法的灵敏度和特异性，检查了检测限、交叉反应性和重现性，并使用临床标本进行了验证。
使用 240 份鼻咽拭子对新检测方法进行临床评估显示，该方法在检测人类 SARS-CoV-2 感染方面具有 100%的灵敏度、特异性和准确性。单重和多重方法的效率和结果均一致。值得注意的是，该试剂盒能够检测到非常低浓度的 SARS-CoV-2 RNA，低至 5 个拷贝/反应。
这是孟加拉国第一个本地开发的多重 rRT-PCR 试剂盒，提供了 COVID-19 的快速和低成本筛查，从孟加拉国的角度来看，这对感染预防和临床管理具有重要价值。
