Paul Deepjyoti, Verma Jyoti, Kumar Shakti, Talukdar Daizee, Jana Pradipta, Narendrakumar Lekshmi, Kumar Roshan, Tanwar Subhash, Gosain Mudita, Porey Karmakar Sonali, Pareek Madhu, Mani Shailendra, Chaudhuri Susmita, Kshetrapal Pallavi, Wadhwa Nitya, Bhatnagar Shinjini, Garg Pramod Kumar, Das Bhabatosh
Functional Genomics Laboratory, Centre for Microbial Research, Translational Health Science and Technology Institute, Faridabad, India.
Maternal and Child Health, Translational Health Science and Technology Institute, Faridabad, India.
Front Microbiol. 2024 Oct 21;15:1459644. doi: 10.3389/fmicb.2024.1459644. eCollection 2024.
Delta and Omicron variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are remarkably contagious, and have been recognized as variants of concern (VOC). The acquisition of spontaneous substitutions or insertion-deletion mutations (indels) in the spike protein-encoding gene substantially increases the binding affinity of the receptor binding domain (RBD)-hACE2 complex and upsurges the transmission of both variants. In this study, we analyzed thousands of genome sequences from 30 distinct SARS-CoV-2 variants, focusing on the unique nucleic acid signatures in the spike gene specific to the Delta and Omicron variants. Using these variant-specific sequences, we synthesized a range of oligonucleotides and optimized a multiplex PCR (mPCR) assay capable of accurately identifying and differentiating between the Delta and Omicron variants. Building on this mPCR assay, we developed a dipstick format by incorporating a tag linker sequence at the 5' end of the forward primer and adding biotin to the 3' end of the oligonucleotides, enhancing the assay's usability and accessibility. Streptavidin-coated latex beads and the dipstick imprinted with a probe for the tag linker sequence in the test strips were used for the detection assay. Our dipstick-based assay, developed as a rapid point-of-care test for identifying and differentiating SARS-CoV-2 variants has the potential to be used in low-resource settings and scaled up to the population level.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的德尔塔变异株和奥密克戎变异株具有极强的传染性，已被认定为值得关注的变异株（VOC）。在刺突蛋白编码基因中获得自发替换或插入缺失突变（indels）会大幅增加受体结合域（RBD）-人血管紧张素转换酶2（hACE2）复合物的结合亲和力，并加剧这两种变异株的传播。在本研究中，我们分析了来自30种不同SARS-CoV-2变异株的数千个基因组序列，重点关注德尔塔变异株和奥密克戎变异株特有的刺突基因中的独特核酸特征。利用这些变异株特异性序列，我们合成了一系列寡核苷酸，并优化了一种多重聚合酶链反应（mPCR）检测方法，该方法能够准确识别和区分德尔塔变异株和奥密克戎变异株。基于这种mPCR检测方法，我们通过在正向引物的5'端掺入一个标签连接序列，并在寡核苷酸的3'端添加生物素，开发了一种试纸条形式，提高了检测方法的实用性和可及性。用链霉亲和素包被的乳胶珠和在试纸条中印上用于检测标签连接序列探针的试纸条进行检测。我们开发的基于试纸条的检测方法作为一种用于识别和区分SARS-CoV-2变异株的快速即时检测方法，有潜力在资源匮乏地区使用并扩大到人群水平。
