Rana Divya Rsjb, Pokhrel Nischal, Dulal Santosh
Hari Khetan Multiple Campus, Tribhuvan University, Birgunj, Nepal.
B and B Medical Institute, Lalitpur, Nepal.
Adv Virol. 2022 May 13;2022:2965666. doi: 10.1155/2022/2965666. eCollection 2022.
The genome sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been evolving via genomic drifts resulting in "emerging/drifting variants" circulating worldwide. The construction of polymerase chain reaction (PCR) assays for the reliable, efficient, and specific diagnosis of the drifting variants of SARS-CoV-2 is specifically governed by the selection and construction of primers and probes. The efficiency of molecular diagnosis is impacted by the identity/homology of the genome sequence of SARS-CoV-2 with other coronaviruses, drifting variants or variants of concern (VOCs) circulating in communities, inherent capacity of mutation(s) of various target genes of SARS-CoV-2, and concentration of genes of interest in host cells. The precise amplicon selection and construction of primers and probes for PCR-based assays can efficiently discriminate specific SARS-CoV-2 drifting variants. The construction of single nucleotide polymorphism (SNP)-specific primers and probes for PCR assays is pivotal to specifically distinguish SARS-CoV-2 variants present in the communities and contributes to better diagnosis and prevention of the ongoing COVID-19 pandemic. In this study, we have utilized -based bioinformatic tools where the alignment for genes, the positions and types of SNPs/mutations of VOCs, and the relative number of SNPs per nucleotide in different genomic regions were investigated. Optimal and specific genome region (amplicon) selection with comparatively lower mutability in the SARS-CoV-2 genome should be prioritized to design/construct PCR assays for reliable and consistent diagnosis in various regions of the world for a longer duration of time. Further, the rational selection of target genes that is at an optimal detectable concentration in biological samples can bolster PCR assays of high analytical sensitivity. Hence, the construction of primers and probes with the rational selection of targeting specific E gene, genomic regions with highly conserved sequences, multiple target genes with relatively lower mutability and detectable level of concentration, SNP-specific binding regions of spike (S gene) protein, and shorter amplicon size (100-150 bp) are vital for the PCR assays to achieve optimal efficiency in the point-of-care laboratory diagnosis of circulating drifting variants of SARS-CoV-2 with optimal accuracy.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的基因组序列一直在通过基因组漂移而演变，导致“新兴/漂移变体”在全球传播。用于可靠、高效和特异性诊断SARS-CoV-2漂移变体的聚合酶链反应（PCR）检测方法的构建，具体取决于引物和探针的选择与构建。分子诊断的效率受到SARS-CoV-2基因组序列与其他冠状病毒、社区中传播的漂移变体或关注变体（VOC）的同一性/同源性、SARS-CoV-2各种靶基因的固有突变能力以及宿主细胞中感兴趣基因的浓度的影响。基于PCR的检测方法中精确的扩增子选择以及引物和探针的构建，可以有效区分特定的SARS-CoV-2漂移变体。用于PCR检测的单核苷酸多态性（SNP）特异性引物和探针的构建，对于特异性区分社区中存在的SARS-CoV-2变体至关重要，有助于更好地诊断和预防正在发生的COVID-19大流行。在本研究中，我们利用了基于生物信息学的工具，研究了基因的比对、VOC的SNP/突变的位置和类型，以及不同基因组区域中每个核苷酸的SNP相对数量。应优先选择SARS-CoV-2基因组中具有相对较低变异性的最佳和特异性基因组区域（扩增子），以设计/构建PCR检测方法，以便在世界各地区进行更长时间的可靠和一致的诊断。此外，合理选择在生物样品中处于最佳可检测浓度的靶基因，可以加强具有高分析灵敏度的PCR检测。因此，合理选择靶向特定E基因、具有高度保守序列的基因组区域、具有相对较低变异性和可检测浓度水平的多个靶基因、刺突（S基因）蛋白的SNP特异性结合区域以及较短的扩增子大小（100-150 bp）来构建引物和探针，对于PCR检测在即时检验实验室诊断SARS-CoV-2循环漂移变体时以最佳准确性实现最佳效率至关重要。
