Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
Kariminejad-Najmabadi Pathology & Genetics Center, Tehran, Iran.
Arch Iran Med. 2023 Feb 1;26(2):69-75. doi: 10.34172/aim.2023.12.
Global real-time monitoring of SARS-CoV-2 variants is crucial to controlling the COVID-19 outbreak. The purpose of this study was to set up a Sanger-based platform for massive SARS-CoV-2 variant tracking in laboratories in low-resource settings.
We used nested RT-PCR assay, Sanger sequencing and lineage assignment for 930-bp of the SARS-CoV-2 spike gene, which harbors specific variants of concern (VOCs) mutations. We set up our platform by comparing its results with whole genome sequencing (WGS) data on 137 SARS-CoV-2 positive samples. Then, we applied it on 1028 samples from March-September 2021.
In total, 125 out of 137 samples showed 91.24% concordance in mutation detection. In lineage assignment, 123 out of 137 samples demonstrated 89.78% concordance, 65 of which were assigned as VOCs and showed 100% concordance. Of 1028 samples screened by our in-house method, 78 distinct mutations were detected. The most common mutations were: S:D614G (21.91%), S:P681R (12.19%), S:L452R (12.15%), S:T478K (12.15%), S:N501Y (8.91%), S:A570D (8.89%), S:P681H (8.89%), S:T716I (8.74%), S:L699I (3.50%) and S:S477N (0.28%). Of 1028 samples, 980 were attributed as VOCs, which include the Delta (B.1.617.2) and Alpha (B.1.1.7) variants.
Our proposed in-house Sanger-based assay for SARS-CoV-2 lineage assignment is an accessible strategy in countries with poor infrastructure facilities. It can be applied in the rapid tracking of SARS-CoV-2 VOCs in the SARS-CoV-2 pandemic.
全球实时监测 SARS-CoV-2 变体对于控制 COVID-19 疫情至关重要。本研究旨在建立一种基于 Sanger 的平台，用于在资源匮乏环境下的实验室中大规模追踪 SARS-CoV-2 变体。
我们使用嵌套 RT-PCR 检测、Sanger 测序和针对 SARS-CoV-2 刺突基因 930 个碱基的谱系赋值，该基因包含特定的关注变体（VOC）突变。我们通过将其结果与 137 个 SARS-CoV-2 阳性样本的全基因组测序（WGS）数据进行比较来建立我们的平台。然后，我们在 2021 年 3 月至 9 月期间应用于 1028 个样本。
在总共 137 个样本中，125 个样本在突变检测中显示出 91.24%的一致性。在谱系赋值方面，137 个样本中有 123 个显示出 89.78%的一致性，其中 65 个被归类为 VOCs，且显示出 100%的一致性。通过我们的内部方法筛选的 1028 个样本中检测到 78 个不同的突变。最常见的突变是：S:D614G（21.91%）、S:P681R（12.19%）、S:L452R（12.15%）、S:T478K（12.15%）、S:N501Y（8.91%）、S:A570D（8.89%）、S:P681H（8.89%）、S:T716I（8.74%）、S:L699I（3.50%）和 S:S477N（0.28%）。在 1028 个样本中，980 个被归类为 VOCs，包括德尔塔（B.1.617.2）和阿尔法（B.1.1.7）变体。
我们提出的用于 SARS-CoV-2 谱系赋值的基于 Sanger 的内部检测方法是基础设施较差的国家的一种可行策略。它可应用于 SARS-CoV-2 大流行中对 SARS-CoV-2 VOCs 的快速追踪。
