Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 pandemic, has infected more than 179 million people worldwide. Testing of infected individuals is crucial for identification and isolation, thereby preventing further spread of the disease. Presently, Taqman™ Reverse Transcription Real Time PCR is considered gold standard, and is the most common technique used for molecular testing of COVID-19, though it requires sophisticated equipments, expertise and is also relatively expensive. Development and optimization of an alternate molecular testing method for the diagnosis of COVID-19, through a two step Reverse Transcription Loop-mediated isothermal AMPlification (RT-LAMP). Primers for LAMP were carefully designed for discrimination from other closely related human pathogenic coronaviruses. Care was also taken that primer binding sites are present in conserved regions of SARS-CoV2. Our analysis shows that the primer binding sites are well conserved in all the variants of concern (VOC) and variants of interest (VOI), notified by World Health Organization (WHO). These lineages include B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427/B.1.429, P.2, B.1.525, P.3, B.1.526 and B.1.617.1. Various DNA polymerases with strand displacement activity were evaluated and conditions were optimized for LAMP amplification and visualization. Different LAMP primer sets were also evaluated using synthetic templates as well as patient samples. In a double blind study, the RT-LAMP assay was validated on more than 150 patient samples at two different sites. The RT-LAMP assay appeared to be 89.2% accurate when compared to the Taqman™ rt-RT-PCR assay.

中文翻译：

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RT-LAMP 检测方法的优化和验证，用于诊断 SARS-CoV2，包括全球主导的 Delta 变体

严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 是 COVID-19 大流行的病原体，已感染全球超过 1.79 亿人。对感染者进行检测对于识别和隔离至关重要，从而防止疾病的进一步传播。目前，Taqman™ 逆转录实时 PCR 被认为是金标准，并且是用于 COVID-19 分子检测的最常用技术，但它需要复杂的设备、专业知识并且也相对昂贵。通过两步逆转录环介导的等温扩增 (RT-LAMP) 开发和优化用于诊断 COVID-19 的替代分子检测方法。LAMP 引物经过精心设计，可与其他密切相关的人类致病冠状病毒区分开来。还注意引物结合位点存在于 SARS-CoV2 的保守区域。我们的分析表明，引物结合位点在世界卫生组织 (WHO) 通知的所有关注变体 (VOC) 和感兴趣的变体 (VOI) 中都非常保守。这些谱系包括 B.1.1.7、B.1.351、P.1、B.1.617.2、B.1.427/B.1.429、P.2、B.1.525、P.3、B.1.526 和 B.1.617 .1. 评估了具有链置换活性的各种 DNA 聚合酶，并优化了 LAMP 扩增和可视化的条件。还使用合成模板和患者样本评估了不同的 LAMP 引物组。在一项双盲研究中，RT-LAMP 检测在两个不同地点的 150 多个患者样本中得到验证。与 Taqman™ rt-RT-PCR 检测相比，RT-LAMP 检测的准确率似乎为 89.2%。