The emergence of SARS-CoV-2 mutations resulting in the S protein amino-acid substitutions N501Y and E484K, which have been associated with enhanced transmissibility and immune escape, respectively, necessitates immediate actions, for which their rapid identification is crucial. For the simultaneous typing of both of these mutations of concern (MOCs), a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed. The assay is highly sensitive with a LOD of 117 copies/reaction, amplification efficiencies >94 % and a linear range of over 5 log₁₀ copies/reaction. Validation of the assay using known SARS-CoV-2-positive and negative samples from human and animals revealed its ability to correctly identify wild type strains, and strains possessing either one or both targeted amino-acid substitutions, thus comprising a useful pre-screening tool for rapid MOC identification. The basic principles of the methodology for the development of the assay are explained in order to facilitate the rapid design of similar assays able to detect emerging MOCs.

SARS-CoV-2 突变的出现导致 S 蛋白氨基酸取代 N501Y 和 E484K，这两种突变分别与增强的传播性和免疫逃逸有关，因此需要立即采取行动，对其快速识别至关重要。为了同时对这两种相关突变 (MOC) 进行分型，开发了一种使用四个锁核酸 (LNA) 修饰的 TaqMan 探针的一步式实时 RT-PCR 测定法。_{该检测具有 117 个拷贝/反应的 LOD、扩增效率 >94% 和超过 5 log 10}的线性范围的高灵敏度副本/反应。使用来自人类和动物的已知 SARS-CoV-2 阳性和阴性样本对该测定法进行验证，表明其能够正确识别野生型菌株，以及具有一个或两个目标氨基酸取代的菌株，从而构成有用的预筛选用于快速 MOC 识别的工具。为了促进能够检测新出现的 MOC 的类似检测的快速设计，解释了检测开发方法的基本原则。