A Smart Single‐Loop‐Mediated Isothermal Amplification Facilitates Flexible SNP Probe Design for On‐Site Rapid Differentiation of SARS‐CoV‐2 Omicron Variants

Rapid on-site typing methods for SARS-CoV-2 variants of concern are crucial for its effective surveillance and control. Herein, a smart single-loop-mediated isothermal amplification (ssLAMP) method with the absence of an inner primer but the addition of a swarm primer for differentiation of SARS-CoV-2 Omicron variants is developed. This unique primer design strategy offers greater flexibility in introducing single nucleotide polymorphism (SNP) identification probes and enables multiple detection assays for SARS-CoV-2 Omicron variants including BA.1, BA.2, BA.3, BA.4, and BA.5. A 3D-printed portable dual fluorescence visualization device and smartphone app are developed to enable point-of-care testing. This assay is rapid (within 90 min), highly sensitive (100 copies/reaction), and specific (identification of SNP) for SARA-CoV-2 Omicron variants. The ssLAMP method identifies five BA.5-positive samples among 97 nasopharyngeal swab samples from the clinic, with a 100% concordance rate with Sanger sequencing. The ssLAMP assay system is expected to be utilized for on-site, highly specific, and rapid visualization detection of SARS-CoV-2 and its variants, with great application potential in pathogen genotyping, early cancer screening, and other areas of SNP mutation detection.