Fluorometric Detection of SARS-CoV-2 Single-Nucleotide Variant L452R Using Ligation-Based Isothermal Gene Amplification

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses have emerged globally. As conventional method, whole-genome sequencing following polymerase chain reaction (PCR) is currently used for diagnosis of SARS-CoV-2 mutations. However, these PCR-based direct DNA methods are time-consuming, complicated, require expensive modules. Here, we developed a fluorometric method accurate detection single missense mutation U to G in spike (S) gene that changes leucine arginine (L452R) genomic RNA. Our single-nucleotide mutations (SNM) viral RNA genome includes sequence-dependent ligation tandem isothermal amplification methods, such as strand displacement (SDA) rolling circle (RCA) generating G-quadruplex (GQ). In presence SNM RNA, both ends probe DNAs occurs between 5′-phosphorylated hairpin linear can discriminate base mismatch. The ligated were then extended generate long-stem subjected (SDA). SDA produces multitudes short ssDNA from DNAs, which serve primers by annealing circular padlock second (RCA). RCA long stretch containing GQ structures. Thioflavin T (ThT) intercalated into emits green fluorescence, allows identification variants. This analysis sensitively distinguished L452R low 10 pM within h. Hence, this using ligation-assisted be applied with high accuracy sensitivity, without need cumbersome sequencing.