Programmable SARS-CoV-2 mutation detection platform based on ligation-triggered isothermal exponential amplification coupled with self-priming amplification

Abstract

In this work, we developed a programmable mutation detection platform with a dual amplification strategy for universal detection of SARS-CoV-2 mutations based on ligation-triggered isothermal exponential amplification reaction(L-EXPAR) and phosphorothioate modified self-priming amplification (PS-SPA). In the presence of mutant target (MT) sequence, it complements with hairpin probe (H1) and hairpin probe (H2) and triggered L-EXPAR at lower temperature. A large number of single-strand DNA (ssDNA) is produced in the circle of ligation, nicking, polymerization, and strand displacement. The ssDNA products containing target sequences can feed back and initiate another L-EXPAR. The ligated H1-H2 complex triggers another isothermal process termed PS-PSA at higher temperature. 20 bases of H1-H2 complex on the 5′ end modified with PS can form a short stem-loop hairpin due to the lower Tm of PS modification. The resulting overhanging 3′ end can also fold back as a new primer to initiate the next round of polymerization. In the circle of PS-SPA, the H1-H2 chain can be extended to be a long hairpin concatemer containing a great number of tandem target sequences. The resulting DNA products can be monitored in real-time fluorescent signaling. In the presence of the SARS-CoV-2 wild type (WT) sequence, the ligation between H1 and H2 cannot happen due to the mismatch of the last base on the 3′ end of H1. The proposed assay displayed a good linear range from 1 fM to 1 nM with a detection limit of femtomole level for the detection of SARS-CoV-2 spike gene D614G mutation and Del246-252 + D253N deletion.