Universal Exponential Amplification Confers Multilocus Detection of Mutation-Prone Virus.

The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has spread rapidly around the world. Accurate and scalable diagnostics are essential for immediate intervention and control of viral transmission. Currently reported diagnostics are rapid and sensitive, yet most are limited by their principle of single-locus identification and suffer from false-negative results because of the mutation-prone nature of RNA viruses. Here, we propose a multilocus detection method for SARS-CoV-2 based on a modified loop-mediated isothermal amplification with a pair of universal primers. The sequence-specific probes are designed to recognize the sequence of nucleocapsid protein (N) and the open reading frame 1ab (Orf1ab) gene from the SARS-CoV-2 genome. In the presence of a target locus, separated probes are ligated to be an intact template, the bipartite ends of which are repetitive sequences for the sequential binding of universal primers to initiate strand displacement. A kind of flap structure-dependent endonuclease is involved in cleaving multicolor TaqMan probes during multiplex amplification, realizing a real-time and multiplex analysis. We evaluated the quantitative performance of the developed method with spiked samples using synthetic target RNA, resulting in a limit of detection as low as 250 aM. Furthermore, the feasibility of multilocus detection was validated using various mutation-prone genes, demonstrating a significant potential for accurate analysis of SARS-CoV-2 and holding great promise for the clinical diagnosis of other infectious diseases.