AND Logic Gate-Based Dual-Specificity DNA Circuit for Isothermal HBV rcDNA Detection

The quantitative analysis of blood HBV DNA is essential for controlling the spread of HBV and improving the prognosis of individuals infected with the virus. Yet the HBV genome exhibits complex variation, such as multiple-site mutations for entecavir resistance, closely adjacent mutation loci, and the need to distinguish missense from nonsense mutations due to genetic code degeneracy and amino acid mutation conservatism, creating challenges for detection specificity and mutation identification. Herein, this study introduced an AND logic gate-based dual-specificity DNA circuit for HBV DNA detection, integrating toehold-mediated DNA strand displacement and RNase H cleavage through a well-designed probe containing an identification element and an amplification element. By this DNA circuit, we achieved enhanced sensitivity (limit of detection: 29.11 fM) and high specificity to discriminate adjacent single-base mismatches with excellent performance in clinical samples. We envision that this innovative and convenient assay advances point-of-care HBV DNA testing.