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

Jingyi Si; Yifan Gao; Zhenzhou Yang; Gang Liu; Xizhong Shen; Qunyan Yao; Changfeng Zhu

doi:10.1021/jacsau.5c01259

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

JACS Au. 2025 Oct 31;5(11):5778-5787. doi: 10.1021/jacsau.5c01259. eCollection 2025 Nov 24.

Authors

Jingyi Si^{1

2}, Yifan Gao^{1

2}, Zhenzhou Yang³, Gang Liu³, Xizhong Shen^{1

2}, Qunyan Yao^{1

4

2}, Changfeng Zhu^{1

2}

Affiliations

¹ Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, Shanghai 200437, China.
² Shanghai Institute of Liver Diseases, Shanghai 200032, China.
³ Key Laboratory of Bioanalysis and Metrology for State Market Regulation, Shanghai Institute of Measurement and Testing Technology, Shanghai 201103, China.
⁴ Department of Gastroenterology and Hepatology, Zhongshan Hospital (Xiamen), Fudan University, Xiamen 361006, China.

Abstract

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.

Keywords: DNA circuit; hepatitis B virus; isothermal amplification; logic gate.