Target recycling amplification (TRA) combined with multiple strand displacement amplification (SDA) for sensitive detection of Epstein-Barr virus microRNA

Objectives: Epstein-Barr virus (EBV) infection is strongly associated with the development of nasopharyngeal carcinoma. However, existing diagnostic methods based on EBV antibodies and plasma DNA exhibit insufficient sensitivity and specificity for early detection. This study aimed to overcome this limitation by developing a highly sensitive and specific method for detecting the EBV-encoded biomarker microRNA miR-BART6-3p. Methods: We designed a probe (EB4) containing a C-rich sequence, a restriction endonuclease half-recognition site, a G-rich stem-loop structure, and a target recognition domain. Based on this probe, an isothermal fluorescence platform was developed by integrating target recycling amplification (TRA) with strand displacement amplification (SDA). The detection mechanism relies on miR-BART6-3p initiating a polymerase-endonuclease cycle, which generates G-quadruplex structures and target-like DNA. The fluorescence signal is produced when Thioflavin T (ThT) binds to these G-quadruplexes. The sensitivity, specificity, and anti-interference capability of the method were systematically evaluated. Results: The assay exhibited a broad linear detection range for miR-BART6-3p, spanning from 1 pM to 100 nM, with an ultra-low detection limit of 0.143 pM, thereby demonstrating significantly enhanced sensitivity compared to conventional methods. The assay also displayed high specificity, effectively differentiating targets with single-base mismatches. Clinical evaluation using serum samples revealed markedly elevated fluorescence signals in EBV-positive patients relative to healthy controls. Furthermore, the platform exhibited strong anti-interference capability, ensuring reliable performance under complex biological conditions. Conclusions: This study successfully developed a one-step, single-probe method for detecting EBV miRNA (miR-BART6-3p) with high sensitivity and specificity. The TRA-SDA platform provides operational simplicity, high interference resistance, and superior diagnostic performance. This innovative approach shows great clinical application prospects as a molecular diagnostic tool for the early detection of nasopharyngeal carcinoma.