Ultra-rapid reverse transcription-free exponential amplification reaction (RTF-EXPAR) technology for hepatitis A virus detection

Abstract Hepatitis A virus (HAV) poses severe food safety threats, yet conventional detection methods suffer from low sensitivity or complex operation. This study established a reverse transcription-free exponential amplification (RTF-EXPAR) coupled with Single-strip nucleic acid test strip for rapid HAV RNA detection. The whole assay completes within 30 min at 50 °C without reverse transcription. After screening three primer pairs, Binder DNA X 2 and Template X′-X′ 2 were confirmed optimal. Specificity tests showed no cross-amplification against other RNA viruses. Serial dilution demonstrated the limit of detection (LOD) reached 1.2 × 10 −2  nmol HAV RNA. Compared with ELISA and RT-PCR, this RTF-EXPAR system requires minimal equipment and delivers visual results rapidly. It is suitable for grassroots medical testing and on-site food safety screening, offering a convenient tool for early HAV surveillance and diagnosis.