A rolling circle mediated exponential amplification reaction with suppressed nonspecific amplification to detect pathogen RNA with high sensitivity.

Respiratory infections caused by pathogens such as influenza virus and SARS-CoV-2 seriously threaten human life and health. RNA has been widely recognized as an important biomarker for diagnosing these pathogens, creating a growing need for rapid and accurate RNA detection methods. Isothermal nucleic acid amplification has emerged as a promising molecular diagnostics approach. Exponential amplification reactions (EXPAR) is a commonly used RNA detection method, known for its simplicity and rapid signal amplification in a short time. However, traditional EXPAR is only suitable for detecting short-sequence RNA, and 3'-end template interactions in the amplification reaction can lead to nonspecific amplification, which greatly limits its practical application. Here, we established an isothermal amplification method comprising a three-way junction (3-WJ) structure and dumbbell probe (DP) for the rapid and sensitive detection of pathogen RNA in a single closed tube, termed the rolling circle mediated exponential amplification reaction (RC-EXPAR). The introduction of the DP eliminated the 3'-end of the template, suppressing nonspecific amplification caused by the 3'-end extension in the reaction. Although the trigger generation by the 3-WJ structure is a linear amplification process, the RC-EXPAR amplifies the triggers exponentially to enhance signal output further and increase sensitivity. The proposed method showed a high sensitivity with a limit of detection (LOD) of 103 copies/mL. Moreover, RC-EXPAR demonstrated strong anti-interference capability in complex biological matrices. This work opens up new ideas for suppressing nonspecific amplification and provides a promising signal amplification strategy for rapid, sensitive, and specific pathogen detection in clinical.