Development of an hAPE1-based RT-RAA method for rapid detection of Sweet potato chlorotic stunt virus.

Sweet potato chlorotic stunt virus (SPCSV) is a major pathogen that threatens sweet potato production. It frequently acts synergistically with other viruses, causing severe yield losses and quality decline. A rapid and efficient detection method is therefore essential for preventing and controlling the spread of this virus. Here, we developed a reverse transcription real-time fluorescence recombinase-aided amplification (RT-RAA) assay using human apurinic/apyrimidinic endonuclease 1 (hAPE1, designated as tool enzyme E10). This enzyme exhibits high AP endonuclease activity but extremely low exonuclease activity, so that amplification products remain intact rather than being degraded as commonly occurs with the traditionally used Exonuclease III (ExoIII), thereby preserving product integrity for subsequent analysis. The established RT-hAPE1-RAA assay for SPCSV can be completed within 10 minutes and has a sensitivity of 100 ag/μL, corresponding to approximately 26 copies/μL of a recombinant pUC57 plasmid (3,484 bp) containing a 774bp SPCSV coat protein gene. Gel electrophoresis confirmed that E10 preserved viral amplification fragments intact, whereas ExoIII caused significant degradation. When testing 43 field-collected sweet potato samples, the method demonstrated a sensitivity of 94.4% and a specificity of 100%, showing high concordance with RT-qPCR results . Furthermore, E10 maintained stable cleavage activity across diverse target sequences, indicating its broad applicability. This work provides a rapid, sensitive, and reliable tool for SPCSV detection and offers technical support for the on-site diagnosis of sweet potato viral diseases.