In Situ Visual Detection of TelMV, EAPV, and PaMoV in Passionfruit Using Reverse Transcription-Recombinase-Aided...

As a tropical fruit of considerable economic importance, passionfruit (Passiflora edulis Sims) is extensively cultivated in the tropical and subtropical regions of China; however, the widespread incidence of viral diseases has significantly hampered the safety of its production. Rapid, sensitive, and visual detection of plant viruses is essential to effectively prevent and manage these viral diseases. In this study, we developed a visual detection system (displaying under blue light or UV) utilizing reverse transcription recombinase-aided amplification (RT-RAA) in conjunction with CRISPR/Cas12a to detect three viruses harmful to passionfruit production: telosma mosaic virus (TelMV), East Asian passiflora virus (EAPV), and passiflora mottle virus (PaMoV). Within this system, the optimal primer concentration for the RT-RAA reaction was determined to be 0.4 μM for all three viruses, with an optimal reaction temperature of 37 °C. The optimal reaction times were established as 20 min for TelMV, 15 min for EAPV, and 30 min for PaMoV. The entire detection process could be completed within 30 min without the need for sophisticated equipment or instruments. For TelMV and EAPV, the detection system demonstrated the capability to detect samples at a dilution of 10⁶, representing an approximately 10⁴-fold improvement over RT-PCR, while for PaMoV, it could identify samples at a dilution of 10⁶, representing an approximately 10²-fold improvement over traditional RT-PCR methods. These results confirm the successful development of the CRISPR/Cas12a-based detection systems. Subsequently, the system was applied for in situ detection of the three target viruses in field settings, yielding results that were fully consistent with laboratory-based RT-PCR assays, a consistency which underscores the system's strong potential for field application in detecting important crop viruses.