Integrated molecular and serological diagnostics for surveillance of the quarantine virus tomato brown rugose fruit virus

Background: The tomato brown rugose fruit virus (Tobamovirus fructirugosum, ToBRFV) is an emerging tobamovirus that has quickly become a significant obstacle to the production of tomatoes and peppers worldwide. It is now classified as a regulated quarantine pathogen. Effective containment requires rapid, reliable, inexpensive, and safe diagnostic protocols for routine screening in laboratories and production systems. Aims: We aimed to thoroughly evaluate integrated molecular and serological diagnostic methods for ToBRFV and develop biosafe positive controls suitable for high-throughput and decentralized applications. Methods: We evaluated the following methods: conventional RT-PCR, one-enzyme RTX-PCR, immunocapture RT-PCR, recombinase polymerase amplification, loop-mediated isothermal amplification with colorimetric detection, Western blotting, dot blot, and tissue blot immunoassay. The non-infectious positive control was prepared using the GoldenBraid 3.0 cloning system. We developed a single-seed assay that enables direct testing of tomato seed stocks. Results: Among the evaluated molecular methods, RTX-PCR was particularly advantageous due to its minimal sample handling, reduced cost, and ability to bypass RNA extraction. The tissue blot immunoassay enabled high-throughput, low-cost screening of hundreds of samples per day using only basic equipment. Although ToBRFV was frequently detected in seeds harvested from infected plants, no systemic infection was observed in progeny seedlings, confirming the low rate of true vertical transmission. A non-infectious positive control was prepared and successfully employed in molecular methods. Conclusions: Our findings provide an integrated diagnostic framework combining molecular, serological, and biosafety tools to effectively monitor and contain ToBRFV in commercial production and phytosanitary settings.