A One-Step RT-PCR-Coupled Cysteamine-Functionalized Gold Nanoparticle Assay for Colorimetric Detection of Tobacco Mosaic Virus

Viral diseases are one of the most destructive threats to global agriculture. Among plant viruses, tobacco mosaic virus (TMV) is a highly contagious pathogen infecting numerous economically vital crops. With no curable treatments, the only strategy to mitigate virus spread is early detection and plant removal. The gold standard for TMV detection is reverse transcriptase-polymerase chain reaction (RT-PCR), followed by agarose gel electrophoresis. To reduce TMV diagnosis time and eliminate the requirement for expensive equipment, this study developed and optimized a one-step RT-PCR-coupled cysteamine-functionalized gold nanoparticle assay for the colorimetric determination of the virus. By integrating cDNA synthesis and PCR amplification into a single tube and analyzing the results using cysteamine- functionalized gold nanoparticles (Au@Cys), the diagnosis turnaround time was significantly reduced. Furthermore, this AuNPs-based colorimetric assay enabled straightforward visual inspection with the naked eye, eliminating the need for costly optical devices. Additionally, our regression equation linking RT-PCR product color values, extracted as mean A value based on the CIELAB color space (green-red axis), and viral load allows for the accurate quantification of TMV infection levels in field samples. Our research lays the groundwork for the further development of more cost-effective, quantitative and rapid plant virus diagnosis methods.