Field‐Deployable RPA/Cas12a Diagnostic Assay for On‐Site Detection of Alternaria Section Alternaria in Sweet Cherry

ABSTRACT Alternaria section Alternaria causes diseases in many fruit and vegetable varieties on a global scale and causes considerable losses. The wide distribution and ecological adaptability in different ecological niches make it important for plant and animal health. Therefore, comprehensive and rapid detection and diagnosis of Alternaria section Alternaria are required, enabling farmers to develop timely, effective, and accurate management strategies to mitigate the impact on agricultural yields and quality. Here, we present the development of a rapid and specific detection method that can be completed within 40 min. Remarkably, this assay can be performed with mini‐UV light or ordinary incandescent light from a mobile phone, allowing detection with the naked eye. The sensitivity of the assay is impressive, with a lowest detection limit of 9.5 × 10 4 fg/μL DNA of the target pathogen, ensuring reliable detection even at low pathogen densities. The evaluation of the specificity of the assay showed that it can detect the target pathogen with no cross‐reactivity with other common fungal pathogens on stone fruits such as cherry and peach. Furthermore, the RPA/Cas12a assay was demonstrated to detect all strains from different geographical regions of China. In simulation of field application, the RPA/Cas12a assay displayed high specificity and sensitivity, accurately distinguishing the diseased fruit inoculated with Alternaria alternata from healthy samples, reaffirming its reliability and practicality for on‐site detection. In summary, the RPA/Cas12a assay offers rapid, sensitive, and specific detection and can be applied directly in the field without compromising sensitivity and specificity.