Rapid and sensitive on-site genetic diagnostics of pest fruit flies using CRISPR-Cas12a

Abstract Bactrocera zonata , a major fruit pest species, is gradually spreading west from its native habitat in East Asia. In recent years it has become a major threat to the Mediterranean area, with the potential of invading Europe, the Americas, and Australia. To prevent its spreading, monitoring efforts in plantation sites and border controls are carried out. Despite these efforts, and due to morphological similarities between B. zonata and other pests in relevant developmental stages, the monitoring process is challenging, time-consuming, and requires external assistance from professional labs. CRISPR-Cas12a genetic diagnostics has been rapidly developing in recent years and provides an efficient tool for the genetic identification of pathogens, viruses, and other genetic targets. Here we design a CRISPR-Cas12a detection assay that differentially detects two major pest species, B. zonata and Ceratitis capitata. Our easy-to-use and affordable assay employs a simple DNA extraction technique together with isothermal amplification, and Cas12a-based detection. We demonstrate the specificity and high sensitivity of this method, and its relevance for on-site applications. This method is highly modular, and the presented target design method can be applied to a wide array of pests. Key Massage Distinguishing different pest fruit flies on-site is crucial for prevention of global spreading but can be difficult We present a genetic identification assay for rapid, on-site detection of pest using CRISPR-Cas12a The method is affordable, quick and easy-to-use, and can be applied in border controls or on-site The design process can be easily tailored for any pest, and can greatly benefit developing countries