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

Abstract

Background: 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 significant threat to the Mediterranean area, with the potential of invading Europe, the Americas, and Australia. To prevent it spreading, monitoring efforts in cultivation 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 laboratories. 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.

Results: We demonstrate the specificity and high sensitivity of this method. Identification of target pests was done using specific and universal primers on pooled samples, enabling differentiation of pests with high certainty. We also demonstrate reaction stability over time for future on-site applications.

Discussion: Our easy-to-use and affordable assay employs a simple DNA extraction technique together with isothermal amplification and Cas12a-based detection. This method is highly modular, and the presented target design method can be applied to a wide array of pests. This approach can be easily adapted to fit local threats and requires minimal training of operators in border controls and other relevant locations, reshaping pest control and making state-of-the-art technologies available worldwide, including in developing countries. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Keywords: Bactrocera zonata; CRISPR-Cas12a; Ceratitis capitata; RPA; genetic detection; pest-control.

Figure 1

Scheme of proposed assay. (a) Comparison of the organs used for larval stage morphological identification of B. zonata (top) and C. capitata (bottom). (b) A scheme of the detection assay. After sample collection from the fruit, larvae are homogenized, then Chelex 100 resin is added to the homogenate and boiled for 10 min. Next, 2 μL of the solution is amplified by RPA. In parallel, a Cas12a‐gRNA complex is assembled (or thawed), then a reporter and the amplicons are added to the solution and incubated for 30 min. Finally, fluorescence is measured to test for the presence of the target DNA.

Figure 2

Pest‐specific identification. (a) A scheme of the gRNA design process. Mitochondrial sequences of relevant pests were aligned, and variable regions were identified and concatenated for gRNA design using CRISPOR. The uniqueness of the gRNA sequences was then validated and tested experimentally (Fig. S1). (b) Cas12a detection of B. zonata in samples of different developmental stages (orange). DNA was obtained from fresh samples using Chelex 100 (Methods). Amplification was performed using RPA with specific primers for B. zonata and Bz1 gRNA used for detection (Table S1). As a negative control, C. capitata larvae were used with Bz1 gRNA (green). (c) Sensitivity of Cas12a‐Bz gRNA detection in varying ratios of pooled larvae. Samples containing a single larva of B. zonata with increasing amounts of C. capitata larvae were prepared. DNA was then extracted and diagnosed as in (b), using B. zonata‐specific primers and Cas12a‐Bz gRNA complexes. As a negative control, C. capitata DNA was used. All experiments were performed with three biological repeats and three technical repeats.

Figure 3

Universal amplification assay. (a) Cas12a detection of B. zonata (orange) and C. capitata (green) from different developmental stages. DNA was obtained using Chelex 100 from fresh samples (Methods). Amplification was performed using RPA with universal primers (Table S1) and detection was achieved using Bz1 gRNA for B. zonata and Cc2 for C. capitata (Table S1). As a negative control (NC), C. capitata and B. zonata larvae were used with noncorresponding gRNAs. (b) Detection sensitivity of Cas12a‐Bz1 gRNA in varying ratios of pooled larvae. Samples containing a single larva of B. zonata with increasing amounts of C. capitata larvae were prepared. DNA was then extracted using Chelex 100, RPA amplified (using VR  forward/reverse primers; Table S1) and diagnosed using Cas12a‐Bz1 gRNA complexes. As a negative control, C. capitata DNA was used. (c) Detection sensitivity of Cas12a‐Cc2 gRNA for C. capitata in varying ratios of pooled larvae. Samples containing a single larva of C. capitata with increasing amounts of B. zonata larvae were prepared. DNA was extracted and amplified as in (b) and diagnosed using Cas12a‐Cc2 gRNA complexes. As a negative control, B. zonata DNA was used. All experiments were performed with three biological repeats and three technical repeats.

Figure 4

Stability of Cas12a complexes in different conditions. Samples were tested at time 0, after 24, 48, and 167 h at room temperature, 4 °C, and –20 °C. Adult B. zonata  RPA‐amplified samples were detected using Cas12‐Bz1 gRNA complexes, and the same samples were detected with Cas12‐ Cc2 gRNA complexes (Table S1) as a negative control. Blank (gray) are detection reactions without DNA.