Rapid visual field detection of Pratylenchus coffeae in yam tissues via RPA-CRISPR/Cas12a

Yam (Dioscorea spp.) is an important crop cultivated worldwide with significant economic value. However, Pratylenchus coffeae is one of the most prevalent pathogenic nematodes causing yam root lesion disease, primarily spreading through seed yams (top tuber portions) or soil. Ensuring that quality of seed yams and cultivated soil free from pathogenic nematodes is crucial for yam production, and an efficient detection method for P. coffeae is essential, especially under field conditions. From November 2022 to November 2024, a simple, rapid, and efficient molecular identification method was developed for P. coffeae detection in yam tissues without requiring expensive laboratory equipment or specialized personnel. The entire detection process was completed in 65 min, including crude nematode DNA extraction (10 min), recombinase polymerase amplification (RPA) (30 min), and CRISPR/Cas12a-based detection (25 min). Compared with PCR and qPCR assays, the proposed diagnostic method demonstrated high sensitivity, with detection as low as 4 copies/μL per reaction, and exhibited high specificity for P. coffeae without cross-reactivity with other common plant-parasitic nematodes. The detection results obtained using RPA-CRISPR/Cas12a were highly consistent with those of conventional PCR assays conducted on 18 seed yams, indicating the method's high applicability and accuracy for field detection. In conclusion, RPA-CRISPR/Cas12a holds great potential as a reliable and rapid diagnostic tool for detecting P. coffeae infections, particularly for seed yam quality control in field conditions where complex equipment is unavailable.