Field-Compatible Detection of Spiroplasma citri Associated with Citrus Stubborn Disease Using CRISPR-Cas12a and Crude...

Citrus stubborn disease (CSD), caused by Spiroplasma citri, presents a significant risk to citrus production, resulting in considerable yield losses when infections remain undetected. Accurate and timely diagnosis is crucial for effective disease management. However, existing nucleic acid-based methods, such as PCR and quantitative PCR (qPCR), require laboratory equipment and are not easily applicable in the field. This study developed a CRISPR-Cas12a-based DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) assay for the rapid, sensitive, and specific detection of S. citri, targeting the spiralin gene. An optimized recombinase polymerase amplification (RPA) primer pair and CRISPR-RNA (crRNA) were utilized for sequence-specific activation of Cas12a, enabling cleavage of fluorescent and lateral flow-compatible reporters. The assay demonstrated a detection limit of 1 attomolar (aM) (around 1.8 genome copies) using a fluorescence plate reader and 10 aM using blue-light visualization and a lateral flow assay (LFA). Specificity testing revealed discrimination against other phytopathogenic spiroplasmas, including S. kunkelii and S. melliferum. Validation in the plate-reader format with DNA extracted from symptomatic citrus samples showed 100% consistency with qPCR results. A 10-min NaOH-Tris crude extraction protocol was also assessed, facilitating straightforward and equipment-free sample preparation. Relative to DETECTR assays with kit-extracted samples, crude extracts preserved full diagnostic sensitivity in fluorescence assays and achieved 70% accuracy in LFA and visual formats in a subset of the same samples. These findings establish a dependable, portable, and highly sensitive diagnostic approach for S. citri, providing a practical tool for on-site detection and enhanced management of citrus stubborn disease.