Field-compatible detection of Spiroplasma citri associated with citrus stubborn disease using CRISPR-Cas12a and crude sample preparation

Authors: SHIH, JUSTIN - Orise Fellow; Yokomi, Raymond; HAJERI, SUBHAS - Alliance Of Pest Control Districts; Roy, Avijit; Wei, Wei

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/17/2026
Publication Date: 2/20/2026
Citation: Shih, J., Yokomi, R.K., Hajeri, S., Roy, A., Wei, W. 2026. Field-compatible detection of Spiroplasma citri associated with citrus stubborn disease using CRISPR-Cas12a and crude sample preparation. Plant Disease. https://doi.org/10.1094/PDIS-08-25-1699-RE.
DOI: https://doi.org/10.1094/PDIS-08-25-1699-RE

Interpretive Summary: Spiroplasma citri is a small, pathogenic bacterium that infects citrus plants and causes a disease known as citrus stubborn disease. This disease presents a significant obstacle for citrus growers worldwide, leading to reduced fruit quality and yield, and consequently, economic losses. To address this challenge, ARS scientists have created a CRISPR-Cas12a-based DETECTR assay (a precision DNA detector). This precise DNA test targets a specific genetic marker for quick identification of S. citri. The method delivers rapid results using two formats: one involving a portable device that produces a glowing signal indicating the presence of the pathogen, and another that uses a simple paper strip, similar to the rapid tests used for COVID-19, making it accessible and easy to implement in various settings. This detection system can distinguish S. citri from other similar bacteria that infect plants, reducing the likelihood of false positives. The test has been proven to perform reliably on samples collected directly from the field and maintains high accuracy even with crude or minimal sample preparation, simplifying field use. The new diagnosis system enables early detection and response to citrus stubborn disease, helping growers, inspectors, biosecurity officials, and researchers prevent outbreaks, minimize economic losses, and safeguard the citrus trade.

Technical Abstract: 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 qPCR, require laboratory equipment and are not easily applicable in the field. This study developed a CRISPR-Cas12a-based DETECTR (DNA endonuclease-targeted CRISPR trans-reporter) 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 the cleavage of fluorescent and lateral flow-compatible reporters. The assay demonstrated a detection limit of 1 attomolar (aM) (around 0.6 genome copies) using a fluorescence plate reader and 10 aM using blue-light visualization and lateral flow assay (LFA). Specificity testing revealed robust discrimination against other phytopathogenic spiroplasmas, including S. kunkelii and S. melliferum. Field validation with DNA extracted from symptomatic citrus samples exhibited 100% consistency with qPCR results. A 10-minute 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 detection accuracy 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.