Development and Evaluation of an RPA-CRISPR/Cas12a Molecular Assay for Rapid Diagnosis of Theileria annulata Infection in Bovines.

Tropical theileriosis, caused by Theileria annulata, continues to be the most significant tick-borne disease affecting cattle, particularly in endemic regions where it leads to significant production losses. Prompt and reliable diagnosis is critically important for timely treatment and disease control; however, conventional diagnostic methods have important limitations. Microscopic examination lacks sensitivity, particularly in carrier animals, while PCR requires specialized laboratory infrastructure. To overcome these limitations, a fluorescence-based RPA-CRISPR/Cas12a assay targeting the multicopy mitochondrial cytb gene was developed for rapid detection of T. annulata. Five RPA primer pairs were designed and evaluated, and primer set-5, generating a 324-bp amplicon, exhibited the best amplification performance. A CRISPR RNA (crRNA) was designed within the selected amplicon to enable specific Cas12a-mediated target recognition. The assay was completed within 50 min and demonstrated no cross-reactivity with DNA from Theileria orientalis, Babesia bigemina, Babesia bovis, Anaplasma marginale, and bovine host DNA, confirming excellent analytical specificity. Sensitivity analysis using recombinant plasmid DNA demonstrated a detection limit of approximately ten copies per reaction. The field evaluation of the assay was done by testing 206 bovine field samples. showed that the developed assay identified 163 positive samples, exceeding the detection rates obtained by PCR (151 positives) and microscopy (116 positives). On comparison with PCR, the assay achieved 100% diagnostic sensitivity, 78.18% specificity, 92.64% positive predictive value, 100% negative predictive value, with an overall agreement of 94.17%. These findings indicate that the RPA-CRISPR/Cas12a platform is a rapid, sensitive, and accurate molecular diagnostic approach with strong potential for field application and point-of-care diagnosis of bovine tropical theileriosis, particularly in resource-constrained settings.