RPA-CRISPR/Cas12-based rapid visual on-site detection method for bovine Cryptosporidium

Four types of Cryptosporidium (C. parvum, C. bovis, C. ryanae, and C. andersoni) and mixed infections were common among cattle in the Inner Mongolia Autonomous Region of China, causing severe harm. For grassroots farms and pastures, the prevention and control of bovine Cryptosporidium requires simultaneous detection of the four major Cryptosporidium species, and a one-time diagnosis of bovine Cryptosporidium without distinguishing between species is necessary to achieve more efficient prevention and treatment. In this study, recombinant polymerase amplification (RPA) was combined with the CRISPR/Cas system to design crRNAs and screen RPA primers targeting the 18S rRNA genes of four bovine Cryptosporidium species that primarily infect cattle.

Detection results were presented using two distinct single-stranded DNA reporter molecules via fluorescence quantification and lateral flow immunochromatography assay (LFIA) test strips. Using specificity and sensitivity assessments on clinical samples, the RPA-CRISPR/Cas12 visual on-site rapid detection method for bovine Cryptosporidium established in this study was completed within 60 min, with a minimum detection limit of 0.1 copies/µL. There was no cross-reactivity with the six pathogens: Haemonchus contortus, coccidia, Cysticercus tenuicollis, trematode, Caenorhabditis elegans, and tapeworm. Four types of bovine Cryptosporidium were specifically detected.

Clinical detection showed an 86% positivity rate for bovine Cryptosporidium, consistent with the Cryptosporidium nested PCR method (National Standard). Therefore, the RPA-CRISPR/Cas12a rapid on-site detection method established in this study has high specificity and sensitivity, convenience, and speed, without requiring specialized operational steps or expensive equipment.