Nucleic acid sequences.

Animal Trypanosomosis (AT) is a significant disease affecting cattle across sub-Saharan Africa, Latin America, and Asia, posing a major threat to economic productivity and animal welfare. The absence of reliable diagnostic tests has led to an over-reliance on widespread pre-emptive drug treatments, which not only compromise animal health but also heighten the risk of drug resistance. The chronic nature of AT, characterized by cyclical low or undetectable parasite levels, and the necessity for field-applicable tests that can distinguish between active infection and prior exposure, present considerable challenges in developing effective diagnostics. In previous work, we identified a parasite-specific small RNA, 7SL-sRNA, which is detectable in the serum of infected cattle, even during the chronic stages of infection. However, existing methods for detecting sRNA require specialized equipment, making them unsuitable for field use. In this study, we have developed both a fluorescence-based and a lateral flow diagnostic test utilizing Cas10 technology for the detection of 7SL-sRNA from Trypanosoma congolense and Trypanosoma brucei. The fluorescence assay detects 10 – 100 fM T. congolense 7SL-sRNA and 1 pM T. brucei 7SL-sRNA, and the lateral flow assay showed a limit of detection of 1 – 10 pM for both species. Either assay can effectively identify active infections in cattle, including during chronic phases (with positive signals observed up to the experimental end point, 63 days post infection). This also highlights the effective use of Cas10 for small RNA detection, paving the way for a cost-effective, user-friendly, and field-deployable diagnostic test for AT, while establishing Cas10 technology for the detection of small RNAs in general.