A Revolutionary CRISPR-Based Detection and Genetic Profiling of Parasitic Infections in Domesticated Animals | [Journal of animal health and production • 2025]

Author(s):

1. Qais R. Lahhob: Collage of Pharmacy, National University of Science and Technology, Dhi Qar, Iraq

2. Mustafa Mudhafar: Department of Medical Physics, Faculty of Medical Applied Sciences, University of Kerbala, Karbala, Iraq; Department of Anesthesia Techniques and Intensive Care, Al-Taff university college, Kerbala, Iraq

3. Hasan A. Alsailawi: Department of Basic Sciences, College of Dentistry, University of Kerbala, Karbala, Iraq; Department of Anesthesia Techniques, AlSafwa University College, Karbala, Iraq

4. Mustafa A. Zaidan: Al-Farahidi university, Baghdad, Iraq.

Abstract:

Detection and treatment for Trypanosoma brucei evansi (T. b. evansi) infections within domesticated animals depend upon the development for fast, very specific, field-deployable diagnostic instruments. Leveraging Cas12b-mediated collateral cleavage for ultrasensitive detection for T. b. evansi, this work provides the creation, and validation for a CRISpen-based Recombinase Polymerase Amplification (RPA) assay, designated as TevRPA-CRISpen. Alongside the RoTat 1.2 VSG gene as the detection marker, the assay was tuned for temperature, reaction conditions, and target specificity. While the One-Pot TevRPA-CRISpen assay attained equivalent sensitivity within a streamlined process, the Two-Pot TevRPA-CRISpen assay showed a 10-fold gain within sensitivity compared to conventional RPA, detecting down to 10 aM for T. b. evansi aDNA through means for infected mouse models, experimental validation employing TevRPA-CRISpen tests revealed active infections alongside 100% concordance alongside TevPCR. Moreover, post-treatment monitoring using these assays consistently separated cured coming from untreated mice, therefore confirming their utility as test-of-cure diagnostics. Alongside no cross-reactivity seen alongside non-target Trypanosoma species, the specificity for both tests achieved 99.8-99.9%. These results show the promise for TevRPA-CRISpen as a transforming diagnostic platform providing a quick, reasonably priced, accurate substitute for field-based and laboratory-based uses.

Page(s): 158-166

DOI: 10.17582/journal.jahp/2025/13.s1.158.166

Published: Journal: Journal of animal health and production, Volume: 13, Issue: 2, Year: 2025

Keywords:

Pointofcare testing , Trypanosoma brucei evansi , TevRPACRISPR , Recombinase polymerase Amplification , Molecular diagnostics , Testofcure , Cas12b

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