CRISPR-Cas-Based Pen-Side Diagnostic Tests for <i>Anaplasma marginale</i> and <i>Babesia bigemina</i>

CRISPR-Cas-Based Pen-Side Diagnostic Tests for <i>Anaplasma marginale</i> and <i>Babesia bigemina</i>

<i>Anaplasma marginale</i> and <i>Babesia bigemina</i> are tick-borne pathogens, posing significant threats to the health and productivity of cattle in tropical and subtropical regions worldwide. Currently, detection of <i>Babesia bigemina</i> and <i>Anaplas...

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Bibliographic Details
Main Authors: Robert Muriuki, Maingi Ndichu, Samuel Githigia, Nicholas Svitek
Format: Article
Language:English
Published: MDPI AG 2024-12-01
Series:Microorganisms
Subjects:
babesiosis
anaplasmosis
pen-side test
point-of-care diagnostics
CRISPR-Cas powered diagnostics
Online Access:https://www.mdpi.com/2076-2607/12/12/2595
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Summary:<i>Anaplasma marginale</i> and <i>Babesia bigemina</i> are tick-borne pathogens, posing significant threats to the health and productivity of cattle in tropical and subtropical regions worldwide. Currently, detection of <i>Babesia bigemina</i> and <i>Anaplasma marginale</i> in infected animals relies primarily on microscopic examination of Giemsa-stained blood or organ smears, which has limited sensitivity. Molecular methods offer higher sensitivity but are costly and impractical in resource-limited settings. Following the development of a pen-side test for detecting <i>Theileria parva</i> infections in cattle, we have created two additional CRISPR-Cas12a assays targeting <i>Anaplasma marginale</i> and <i>Babesia bigemina</i>. The assays target the major surface protein 5 (MSP5) for <i>A. marginale</i> and rhoptry-associated protein 1a (RAP1a) for <i>B. bigemina</i>. These additional tests involve a 20 min recombinase polymerase amplification (RPA) reaction followed by a 60 min CRISPR-Cas12a detection with a lateral strip readout. Results demonstrate high specificity, with no cross-reactivity against other tick-borne parasites, and a limit of detection down to 10<sup>2</sup> DNA copies/µL of each target marker. The findings pave the way for sensitive and user-friendly pen-side tests to diagnose <i>A. marginale</i> and <i>B. bigemina</i> infections.
ISSN:2076-2607

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