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<p>Canine babesiosis and canine monocytic ehrlichiosis are important tick-borne diseases caused by <i>Babesia gibsoni (B. gibsoni)</i> and <i>Ehrlichia canis (E. canis)</i> in dogs. Early diagnosis is important for effective clinical management, as these infections can result in other complications if untreated. This study aimed to develop a CRISPR-Cas12a-based detection assay for <i>B. gibsoni</i> and <i>E. canis</i> in a fluorometer platform. Custom designed guide RNAs (gRNAs) were synthesised with the spacer sequence targeting the <i>B. gibsoni18S rRNA</i> and <i>E. canis p43</i> genes, located 20&#xa0;bp downstream of the PAM site (5’-TTTV- 3’). Following PCR amplification of the short fragments encompassing the above target regions, the specific gRNA binding the <i>Lba</i>Cas12a-gRNA complex initiated the collateral cleavage of FAM- labelled AT rich <i>ssDNA</i> probe for detection using a fluorometer and Biotin-digoxigenin (DIG) labelled GT-rich <i>ssDNA</i> probe for visual detection in LFA.The limit of detection (LOD) using the fluorometer based detection platform was 6 × 10<sup>8</sup>and 6 × 10<sup>9</sup> for <i>B. gibsoni</i> and <i>E. canis</i> respectively. The LOD was comparable to that of real-time PCR, but more sensitive than point-of-care methods such as LFA, indicating its potential applicability in veterinary clinical settings.</p>

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CRISPR-Cas12a-based rapid detection of Babesia gibsoni and Ehrlichia canis in dogs using fluorometer platform

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月11日

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  • Azhahianambi Palavesam,
  • B. N. Karthik Raj,
  • N. Madan,
  • S. Sakthi Lalitha Sri,
  • R. Babitha,
  • K. G. Tirumurugaan

摘要

Canine babesiosis and canine monocytic ehrlichiosis are important tick-borne diseases caused by Babesia gibsoni (B. gibsoni) and Ehrlichia canis (E. canis) in dogs. Early diagnosis is important for effective clinical management, as these infections can result in other complications if untreated. This study aimed to develop a CRISPR-Cas12a-based detection assay for B. gibsoni and E. canis in a fluorometer platform. Custom designed guide RNAs (gRNAs) were synthesised with the spacer sequence targeting the B. gibsoni18S rRNA and E. canis p43 genes, located 20 bp downstream of the PAM site (5’-TTTV- 3’). Following PCR amplification of the short fragments encompassing the above target regions, the specific gRNA binding the LbaCas12a-gRNA complex initiated the collateral cleavage of FAM- labelled AT rich ssDNA probe for detection using a fluorometer and Biotin-digoxigenin (DIG) labelled GT-rich ssDNA probe for visual detection in LFA.The limit of detection (LOD) using the fluorometer based detection platform was 6 × 108and 6 × 109 for B. gibsoni and E. canis respectively. The LOD was comparable to that of real-time PCR, but more sensitive than point-of-care methods such as LFA, indicating its potential applicability in veterinary clinical settings.