Frontiers in Microbiology (Mar 2025)

Rapid detection of feline parvovirus using RAA-CRISPR/Cas12a-based lateral flow strip and fluorescence

  • Han Chen,
  • Hailing Zhang,
  • Jie Guo,
  • Xiangshu Meng,
  • Mengfan Yao,
  • Longbin He,
  • Xiaoxuan Nie,
  • Han Xu,
  • Chao Liu,
  • Jian Sun,
  • Jian Sun,
  • Fei Wang,
  • Yuelong Sun,
  • Zhong Jiang,
  • Yanliang He,
  • Jianlou Zhang,
  • Jianke Wang

DOI
https://doi.org/10.3389/fmicb.2025.1501635
Journal volume & issue
Vol. 16

Abstract

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Feline parvovirus (FPV) causes severe gastroenteritis and leukopenia in cats, with high morbidity and mortality, necessitating a rapid and effective antigen diagnostic test with high sensitivity and specificity. In this study, a diagnostic platform based on a combination of Recombinase-Aided Amplification (RAA) and CRISPR/Cas12a was established for detecting FPV. Cas12a recombinant protein was purified using Nickel-Nitriloacetic Acid resin after heterologous expression in Escherichia coli. The results of RAA-CRISPR/Cas12a can be detected with a fluorescence reader or lateral flow strips (LFS) for on-site detection. The RAA-CRISPR/Cas12a-LFS had a detection limit of 2.1 × 100 copies of recombinant plasmids per reaction, compared with 2.1 × 103 copies for conventional PCR analysis. Furthermore, no cross-reactivity was observed for the RAA-CRISPR/Cas12a assay with feline coronavirus, feline herpesvirus, and feline calicivirus, demonstrating reasonable specificity. Additionally, 43 cat fecal samples with suspected clinical signs were assayed with RAA-CRISPR/Cas12a-LFS and conventional PCR in parallel. The RAA-CRISPR/Cas12a-LFS showed a 100% coincident rate with PCR. In summary, a novel, visual, sensitive, and specific detection assay based on RAA and CRISPR/Cas12a was developed for FPV.

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