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LAMP assay coupled with a CRISPR/Cas12a system for the rapid and ultrasensitive detection of porcine circovirus-like virus in the field

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Abstract

A recent outbreak of porcine circovirus-like virus (PCLV), a virus that may be associated with porcine diarrhea, has been reported in swine herds in China. The virus is spreading rapidly, causing huge economic losses to the swine farming industry. To achieve the rapid, inexpensive, and sensitive detection of PCLV, we combined loop-mediated isothermal amplification (LAMP) and the CRISPR/Cas12a system, whose fluorescence intensity readout can detect PCLV ORF4 gene levels as low as 10 copies. To overcome the need for sophisticated equipment, lateral flow strip reading technology was introduced for the first time in a LAMP-Cas12a-based system to detect PCLV. The lateral flow strip (LFS) results were readout by the naked eye, and the method was highly sensitive with a detection limit of 10 copies, with a detection time of about 60 min. In addition, the method is highly specific and has no cross-reactivity with other related viruses. In conclusion, LAMP-CRISPR/Cas12a-based assays have the advantages of rapidity, accuracy, portability, low cost, and visualization of the results. They therefore have great potential, especially for areas where specialized equipment is lacking, and can expect to be an ideal method for early diagnosis and on-site detection of PCLV.

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Acknowledgements

We thank International Science Editing (http://www.internationalscienceediting.com ) for editing this manuscript.

Funding

This work was supported financially by the Natural Science Foundation of China (no. 31972642).

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Author notes

  1. Zhaorong Yu and Hua Liu contributed equally to this work.

Authors and Affiliations

  1. Anhui Province Engineering Laboratory for Animal Food Quality and Bio-safety, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, People’s Republic of China

    Zhaorong Yu, Hua Liu, Zhe Chen, Ying Shao, Zhipeng Wang, Fanyu Cheng, Yu Zhang, Zhenyu Wang, Jian Tu, Xiangjun Song & Kezong Qi

  2. Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui, 230036, People’s Republic of China

    Zhaorong Yu, Hua Liu, Zhe Chen, Ying Shao, Zhipeng Wang, Fanyu Cheng, Yu Zhang, Zhenyu Wang, Jian Tu, Xiangjun Song & Kezong Qi

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  1. Zhaorong Yu
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  2. Hua Liu
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Contributions

Zhaorong Yu, Hua Liu, Zhe Chen, and Ying Shao contributed conception and design of the study. Zhaorong Yu wrote the manuscript. Yu Zhang and Zhipeng Wang performed the experiments. Fanyu Cheng and Zhenyu Wang analyzed the data. Jian Tu, Xiangjun Song, and Kezong Qi supervised the study, interpreted the data, and acquired the research funds. All authors read and approved the final manuscript. All authors have declared that no competing interests exist.

Corresponding authors

Correspondence to Xiangjun Song or Kezong Qi.

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All animal experiments complied with the ethical standards of Anhui Agricultural University.

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Yu, Z., Liu, H., Chen, Z. et al. LAMP assay coupled with a CRISPR/Cas12a system for the rapid and ultrasensitive detection of porcine circovirus-like virus in the field. Anal Bioanal Chem 416, 363–372 (2024). https://doi.org/10.1007/s00216-023-05020-y

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