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Comparison of dye-based and probe-based RT-LAMP in detection of canine astrovirus

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Abstract

A rapid and sensitive assay is essential for reliable surveillance and diagnosis of canine astrovirus (CaAstV). In this study, two real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays with high sensitivity, rapidity, and reliability were developed using fluorescence dye and FRET-based assimilating probes for real-time detection of CaAstV. These assays specifically amplified the ORF2 gene of CaAstV and did not amplify any sequences from canine enterovirus. The limit of detection (LOD) of both the probe-based and dye-based RT-LAMPs was 100 copies/μL. Fluorescence signals were generated within 30 min for the lowest concentration of a standard RNA sample, which was significantly faster than that achieved by real-time fluorescence quantitative PCR (qRT-PCR) assay. When clinical samples were tested, the positive and negative agreement of the dye-based RT-LAMP assay with qRT-PCR was 87.5% (14/16) and 93.55% (29/31), respectively. The positive and negative agreement of the probe-based RT-LAMP assay with qRT-PCR was 94.11% (16/17) and 96.55% (28/29), respectively. The RT-LAMP assays developed in this study showed strong potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of CaAstV in clinical samples.

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Data availability

Data available on request from the authors.

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Funding

This work was supported by a grant from Shanghai Agriculture Applied Technology Development Program, China (Grant nos. (X2022-02-08-00-12-F01122) 2022-02-08-00-12-F01122).

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Author notes

  1. These authors contributed equally: Haixiao Shen and Dequan Yang.

Authors and Affiliations

  1. Shanghai Animal Disease Control Center, Shanghai, China

    Haixiao Shen, Dequan Yang, Xin Li, Houbin Ju, Feifei Ge, Xianchao Yang, Jian Wang, Luming Xia & Hongjin Zhao

  2. Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China

    Haixiao Shen & Ping Jiang

Authors
  1. Haixiao Shen
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  2. Dequan Yang
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  3. Xin Li
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Contributions

PJ and HZ conceived and designed the study. HS, FG, HJ, DY, XY, and XL performed the experiments. LX and JW analyzed the data. HS and DY wrote the manuscript, and all authors read and approved the final manuscript.

Corresponding authors

Correspondence to Hongjin Zhao or Ping Jiang.

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The authors declare no conflict of interest.

Ethical approval

This study was conducted according to animal welfare guidelines of the World Organization for Animal Health and approved by Shanghai Municipal Commission of Agriculture (permit number: 2013 [17]).

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Handling Editor: Akbar Dastjerdi.

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Shen, H., Yang, D., Li, X. et al. Comparison of dye-based and probe-based RT-LAMP in detection of canine astrovirus. Arch Virol 169, 21 (2024). https://doi.org/10.1007/s00705-023-05913-4

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