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A sensitive fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies to detect the H1N1 virus

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Abstract

We propose a sensitive H1N1 virus fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies. Products are generated via the hybridization of single-stranded DNA (ssDNA) probes containing T7 promoter and crRNA templates to a target RNA sequence using SplintR ligase. This generates large crRNA quantities in the presence of T7 RNA polymerase. At such crRNA quantities, ternary Cas13a, crRNA, and activator complexes are successfully constructed and activate Cas13a to enhance fluorescence signal outputs. The biosensor sensitively and specifically monitored H1N1 viral RNA levels down to 3.23 pM and showed good linearity when H1N1 RNA concentrations were 100 pM–1 µM. Biosensor specificity was also excellent. Importantly, our biosensor may be used to detect other viral RNAs by altering the sequences of the two probe junctions, with potential applications for the clinical diagnosis of viruses and other biomedical studies.

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Funding

This work was financially supported by the Science and Technology Development Plans of Jilin Province (20230203143SF).

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Authors and Affiliations

  1. College of Life Science, Jilin Agricultural University, Changchun, 130118, China

    Lulu Xue, Mengyao Xu, Yao Xu & Zehong Li

  2. Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, 130122, China

    Shengjun Bu, Jiaqi Wei, Hongyu Zhou, Zhuo Hao & Jiayu Wan

  3. School of Life Science and Technology, Changchun University of Science and Technology, Changchun, 130022, China

    Shengjun Bu

Authors
  1. Lulu Xue
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  2. Shengjun Bu
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  3. Mengyao Xu
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  4. Jiaqi Wei
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  5. Hongyu Zhou
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  6. Yao Xu
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  7. Zhuo Hao
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  8. Zehong Li
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  9. Jiayu Wan
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Contributions

Lulu Xue: experiments, investigation, methodology, data curation, writing—original draft. Shengjun Bu: methodology, statistical analysis, software. Mengyao Xu: conceptualization, methodology. Jiaqi Wei: investigation, software. Hongyu Zhou: validation. Yao Xu: validation. Zhuo Hao: data curation. Zehong Li: conceptualization, methodology. Jiayu Wan: writing—review, supervision.

Corresponding authors

Correspondence to Zehong Li or Jiayu Wan.

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Declarations

All animal procedures were performed in accordance with the Guidelines of Animal Ethics Committee of Changchun Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences, and approved by the Animal Ethics Committee of Changchun Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences.

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The authors declare no competing interests.

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Xue, L., Bu, S., Xu, M. et al. A sensitive fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies to detect the H1N1 virus. Anal Bioanal Chem (2024). https://doi.org/10.1007/s00216-024-05269-x

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  • DOI: https://doi.org/10.1007/s00216-024-05269-x

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