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Multi-aptamer–mediated hairpin allosteric and aptamer-assisted CRISPR system for detection of S. pneumoniae and S. aureus

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Abstract

A novel nucleic acid aptamer nanoprobes-mediated hairpin allosteric and aptamer-assisted CRISPR system for detection of Streptococcus pneumoniae and Staphylococcus aureus is presented. In this fluorescence assay system, utilizing the hairpin allosteric effect caused by the aptamer binding to the target bacteria, the detection of S. pneumoniae is first achieved through changes in fluorescence due to FRET. Subsequently, a Cas12a protein mixture is added to detect S. aureus. The amplified output signal is triggered by two methods to ensure the sensitivity of the method: the synergistic FRET effect is achieved by the assembly of multi-aptamer through the conjugation of streptavidin–biotin, and the trans-cleavage function of CRISPR/Cas 12a. Under the optimized conditions, the proposed hairpin allosteric aptasensor could achieve high sensitivity (a detection limit of 135 cfu/mL) and broad-concentration quantification (dynamic range of 103–107 cfu/mL) of S. pneumoniae. The aptamer-assisted CRISPR system for S. aureus detection showed good linearity (R2 = 0.996) in the concentration range 102–108 cfu/mL, with a detection limit of 39 cfu/mL. No cross-reactivity with other foodborne pathogenic bacteria was observed in both systems. Taking only 55 min, this method of multiple pathogen detection proved to be promising.

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Funding

The authors acknowledge the support from the Key Project of Sichuan Province (No. 22ZDYF1033), the central government guides local science and technology development funds to be directed transfer payment projects (No. 22ZYZF0007), the National Natural Science Foundation of China (No. 2022NSFSC1426), and Wine City Excellence—Science and Technology Innovation Team ([2021] 162).

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Author notes

  1. Limei Zhang and Xuejing Xu contributed equally to this work.

Authors and Affiliations

  1. Department of Laboratory Medicine, the Affiliated Hospital of Southwest Medical University, Luzhou, 646000, Sichuan, China

    Limei Zhang, Xuejing Xu, Linhong Cao, Zixin Zhu, Yinhuan Ding, Hui Jiang, Baolin Li & Jinbo Liu

  2. Sichuan Province Engineering Technology Research Center of Molecular Diagnosis of Clinical Diseases, Luzhou, 646000, China

    Limei Zhang, Xuejing Xu, Linhong Cao, Zixin Zhu, Yinhuan Ding, Baolin Li & Jinbo Liu

  3. Molecular Diagnosis of Clinical Diseases Key Laboratory of Luzhou, Luzhou, 646000, China

    Limei Zhang, Xuejing Xu, Linhong Cao, Zixin Zhu, Yinhuan Ding, Baolin Li & Jinbo Liu

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  1. Limei Zhang
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Contributions

Limei Zhang: conceptualization, data curation, writing—original draft, writing—review and editing. Xuejing Xu: writing—review and editing, visualization. Linhong Cao: writing—review and editing. Zixin Zhu: writing—review and editing. Yinhuan Ding: funding support. Hui Jiang: data curation. Baolin Li: writing—review and editing, supervision. Jinbo Liu: funding support, supervision.

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Correspondence to Baolin Li or Jinbo Liu.

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Zhang, L., Xu, X., Cao, L. et al. Multi-aptamer–mediated hairpin allosteric and aptamer-assisted CRISPR system for detection of S. pneumoniae and S. aureus. Microchim Acta 191, 29 (2024). https://doi.org/10.1007/s00604-023-06094-2

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