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Cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of Shigella flexneri

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Abstract

Pathogen infections including Shigella flexneri have posed a significant threat to human health for numerous years. Although culturing and qPCR were the gold standards for pathogen detection, time-consuming and instrument-dependent restrict their application in rapid diagnosis and economically less-developed regions. Thus, it is urgently needed to develop rapid, simple, sensitive, accurate, and low-cost detection methods for pathogen detection. In this study, an immunomagnetic beads-recombinase polymerase amplification-CRISPR/Cas12a (IMB-RPA-CRISPR/Cas12a) method was built based on a cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of S. flexneri in the laboratory. Firstly, S. flexneri was specifically captured and enriched by IMB (Shigella antibody-coated magnetic beads), and the genomic DNA was released and used as the template in the RPA reaction. Then, the RPA products were mixed with the pre-loaded CRISPR/Cas12a for fluorescence visualization. The results were observed by naked eyes under LED blue light, with a sensitivity of 5 CFU/mL in a time of 70 min. With no specialized equipment or complicated technical requirements, the IMB-RPA-CRISPR/Cas12a diagnostic method can be used for visual, rapid, and simple detection of S. flexneri and can be easily adapted to monitoring other pathogens.

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All data generated or analyzed during this study are included in this published article and its supplementary information files.

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Funding

This study was financially supported by the National Key Research and Development Program of China (2018YFE0107500); Qin Chuangyuan recruited high-level innovation and entrepreneurship talents project of Science and Technology Department of Shaanxi Province (QCYRCXM-2022–56); Foreign expert service project of Science and Technology Department of Shaanxi Province (2023WGZJ-YB-39); Medical Research project of Xi'an Science and Technology Bureau (22YXYJ0120); the CAMS Initiative for Innovative Medicine (CAMS-2021-I2M-1–060); the Sichuan Science and Technology Program (2021YFH0100); Central government directed special funds for local science and technology development project (2021ZYD0085); and Sichuan Science and Technology Program (2021056).

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Author notes

  1. Yaoqiang Shi and Qi Tan contributed equally to this work.

Authors and Affiliations

  1. Provincial Key Laboratory for Transfusion-Transmitted Infectious Diseases, Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, 610052, Sichuan, China

    Yaoqiang Shi, Qi Tan, Tao Gong, Qing-yuan Li, Ya Zhu, Xiaoqiong Duan, Chunhui Yang, Shilin Li, Yujia Li & Limin Chen

  2. The Joint Laboratory On Transfusion-Transmitted Diseases (TTDs) Between Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Nanning Blood Center, Nanning Blood Center, Nanning, 530007, China

    Limin Chen

  3. The Hospital of Xidian Group, Xi’an, 710077, China

    He Xie & Limin Chen

  4. Clinical Laboratory Department, Yan’an Hospital Affiliated to Kunming Medical University, Kunming, 650051, Yunnan, China

    Jia-wei Ding

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  1. Yaoqiang Shi
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Corresponding authors

Correspondence to Yujia Li or Limin Chen.

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Ethical approval

All animal studies were performed in accordance with the guidelines approved by the Institutional Ethics Review Committee of the Institute of Blood Transfusion, Chinese Academy of Medical Sciences, and Peking Union Medical College (No: 2022007).

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Shi, Y., Tan, Q., Gong, T. et al. Cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of Shigella flexneri. Microchim Acta 191, 271 (2024). https://doi.org/10.1007/s00604-024-06309-0

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