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Three-way junction structure-mediated reverse transcription-free exponential amplification reaction for pathogen RNA detection

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Abstract

Since RNA is an important biomarker of many infectious pathogens, RNA detection of pathogenic organisms is crucial for disease diagnosis and environmental and food safety. By simulating the base mismatch during DNA replication, this study presents a novel three-way junction structure-mediated reverse transcription-free exponential amplification reaction (3WJ-RTF-EXPAR) for the rapid and sensitive detection of pathogen RNA. The target RNA served as a switch to initiate the reaction by forming a three-way junction (3WJ) structure with the ex-trigger strand and the ex-primer strand. The generated trigger strand could be significantly amplified through EXPAR to open the stem-loop structure of the molecular beacon to emit fluorescence signal. The proofreading activity of Vent DNA polymerase, in combination with the unique structure of 2+1 bases at the 3′-end of the ex-primer strand, could enhance the role of target RNA as a reaction switch to reduce non-specific amplification and ensure excellent specificity to differentiate target pathogen from those causing similar symptoms. Furthermore, detection of target RNA showed a detection limit of 1.0×104 copies/mL, while the time consumption was only 20 min, outperforming qRT-LAMP and qRT-PCR, the most commonly used RNA detection methods in clinical practice. All those indicates the great application prospects of this method in clinical diagnostic.

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Funding

We highly appreciate the financial support of the Science and Technology Benefiting the People Demonstration Project of Qingdao (22-2-7-smjk-2-nsh), the Natural Science Foundation of Shandong Province (ZR2023MC064), and the Key Project of Shandong Province Natural Science Foundation (ZR2020KH030). This work is supported by the Taishan Industrial Experts Program.

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Author notes

  1. Xinguang Zhang and Yang Li contributed equally to this work.

Authors and Affiliations

  1. Qingdao Nucleic Acid Rapid Testing International Science and Technology Cooperation Base, College of Life Sciences, Qingdao University, Qingdao, 266071, People’s Republic of China

    Xinguang Zhang, Yang Li, Chao Jiang, Yuting Shan, Yao Liu, Qunqun Guo & Chao Shi

  2. Department of Pathogenic Biology, School of Basic Medicine, Qingdao University, Qingdao, 266071, People’s Republic of China

    Chao Shi

  3. Department of Clinical Laboratory, the Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, 266071, People’s Republic of China

    Qing Wang & Chao Shi

  4. Shandong Provincial Key Laboratory of Biochemical Engineering, Key Laboratory of Nucleic Acid Rapid Detection, Sino-UAE International Cooperative Joint Laboratory of Pathogenic Microorganism Rapid Detection, College of Biological Engineering, Qingdao Nucleic Acid Rapid Detection Engineering Research Center, Qingdao University of Science and Technology, QingdaoQingdao, 266042, China

    Cuiping Ma

  5. Qingdao JianMa Gene Technology Co., Ltd, Qingdao, 266114, People’s Republic of China

    Chao Shi

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  1. Xinguang Zhang
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Contributions

Xinguang Zhang, Chao Jiang, Yuting Shan, and Yao Liu performed the experiments. Yang Li, Xinguang Zhang, Chao Jiang, Yuting Shan, and Yao Liu analyzed the data. Yang Li, Qing Wang, Qunqun Guo, Cuiping Ma, and Chao Shi designed the study. Yang Li and Xinguang Zhang wrote the manuscript. All authors contributed to the writing of the paper, had primary responsibility for the final content, and read and approved the final manuscript.

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Correspondence to Qunqun Guo or Chao Shi.

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The authorized Human Health and Ethics Committee of the Affiliated Hospital of Qingdao University approved this study (Approval No. QDU-HEC-2023260). All volunteers providing nasopharyngeal swabs signed informed consent forms. All methods were carried out in accordance with the relevant guidelines and regulations.

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Zhang, X., Li, Y., Wang, Q. et al. Three-way junction structure-mediated reverse transcription-free exponential amplification reaction for pathogen RNA detection. Anal Bioanal Chem 416, 3161–3171 (2024). https://doi.org/10.1007/s00216-024-05264-2

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