One copy in one-pot for rapid and accurate SFTSV testing by LAC12b-2M
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Ya Pang
Abstract
Objectives
Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne pathogen that can cause a fatality rate as high as 12–50 %, posing a significant threat to public health. SFTSV is prevalent in mountainous and hilly regions with relatively poor medical conditions. Therefore, there is an urgent need to develop a new convenient, rapid and sensitive method for SFTSV detection in low-resource environments.
Methods
We developed a one-pot and visualized method for SFTSV detection using loop-mediated isothermal amplification assisted by CRISPR/Cas12b with G478A/K396A double mutations (LAC12b-2M). The specificity, sensitivity, accuracy and limit of detection (LOD) of LAC12b-2M were evaluated using clinical reverse transcription-quantitative PCR (RT-qPCR) as the reference method, with gradient dilutions of strong positive SFTSV RNA samples and 215 clinical serum samples from two cohorts.
Results
LAC12b-2M is sensitive to detect SFTSV with a LOD of 1 copy/μL at 61 °C within 30 min. Compared to clinical RT-qPCR, LAC12b-2M demonstrated a sensitivity of 98.8 % (82/83), a specificity of 100.0 % (96/96), and an accuracy of 99.4 % (178/179) in cohort 1 (n=179), and an accuracy of 100.0 % in cohort 2 (n=36).
Conclusions
Our LAC12b-2M method holds promise for point-of-care SFTSV testing in different healthcare settings, particularly in low-resource region where SFTSV is prevalent.
Funding source: National Natural Science Foundation of China
Award Identifier / Grant number: 82372338
Funding source: the transformaion project of scientific and technological achievements, Zhongnan Hospital of Wuhan University
Award Identifier / Grant number: 2025LCYJZX-MS007
Funding source: the translational medicine and interdisciplinary research joint fund of Zhongnan Hospital of Wuhan University
Award Identifier / Grant number: ZNJC202302
Acknowledgments
We would like to thank Prof. Hao Yin and Prof. Ying Zhang at the Medical Research Institute of Wuhan University, and Prof. Fei Deng at Key Laboratory of Virology and Biosafety and National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences for their technical supports.
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Research ethics: All studies were performed in accordance with ethical regulations and approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (2024046K) and the Ethical Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (TJ‐IRB20230632). The experiments were conducted following the Declaration of Helsinki (as revised in 2013).
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Informed consent: Not applicable.
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Author contributions: Y. Pang, method development and drafting manuscript; Y. Duan, clinical sample collection and acquisition of data; Y. Sun, virus culturing; Tong Zhou, analysis and interpretation of data; Anling Li, clinical sample testing of cohort 1; Ruoxi Ran, virus isolation and genotyping; Hongyan Hou, clinical sample collection and testing of cohort 2; Song-Mei Liu, the conception and design, drafting and revising manuscript, financial support. All authors approved the final manuscript.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: The authors state no conflict of interest.
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Research funding: This work was supported by grants from the National Natural Science Foundation of China (82372338), the translational medicine and interdisciplinary research joint fund of Zhongnan Hospital of Wuhan University (ZNJC202302) and the transformaion project of scientific and technological achievements, Zhongnan Hospital of Wuhan University (2025LCYJZX-MS007).
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Data availability: Not applicable.
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Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2025-0791).
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