Home Medicine One copy in one-pot for rapid and accurate SFTSV testing by LAC12b-2M
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One copy in one-pot for rapid and accurate SFTSV testing by LAC12b-2M

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Published/Copyright: January 14, 2026
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 64 Issue 4

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.

Keywords: SFTSV; loop-mediated isothermal amplification; CRISPR/Cas12b-2M; one-pot; point-of-care testing
Corresponding author: Song-Mei Liu, Department of Clinical Laboratory, Center for Gene Diagnosis & Program of Clinical Laboratory, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan, 430071, China, E-mail:
Ya Pang and Yongwei Duan contributed equally to this work and share first authorship.

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.

  1. 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).

  2. Informed consent: Not applicable.

  3. 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.

  4. Use of Large Language Models, AI and Machine Learning Tools: None declared.

  5. Conflict of interest: The authors state no conflict of interest.

  6. 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).

  7. Data availability: Not applicable.

References

1. Yu, XJ, Liang, MF, Zhang, SY, Liu, Y, Li, JD, Sun, YL, et al.. Fever with thrombocytopenia associated with a novel bunyavirus in China. N Engl J Med 2011;364:1523–32. https://doi.org/10.1056/nejmoa1010095.Search in Google Scholar PubMed PubMed Central

2. Liu, Q, He, B, Huang, SY, Wei, F, Zhu, XQ. Severe fever with thrombocytopenia syndrome, an emerging tick-borne zoonosis. Lancet Infect Dis 2014;14:763–72. https://doi.org/10.1016/s1473-3099(14)70718-2.Search in Google Scholar

3. Li, H, Zhang, LK, Li, SF, Zhang, SF, Wan, WW, Zhang, YL, et al.. Calcium channel blockers reduce severe fever with thrombocytopenia syndrome virus (SFTSV) related fatality. Cell Res 2019;29:739–53. https://doi.org/10.1038/s41422-019-0214-z.Search in Google Scholar PubMed PubMed Central

4. Fang, Y, Shen, S, Zhang, J, Xu, L, Wang, T, Fan, L, et al.. Thrombocytopenia in severe fever with thrombocytopenia syndrome due to platelets with altered function undergoing cell death pathways. J Infect Dis 2025;231:e183–94. https://doi.org/10.1093/infdis/jiae355.Search in Google Scholar PubMed PubMed Central

5. Cui, H, Shen, S, Chen, L, Fan, Z, Wen, Q, Xing, Y, et al.. Global epidemiology of severe fever with thrombocytopenia syndrome virus in human and animals: a systematic review and meta-analysis. Lancet Reg Health West Pac 2024;48:101133. https://doi.org/10.1016/j.lanwpc.2024.101133.Search in Google Scholar PubMed PubMed Central

6. Sheng, R, Cheng, T, Wang, Y, Wen, H. Molecular evolution and geographic migration of severe fever with thrombocytopenia syndrome virus in Asia. PLoS Pathog 2025;21:e1012970. https://doi.org/10.1371/journal.ppat.1012970.Search in Google Scholar PubMed

7. Xie, J, Li, H, Zhang, X, Yang, T, Yue, M, Zhang, Y, et al.. Akkermansia muciniphila protects mice against an emerging tick-borne viral pathogen. Nat Microbiol 2023;8:91–106. https://doi.org/10.1038/s41564-022-01279-6.Search in Google Scholar PubMed

8. Wang, Y, Han, S, Ran, R, Li, A, Liu, H, Liu, M, et al.. A longitudinal sampling study of transcriptomic and epigenetic profiles in patients with thrombocytopenia syndrome. Nat Commun 2021;12:5629. https://doi.org/10.1038/s41467-021-25804-z.Search in Google Scholar PubMed PubMed Central

9. Chen, Q, Yang, D, Zhang, Y, Zhu, M, Chen, N, Yushan, Z. Transmission and mortality risk assessment of severe fever with thrombocytopenia syndrome in China: results from 11-years’ study. Infect Dis Poverty 2022;11:93. https://doi.org/10.1186/s40249-022-01017-4.Search in Google Scholar PubMed PubMed Central

10. Li, XK, Lu, QB, Chen, WW, Xu, W, Liu, R, Zhang, SF, et al.. Arginine deficiency is involved in thrombocytopenia and immunosuppression in severe fever with thrombocytopenia syndrome. Sci Transl Med 2018;10. https://doi.org/10.1126/scitranslmed.aat4162.Search in Google Scholar PubMed

11. Dolskiy, AA, Grishchenko, IV, Yudkin, DV. Cell cultures for virology: usability, advantages, and prospects. Int J Mol Sci 2020;21. https://doi.org/10.3390/ijms21217978.Search in Google Scholar PubMed PubMed Central

12. Leland, DS, Ginocchio, CC. Role of cell culture for virus detection in the age of technology. Clin Microbiol Rev 2007;20:49–78. https://doi.org/10.1128/cmr.00002-06.Search in Google Scholar PubMed PubMed Central

13. Torres, I, Poujois, S, Albert, E, Colomina, J, Navarro, D. Evaluation of a rapid antigen test (Panbio™ COVID-19 Ag rapid test device) for SARS-CoV-2 detection in asymptomatic close contacts of COVID-19 patients. Clin Microbiol Infect 2021;27:636.e1–4. https://doi.org/10.1016/j.cmi.2020.12.022.Search in Google Scholar PubMed PubMed Central

14. Freire-Paspuel, B, Garcia-Bereguiain, MA. Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis. Int J Infect Dis 2021;102:14–6. https://doi.org/10.1016/j.ijid.2020.10.047.Search in Google Scholar PubMed PubMed Central

15. Zhao, Y, Chen, F, Li, Q, Wang, L, Fan, C. Isothermal amplification of nucleic acids. Chem Rev 2015;115:12491–545. https://doi.org/10.1021/acs.chemrev.5b00428.Search in Google Scholar PubMed

16. De Felice, M, De Falco, M, Zappi, D, Antonacci, A, Scognamiglio, V. Isothermal amplification-assisted diagnostics for COVID-19. Biosens Bioelectron 2022;205:114101. https://doi.org/10.1016/j.bios.2022.114101.Search in Google Scholar PubMed PubMed Central

17. Chaouch, M. Loop-mediated isothermal amplification (LAMP): an effective molecular point-of-care technique for the rapid diagnosis of coronavirus SARS-CoV-2. Rev Med Virol 2021;31:e2215. https://doi.org/10.1002/rmv.2215.Search in Google Scholar PubMed PubMed Central

18. Bonney, LC, Watson, RJ, Slack, GS, Bosworth, A, Wand, NIV, Hewson, R. A flexible format LAMP assay for rapid detection of ebola virus. PLoS Neglected Trop Dis 2020;14:e0008496. https://doi.org/10.1371/journal.pntd.0008496.Search in Google Scholar PubMed PubMed Central

19. Kim, SH, Lee, SY, Kim, U, Oh, SW. Diverse methods of reducing and confirming false-positive results of loop-mediated isothermal amplification assays: a review. Anal Chim Acta 2023;1280:341693. https://doi.org/10.1016/j.aca.2023.341693.Search in Google Scholar PubMed

20. Dong, M, Kshirsagar, A, Politza, AJ, Guan, W. High fidelity machine-learning-assisted false positive discrimination in loop-mediated isothermal amplification using nanopore-based sizing and counting. ACS Nano 2024;18:7170–9. https://doi.org/10.1021/acsnano.3c12053.Search in Google Scholar PubMed PubMed Central

21. Mukama, O, Wu, J, Li, Z, Liang, Q, Yi, Z, Lu, X, et al.. An ultrasensitive and specific point-of-care CRISPR/Cas12 based lateral flow biosensor for the rapid detection of nucleic acids. Biosens Bioelectron 2020;159:112143. https://doi.org/10.1016/j.bios.2020.112143.Search in Google Scholar PubMed

22. Chen, JS, Ma, E, Harrington, LB, Da Costa, M, Tian, X, Palefsky, JM, et al.. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 2018;360:436–9. https://doi.org/10.1126/science.aar6245.Search in Google Scholar PubMed PubMed Central

23. Li, SY, Cheng, QX, Liu, JK, Nie, XQ, Zhao, GP, Wang, J. CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA. Cell Res 2018;28:491–3. https://doi.org/10.1038/s41422-018-0022-x.Search in Google Scholar PubMed PubMed Central

24. Gootenberg, JS, Abudayyeh, OO, Kellner, MJ, Joung, J, Collins, JJ, Zhang, F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 2018;360:439–44. https://doi.org/10.1126/science.aaq0179.Search in Google Scholar PubMed PubMed Central

25. Teng, F, Guo, L, Cui, T, Wang, XG, Xu, K, Gao, Q, et al.. CDetection: CRISPR-Cas12b-Based DNA detection with sub-attomolar sensitivity and single-base specificity. Genome Biol 2019;20:132. https://doi.org/10.1186/s13059-019-1742-z.Search in Google Scholar PubMed PubMed Central

26. Joung, J, Ladha, A, Saito, M, Kim, NG, Woolley, AE, Segel, M, et al.. Detection of SARS-CoV-2 with SHERLOCK one-pot testing. N Engl J Med 2020;383:1492–4. https://doi.org/10.1056/nejmc2026172.Search in Google Scholar

27. Tong, X, Zhang, K, Han, Y, Li, T, Duan, M, Ji, R, et al.. Fast and sensitive CRISPR detection by minimized interference of target amplification. Nat Chem Biol 2024;20:885–93. https://doi.org/10.1038/s41589-023-01534-9.Search in Google Scholar PubMed

28. Xu, H, Lin, G, Chen, R, Cai, Z, Sun, Y, Zhang, X, et al.. CRISPR/Cas12b assisted loop-mediated isothermal amplification for easy, rapid and sensitive quantification of chronic HBV DNA in one-pot. Anal Chim Acta 2024;1310:342702. https://doi.org/10.1016/j.aca.2024.342702.Search in Google Scholar PubMed

29. Liu, T, Liu, Q, Chen, F, Shi, Y, Maimaiti, G, Yang, Z, et al.. An accurate and convenient method for Mycoplasma pneumoniae via one-step LAMP-CRISPR/Cas12b detection platform. Front Cell Infect Microbiol 2024;14:1409078. https://doi.org/10.3389/fcimb.2024.1409078.Search in Google Scholar PubMed PubMed Central

30. Jing, W, Zhang, T, Min, X, Li, X, Jin, K, Feng, M, et al.. CHAMP: a centrifugal microfluidics-based CRISPR/Cas12b-Combined real-time LAMP one-pot method for Mycoplasma pneumoniae infection diagnosis. ACS Omega 2024;9:38989–97. https://doi.org/10.1021/acsomega.4c05489.Search in Google Scholar PubMed PubMed Central

31. Soh, JH, Balleza, E, Abdul Rahim, MN, Chan, HM, Mohd Ali, S, Chuah, JKC, et al.. CRISPR-Based systems for sensitive and rapid on-site COVID-19 diagnostics. Trends Biotechnol 2022;40:1346–60. https://doi.org/10.1016/j.tibtech.2022.06.002.Search in Google Scholar PubMed PubMed Central

32. Bustin, SA, Ruijter, JM, van den Hoff, MJB, Kubista, M, Pfaffl, MW, Shipley, GL, et al.. Miqe 2.0: revision of the minimum information for publication of quantitative real-time PCR experiments guidelines. Clin Chem 2025;71:634–51. https://doi.org/10.1093/clinchem/hvaf043.Search in Google Scholar PubMed

33. Bustin, SA, Benes, V, Garson, JA, Hellemans, J, Huggett, J, Kubista, M, et al.. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009;55:611–22. https://doi.org/10.1373/clinchem.2008.112797.Search in Google Scholar PubMed

34. Land, KJ, Boeras, DI, Chen, XS, Ramsay, AR, Peeling, RW. REASSURED diagnostics to inform disease control strategies, strengthen health systems and improve patient outcomes. Nat Microbiol 2019;4:46–54. https://doi.org/10.1038/s41564-018-0295-3.Search in Google Scholar PubMed PubMed Central

35. Plebani, M, Nichols, JH, Luppa, PB, Greene, D, Sciacovelli, L, Shaw, J, et al.. Point-of-care testing: State-of-The art and perspectives. Clin Chem Lab Med 2025;63:35–51. https://doi.org/10.1515/cclm-2024-0675.Search in Google Scholar PubMed

Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2025-0791).

Received: 2025-06-25
Accepted: 2025-10-28
Published Online: 2026-01-14
Published in Print: 2026-03-26

© 2025 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2025-0791
Keywords for this article
SFTSV; loop-mediated isothermal amplification; CRISPR/Cas12b-2M; one-pot; point-of-care testing

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Reshaping laboratory medicine through technological advances
  4. Reviews
  5. Capillary blood in core laboratories: current and future challenges
  6. Artificial intelligence and machine learning in thrombosis and hemostasis: a scoping review of clinical and laboratory applications, challenges, and future directions
  7. Opinion Papers
  8. Hierarchy of reference interval models: advancing laboratory data interpretation
  9. Reimagining External Quality Assessment for precision medicine: a perspective from biochemistry laboratories
  10. Science, Quality and Value of Laboratory Medicine
  11. Guidelines and Recommendations
  12. Recommendations from the IFCC Working Group on Laboratory Errors and Patient Safety for the Global Adoption of an Essential Quality Indicators Panel in Laboratory Medicine
  13. Addressing the silent epidemic of recreational nitrous oxide use: a position, call to action and recommendations by the European Federation of Clinical Chemistry and Laboratory Medicine Committee on Biological Markers of Nitrous Oxide Abuse
  14. General Clinical Chemistry and Laboratory Medicine
  15. Assessment of drone transport for biological samples: a real-world experience at a tertiary hospital
  16. Impact of an autonomous delivery robot on sample turnaround time in a clinical laboratory: an early evaluation of first implementation
  17. Implementation of an automated alert system of critical results in hospitalized and emergency patients
  18. Comparison of blood sample quality and test results between robotic and manual venipuncture: a pilot study
  19. At-home blood collection for clinical chemistry analyses in a kidney transplant population: a feasibility study
  20. Clinical validation of a DBS-based LC-MS/MS method for 25-hydroxyvitamin D: from lab sampling to home sampling
  21. Comparative analysis of three platforms for serum NfL quantification in healthy controls and MS patients
  22. Uracil in plasma: comparison of two in-house-developed LC-MS/MS methods
  23. Assessment for potential bias in multiplexed IL-10 and TNF-α from plex count
  24. Hematology and Coagulation
  25. A specific-neonatal hemolysis correction model for accurate potassium assessment in blood samples with in vitro hemolysis
  26. Cancer Diagnostics
  27. Analytical verification and comparative assessment of the new Atellica IM high-sensitivity prostate specific antigen assay
  28. Extended verification of an automated MALDI-TOF mass spectrometry system for high throughput serum M-protein measurement
  29. Cardiovascular Diseases
  30. Performance evaluation of a new high-sensitivity cardiac troponin T assay: hs-cTnT (CLIA) assay
  31. Infectious Diseases
  32. Prognostic value of suPAR in sepsis: a potential tool to support patient management in the Emergency Department
  33. Contribution of SuPAR for patients in a situation of uncertainty downstream of emergencies
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  35. Corrigendum
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  42. Innovative closed tube protocol reveals a super critical early preanalytical phase of whole blood glucose stability in routine matrices
  43. Spun citrate samples as a suitable alternative for platelet measurement. Is recollection necessary? A preliminary study
  44. Mass spectrometry reveals limitations of serum immunofixation electrophoresis in monitoring lambda light chain myeloma
  45. A study of the performance of different methods for measuring serum lithium
  46. Congress Abstracts
  47. 47th Annual Conference of the Association for Clinical Biochemists in Ireland (ACBI)
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