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LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test

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Abstract

A CRISPR-based nucleic acid detection platform, termed LACD (loop-mediated isothermal amplification coupled with CRISPR-Cas12a-mediated diagnostic) has been developed. In the LACD system, the core primer used in conventional LAMP (forward inner primer or backward inner primer) was engineered to contain a PAM (protospacer adjacent motif) site (TTTT) at the linker region. As a result, the LAMP amplicons contained a specific PAM site for CRISPR-Cas12a recognition. At the CRISPR-mediated detection stage, the resulting LAMP products can activate the corresponding CRISPR-Cas12a effector upon the formation of the CRISPR-Cas12a/gRNA/target DNA complex. The single-strand DNA (ssDNA) reporter molecules are then rapidly cleaved due to the CRISPR-Cas12a’s trans-enzyme activity. The ssDNA degradation can then be visualized on a lateral flow biosensor or measured  by a real-time fluorescence instrument. Our LACD assay allows any target sequence to be detected (even targets which do not contain any PAM sites) as long as they met the design requirement for LAMP. The feasibility of the LACD methodology for nucleic acid detection was validated on the Mycobacterium tuberculosis complex (MTC). This proof-of-concept assay can be reconfigured to detect a variety of target sequences by redesigning the engineered LAMP primers.

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Acknowledgements

We would like to thank Laurence Don Wai Luu (School of Biotechnology and Biomolecular Science, University of New South Wales, Sydney, Australia) for linguistic assistance during the revision of this manuscript.

Funding

This work was supported by Cultivation Fund Project of the National Natural Science Foundation in Beijing Children’s Hospital, Capital Medical University (GPMS201904), and Qian Ke He Support Plan [2019]2822.

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Author notes

  1. Yi Wang, Jieqiong Li, Shijun Li and Xiong Zhu contributed equally to this work.

Authors and Affiliations

  1. Experimental Research Center, Capital Institute of Pediatrics, Beijing, 100020, People’s Republic of China

    Yi Wang

  2. Department of Respiratory Disease, Beijing Pediatric Research Institute, Beijing Children’s Hospital, National Center for Children’s Health, Capital Medical University, Beijing, 100045, People’s Republic of China

    Jieqiong Li

  3. Laboratory of Infectious Disease of Experimental Center, Guizhou Provincial Center for Disease Control and Prevention, Guiyang, 550050, China

    Shijun Li & Junfei Huang

  4. Central & Clinical Laboratory of Sanya People’s Hospital, Sanya, Hainan, 572000, People’s Republic of China

    Xiong Zhu & Xiaoxia Wang

  5. Public Health School, Guizhou Medical University, Guiyang, 550025, Guizhou, People’s Republic of China

    Xinggui Yang

  6. Department of Otolaryngology, Head and Neck Surgery, Children’s Hospital Capital Institute of Pediatrics, Beijing, 100020, People’s Republic of China

    Jun Tai

Authors
  1. Yi Wang
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  2. Jieqiong Li
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  3. Shijun Li
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  5. Xiaoxia Wang
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  6. Junfei Huang
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  7. Xinggui Yang
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  8. Jun Tai
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Correspondence to Yi Wang or Jun Tai.

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Wang, Y., Li, J., Li, S. et al. LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test. Microchim Acta 188, 347 (2021). https://doi.org/10.1007/s00604-021-04985-w

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  • DOI: https://doi.org/10.1007/s00604-021-04985-w

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