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Highly specific and sensitive detection of the Mycobacterium tuberculosis complex using multiplex loop-mediated isothermal amplification combined with a nanoparticle-based lateral flow biosensor

  • Clinical Microbiology - Research Paper
  • Published:
Brazilian Journal of Microbiology Aims and scope Submit manuscript

Abstract

Tuberculosis (TB) is the deadliest infectious caused by Mycobacterium tuberculosis complex (MTBC). Because most TB cases occur within low-income populations, developing a specific, sensitive, cost-saving, and rapid point-of-care test for the early diagnosis of TB is important for achieving the WHO’s End Tuberculosis Strategy. In the current study, a novel nucleic acid detection strategy that includes multiplex loop-mediated isothermal amplification combined with a nanoparticle-based lateral flow biosensor (mLAMP-LFB) was used to detect MTBC. The two sets of LAMP primers specific to the IS6110 and gyrB genes of MTBC were successfully designed and validated for the detection of MTBC. The preferred reaction conditions for this assay were confirmed to be 65 °C for 40 min, and the amplification products could be visually identified through LFB within 2 min. The full assay process, including genomic DNA template extraction, LAMP reaction, and product detection, could be completed in 80 min. The limit detection of the assay was 100 fg of DNA in pure culture. The specificity of the assay was 100%, and it had no cross-reactions to other strains. Thus, the m-LAMP-LFB technology established in the present study was an objective, rapid, simple, and sensitive assay for MTBC identification, which could be applied in a clinical setting, especially in resource-constrained regions of the world.

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Data availability

The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request.

Abbreviations

MTBC:

M. tuberculosis Complex

WHO:

World Health Organization

ATCC:

American Type Culture Collection

LAMP:

Loop-mediated isothermal amplification

LFB:

Lateral flow biosensor

MG:

Malachite green

PCR:

Polymerase chain reaction

LoD:

Limit of detection

GZCDC:

Guizhou Provincial Center for Disease Control and Prevention

FAM:

Carboxyfluorescein

Dig:

Digoxigenin

mer:

Monomeric unit

nt:

Nucleotide

CL:

Control line

TL1:

Test line 1

TL2:

Test line 2

NC:

Negative control

BC:

Blank control

DW:

Distilled water

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Acknowledgements

We acknowledge the medical staff at the Guizhou Provincial Center for Disease Control and Prevention for their cooperation in this study.

Funding

This study was funded by grants from the Science and Technology Department of Guizhou Province (No. [2019]1186, [2019]2822), [2018]-5606, and [2018]-5767).

Author information

Authors and Affiliations

  1. Laboratory of Infectious Disease of Experimental Center, Guizhou Provincial Center for Disease Control and Prevention, 73 Bageyan Road, Guiyang, 550004, Guizhou, People’s Republic of China

    Xu Chen, Junfei Huang, Xingui Yang, Yijiang Chen, Wenlin Zheng, Wei Chen, Huijuan Chen & Shijun Li

  2. The Second Affiliated Hospital, Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou, 550003, People’s Republic of China

    Xu Chen & Ziyu Xiao

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

    Ziyu Xiao, Xingui Yang & Shijun Li

Authors
  1. Xu Chen
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  2. Junfei Huang
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  3. Ziyu Xiao
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  4. Xingui Yang
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  5. Yijiang Chen
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  7. Wei Chen
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  8. Huijuan Chen
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  9. Shijun Li
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Contributions

XC and SL designed and conceived the experiments. XC, JH, and SL designed and analyzed the LAMP primers. XC, JH, ZX, XY, and YC performed the experiments. YC, WZ, WC, and HC collected and analyzed the data. XC and SL wrote and revised the manuscript. XC and SL revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Shijun Li.

Ethics declarations

Ethics approval

The study was approved by the Human Ethics Committee of the Guizhou Provincial Center for Disease Control and Prevention and complied with the Declaration of Helsinki. All data/isolates were analyzed anonymously. The patients with tuberculosis included in the present research were given a subject information sheet, and they all provided written informed consent before participating in the study.

Competing interests

The authors declare no competing interests.

Additional information

Responsible Editor: Fernando R. Pavan

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Chen, X., Huang, J., Xiao, Z. et al. Highly specific and sensitive detection of the Mycobacterium tuberculosis complex using multiplex loop-mediated isothermal amplification combined with a nanoparticle-based lateral flow biosensor. Braz J Microbiol 52, 1315–1325 (2021). https://doi.org/10.1007/s42770-021-00520-4

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  • DOI: https://doi.org/10.1007/s42770-021-00520-4

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