TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis - 19/06/21

Doi : 10.1016/j.jinf.2021.04.032

I Kuan Sam ^a,^{^{1
These authors contributed equally to this work.}}, Ying-ying Chen ^a,^{^{1
These authors contributed equally to this work.}}, Jun Ma ^b,^{^{1
These authors contributed equally to this work.}}, Shi-yuan Li ^c,^{^{1
These authors contributed equally to this work.}}, Ruo-yan Ying ^b, Lin-xian Li ^d, Ping Ji ^a, Shu-jun Wang ^a, Jie Xu ^e, Yu-jie Bao ^e, Guo-ping Zhao ^f,^g, Hua-jun Zheng ^g, Jin Wang ^d,^⁎ , Wei Sha ^b,^⁎ , Ying Wang ^a,^g,^h,^⁎
^a Shanghai Institute of Immunology, Department of Microbiology and Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^b Clinic and Research Center of Tuberculosis, Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China
^c Shanghai TOLO Biotechnology Company Limited, Shanghai 200233, China
^d Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518035, China
^e Department of Infectious Disease, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^f Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai 200032, China
^g Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai 201203, China
^h Shanghai Key Laboratory of Emergency Prevention, Diagnosis and Treatment of Respiratory Infectious Diseases (20dz2261100), Shanghai, China

^⁎Corresponding authors.^⁎⁎Corresponding author at: Shanghai Institute of Immunology, Department of Microbiology and Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.Shanghai Institute of Immunology, Department of Microbiology and ImmunologyShanghai Jiao Tong University School of MedicineShanghai200025China

Highlights

•	TB-QUICK combines LAMP amplification and CRISPR-Cas12b reaction for M. tuberculosis detection.
•	TB-QUICK detection is isothermal, ultra-sensitive and rapid.
•	TB-QUICK exhibits 86.8% sensitivity and 95.2% specificity in pulmonary TB specimens.
•	TB-QUICK is of great advantages for smear-negative TB patients.

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Summary

Objectives

Tuberculosis (TB) remains one of the public health problems worldwide. Rapid, sensitive and cost-effective diagnosis of Mycobacterium tuberculosis (M.tb) is critical for TB control.

Methods

We developed a novel M.tb DNA detection platform (nominated as TB-QUICK) which combined loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12b detection. TB-QUICK was performed on pulmonary or plasma samples collected from 138 pulmonary TB (PTB) patients, 21 non-TB patients and 61 close contacts to TB patients. Acid-fast bacillus (AFB) smear, M.tb culture and GeneXpert MTB/RIF (Xpert) assays were routinely conducted in parallel.

Results

By targeting M.tb IS6110, TB-QUICK platform could detect as low as 1.3 copy/μL M.tb DNA within 2 h. In pulmonary TB samples, TB-QUICK exhibited improved overall sensitivity of 86.8% over M.tb culture (66.7%) and Xpert (70.4%), with the specificity of 95.2%. More significantly, TB-QUICK exhibited a superior sensitivity in AFB-negative samples (80.5%) compared to Xpert (57.1%) and M.tb culture (46.2%). In the detection of plasma M.tb DNA by TB-QUICK, 41.2% sensitivity for AFB-positive and 31.7% for AFB-negative patients were achieved.

Conclusion

In conclusion, TB-QUICK exhibits rapidity and sensitivity for M.tb DNA detection with the superiority in smear-negative paucibacillary TB patients. The clinical application of TB-QUICK in TB diagnosis needs to be further validated in larger cohort.

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Graphical abstract

Image, graphical abstract

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Keywords : Tuberculosis, CRISPR-Cas12b, Mycobacterium tuberculosis, IS6110, Diagnosis, TB-QUICK

Plan

Introduction

Materials and methods

Study subjects and clinical samples

Construction of pUC18-IS6110 plasmid

Preparation of IS6110 sgRNAs

DNA preparation

Establishment of LAMP-CRISPR-Cas12b detection assay

Determination of limit of detection (LOD)

Data procession and statistical analysis

Results

Establishment of IS6110-based CRISPR-Cas12b-assisted nucleic acid detection system

Detection of M.tb DNA in pulmonary samples from TB patients by the TB-QUICK assay

Detection of circulating bacterial DNA by the TB-QUICK assay

Discussion and conclusion

CRediT authorship contribution statement

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Vol 83 - N° 1

P. 54-60 - juillet 2021 Retour au numéro

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TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis - 19/06/21

Highlights

Summary

Objectives

Methods

Results

Conclusion

Graphical abstract

Plan

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