Development of CRISPR/Cas12b-Based Multiple Cross Displacement Amplification Technique for the Detection of
            <i>Mycobacterium tuberculosis</i>
            Complex in Clinical Settings

Yang, Xinggui; Huang, Junfei; Chen, Yijiang; Xia, Ying; Tan, Qinqin; Chen, Xu; Zeng, Xiaoyan; Lei, Shiguang; Wang, Yi; Li, Shijun

doi:10.1128/spectrum.03475-22

Cited by 8 publications

(10 citation statements)

References 45 publications

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“…Of note, our new devised MTB-MCDA-CRISPR assay correctly discerned all the sputum smear microscopy test negative TB patient samples, further emphasizing the advantages of our assay in paucibacillary patients. Recently, besides the novel detection platform devised here, several CRISPR-based assays for MTB detection, including the TB-QUICK detection platform ( Sam et al., 2021 ), the LACD detection platform ( Wang et al., 2021 ), the CRISPR-MCDA system and more ( Yang et al., 2023 ), also have been developed for better diagnosing and controlling of MTB infection, all of which have been validated with excellent performance in clinical practices, further illustrating the importance of diagnosis of MTB with rapidity and accuracy for public health. Given the limited amount of sample size, the efficiency of our MTB-MCDA-CRISPR detection system for clinical practice need to be further verified.…”

Section: Discussionmentioning

confidence: 99%

A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application

Jia

Wang

Liu

et al. 2023

Front. Cell. Infect. Microbiol.

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Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.

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Section: Discussionmentioning

confidence: 99%

A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application

Jia

Wang

Liu

et al. 2023

Front. Cell. Infect. Microbiol.

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“…1f ). This LOD surpasses that of Huang et al’s CRISPR-TB method (0.25 copies/μL) ( 5 ) but remains below the typical range of LODs reported in various CRISPR-based TB detection studies (1–298 copies/μL) ( 4 – 8 , 21 – 24 ). Considering the variation in the number of IS 6110 copies in different Mtb complex strains’ genomes, related to strain evolution ( 31 ), we employed a single-copy plasmid template by cloning the IS 6110 target segment into the pUC57 plasmid.…”

Section: Discussionmentioning

confidence: 53%

“…There have been nine published studies utilizing CRISPR Cas protein trans-cleavage activity for TB detection ( 4 – 8 , 21 – 24 ). These studies employed a two-step approach, involving the pre-amplification of the target sequence using techniques such as PCR, LAMP, or RPA, followed by the transfer of the amplified products to the CRISPR reaction system.…”

Section: Discussionmentioning

confidence: 99%

“…AFB smear microscopy lacks specificity in distinguishing Mtb from nontuberculous mycobacteria (NTM). While culture is considered a “gold standard” with moderate sensitivity (39%–70%), its utility is limited by infrastructure and lengthy incubation (2–8 weeks) ( 2 – 8 ). Advancements in molecular diagnostics have led to the rapid evolution of TB testing ( 9 ), exemplified by Xpert MTB/RIF (Cepheid, USA).…”

Section: Introductionmentioning

confidence: 99%

“…However, this approach raises concerns about the potential for cross-contamination and operational complexity. To our knowledge, current CRISPR-based TB detection systems all involve a two-step process ( 4 – 8 , 21 – 24 ). To address these limitations, we have developed an innovative TB detection method, which we have named TB One-Pot.…”

Section: Introductionmentioning

confidence: 99%

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Rapid detection of Mycobacterium tuberculosis in sputum using CRISPR-Cas12b combined with cross-priming amplification in a single reaction

Peng,

Fang,

Cai

et al. 2024

J Clin Microbiol

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The diagnosis of tuberculosis (TB) still lacks a rapid, user-friendly method. Recent research has revealed the significant diagnostic potential of the CRISPR system in TB detection. However, most current CRISPR-based diagnostic tests involve a two-step process, including nucleic acid amplification followed by CRISPR-guided sequence-specific detection, which raises concerns about cross-contamination and operational complexity. In this study, we introduced TB One-Pot, which combines CRISPR-Cas12b-mediated trans-cleavage with cross-priming amplification, enabling one-pot detection in a single reaction. TB One-Pot achieves a minimum detection limit of 0.8 copies/μL for H37Rv genomic DNA and a quantification limit of 50 CFU/mL. It exhibited no cross-reactivity with common nontuberculous mycobacteria and respiratory pathogens. TB One-Pot employs real-time fluorescence, enabling the process from nucleic acid extraction to result in reporting in just 80 minutes. It also includes a UV-based visual check, eliminating the need for specialized equipment. A retrospective cohort study evaluated the diagnostic performance of this method using a composite reference standard as the gold standard. TB One-Pot demonstrated a sensitivity of 67.2% and an area under the curve (AUC) of 0.820 in sputum samples, surpassing conventional methods such as culture and acid-fast bacilli (AFB) smear. Comparative analysis with Xpert (sensitivity:64.2%, AUC:0.821) showed no statistically significant difference ( P > 0.05). TB One-Pot exhibited a specificity of 96.7%, similar to Xpert, culture, and AFB smear ( P > 0.05). TB One-Pot excels in speed, sensitivity, specificity, and practical requirements. Ongoing refinements aim to improve sensitivity, making it a promising solution for overcoming limitations in molecular TB diagnosis, particularly in resource-constrained settings. IMPORTANCE In this study, we successfully established a new One-Pot method, named TB One-Pot, for detecting Mtb in sputum by combining CRISPR-cas12b-mediated trans-cleavage with cross-priming amplification (CPA). Our study evaluated the diagnostic performance of TB One-Pot in clinical sputum samples for tuberculosis. The findings provide evidence for the potential of TB One-Pot as a diagnostic tool for tuberculosis.

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Development and evaluation of rapid and accurate one-tube RPA-CRISPR-Cas12b-based detection of mcr-1 and tet(X4)

Wang,

Chen,

Pan

et al. 2024

Appl Microbiol Biotechnol

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The emergence and quick spread of the plasmid-mediated tigecycline resistance gene tet(X4) and colistin resistance gene mcr-1 have posed a great threat to public health and raised global concerns. It is imperative to develop rapid and accurate detection systems for the onsite surveillance of mcr-1 and tet(X4). In this study, we developed one-tube recombinase polymerase amplification (RPA) and CRISPR-Cas12b integrated mcr-1 and tet(X4) detection systems. We identified mcr-1- and tet(X4)-conserved and -specific protospacers through a comprehensive BLAST search based on the NCBI nt database and used them for assembling the detection systems. Our developed one-tube RPA-CRISPR-Cas12b-based detection systems enabled the specific detection of mcr-1 and tet(X4) with a sensitivity of 6.25 and 9 copies within a detection time of ~ 55 and ~ 40 min, respectively. The detection results using pork and associated environmental samples collected from retail markets demonstrated that our developed mcr-1 and tet(X4) detection systems could successfully monitor mcr-1 and tet(X4), respectively. Notably, mcr-1- and tet(X4)-positive strains were isolated from the positive samples, as revealed using the developed detection systems. Whole-genome sequencing of representative strains identified an mcr-1-carrying IncI2 plasmid and a tet(X4)-carrying IncFII plasmid, which are known as important vectors for mcr-1 and tet(X4) transmission, respectively. Taken together, our developed one-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems show promising potential for the onsite detection of mcr-1 and tet(X4). Key points • One-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems were developed based on identified novel protospacers. • Both detection systems exhibited high sensitivity and specification with a sample-to-answer time of less than 1 h. • The detection systems show promising potential for onsite detection of mcr-1 and tet(X4).

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Development of CRISPR/Cas12b-Based Multiple Cross Displacement Amplification Technique for the Detection of Mycobacterium tuberculosis Complex in Clinical Settings

Abstract: The Mycobacterium tuberculosis complex pathogen is a crucial infectious agent of tuberculosis. Hence, improving the capability of MTC detection is one of the most urgently required strategies for preventing and controlling TB.

Cited by 8 publications

References 45 publications

A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application

A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application

Rapid detection of Mycobacterium tuberculosis in sputum using CRISPR-Cas12b combined with cross-priming amplification in a single reaction

Development and evaluation of rapid and accurate one-tube RPA-CRISPR-Cas12b-based detection of mcr-1 and tet(X4)

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