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

Wang, Yi; Li, Jieqiong; Li, Shijun; Zhu, Xiong; Wang, Xiaoxia; Huang, Junfei; Yang, Xinggui; Tai, Jun

doi:10.1007/s00604-021-04985-w

Cited by 54 publications

(49 citation statements)

References 19 publications

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“…With dCas9, the first approach for TB detection was initiated following which the CRISPR/Cas12 effector was utilized as a pulmonary and extra-pulmonary TB-detection platform [ 9 , 59 , 68 , 110 , 111 ]. CRISPR-based detection eliminates pathogen cultures and provides faster results without requiring an expert technician.…”

Section: Broad Application Of Crispr/cas Systemmentioning

confidence: 99%

“…With specifically designed primers that meet the design requirements of LAMP or RPA, PAM sequences can be delivered to the target during pre-amplification. Any target (with or without the PAM sequence) can theoretically be detected using PAM-introduced primers for pre-amplification [ 68 , 125 ]. Furthermore, cross-contaminations can be avoided with visual or smartphone-based applications for result readouts [ 53 , 109 ].…”

Section: Advancements In Crispr Detectionmentioning

confidence: 99%

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Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection

Bhardwaj

Kant

Behera

et al. 2022

IJMS

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The early management, diagnosis, and treatment of emerging and re-emerging infections and the rising burden of non-communicable diseases (NCDs) are necessary. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system has recently acquired popularity as a diagnostic tool due to its ability to target specific genes. It uses Cas enzymes and a guide RNA (gRNA) to cleave target DNA or RNA. The discovery of collateral cleavage in CRISPR-Cas effectors such as Cas12a and Cas13a was intensively repurposed for the development of instrument-free, sensitive, precise and rapid point-of-care diagnostics. CRISPR/Cas demonstrated proficiency in detecting non-nucleic acid targets including protein, analyte, and hormones other than nucleic acid. CRISPR/Cas effectors can provide multiple detections simultaneously. The present review highlights the technical challenges of integrating CRISPR/Cas technology into the onsite assessment of clinical and other specimens, along with current improvements in CRISPR bio-sensing for nucleic acid and non-nucleic acid targets. It also highlights the current applications of CRISPR/Cas technologies.

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Section: Broad Application Of Crispr/cas Systemmentioning

confidence: 99%

Section: Advancements In Crispr Detectionmentioning

confidence: 99%

Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection

Bhardwaj

Kant

Behera

et al. 2022

IJMS

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“…Recognition and cleavage of the target transform the RNP complex into a non-specific nuclease that collaterally cleaves singlestranded DNA (ssDNA) in vitro [44,107]. This collateral cleavage property and programmability of CRISPR-Cas12a offer an adaptation for highly specific and sensitive detection of DNA [44,46,53,[108][109][110]. Therefore, we aimed to apply this CRISPR diagnostic platform to the detection of B. pseudomallei DNA.…”

Section: Designing Crispr-cas12a Specific To B Pseudomalleimentioning

confidence: 99%

Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a

et al. 2022

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Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.

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“…The TB-QUICK assay showed comparable sensitivity in detecting AFB-positive TB patients with MTB culture and Xpert assays (100%, 100%, and 95.8%, respectively), with dramatically higher sensitivity in detecting AFB-negative TB patients, making it more advantageous for the diagnosis of paucibacillary TB patients. A similar study targeting the MTB complex was conducted by coupling LAMP with CRISPR/Cas12a, using the end-point detection of either fluorescent detection or lateral flow test [ 13 ]. The established method, with an LOD of about 10 copies/reaction and a specificity of 100%, performed better than conventional culture, AFB smear test, and Xpert MTB/FIR in detection of sputum samples with a low MTB complex pathogen load.…”

Section: Crispr-based Diagnostics In Tb Diagnosismentioning

confidence: 99%

CRISPR-Based Diagnostics: A Potential Tool to Address the Diagnostic Challenges of Tuberculosis

Li²,

Li³

et al. 2022

Pathogens

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Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, which infects more than 23% of the world’s population. With the emergence of drug-resistant TB (DR-TB) and latent TB infection (LTBI), rapid diagnosis of DR-TB and LTBI has become a challenge for the prevention and control of TB. Herein, we highlight these challenges and discuss emerging clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics in TB detection. Currently, the clinical diagnosis of M. tuberculosis infection mainly depends on pathogenic and molecular biological methods, including sputum smear, sputum culture, and Xpert. Although CRISPR-based diagnostics have not been applied to the clinical diagnosis of TB, they have shown exciting preponderances in TB diagnosis compared with traditional methods, including higher sensitivity, less sample input, and shorter turnaround time. CRISPR-based diagnostics represent a potential tool to address the challenges and natural weaknesses associated with traditional TB diagnosis methods. Based on the currently available data, we suggest that future CRISPR-based TB diagnostics should be developed in the direction of automation, modularization, diversification, and intelligence. By combining the CRISPR platform with various systems, such as microfluidic chips, droplet microfluidics, electrochemical techniques, and optical systems, the specificity and sensitivity of TB diagnosis may be revolutionized.

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

Cited by 54 publications

References 19 publications

Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection

Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection

Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a

CRISPR-Based Diagnostics: A Potential Tool to Address the Diagnostic Challenges of Tuberculosis

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