Ultrafast visual nucleic acid detection with CRISPR/Cas12a and rapid PCR in single capillary

Wang, Rui; Chen, Rui; Qian, Cheng; Pang, Yanan; Wu, Jian; Li, Fuyou

doi:10.1016/j.snb.2020.128618

Cited by 31 publications

(26 citation statements)

References 35 publications

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“…The corresponding target nucleic acid sequence can also be detected by CRISPR technology alone, but its minimum detection limit can only reach the femtomolar level ( Gootenberg et al, 2017 ), which is far from meeting the needs of clinical detection. Nucleic acid amplification reagents are mixed with Cas enzyme to perform nucleic acid amplification and detection at the same time and avoid opening the lid ( Wang et al, 2021a ; Wu et al, 2020 ; Zhang et al, 2020 ). However, this reduces the nucleic acid detection sensitivity, mainly because some components of the CRISPR reagent buffer have a certain inhibitory effect on nucleic acid amplification ( Arizti-Sanz et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

“…The innovation of experimental consumables is also an important approach to solve the above-mentioned problems. For example, Wang et al separated the two reaction systems by pre-adding the rapid PCR reagent and CRISPR reagent to the bottom and top of the capillary tube, respectively, to realize nucleic acid detection without opening the cap ( Wang et al, 2021a ). Wu et al proposed a Cas12a-PB sensor composed of a chamber for CRISPR detection, opened on one side of the PMMA plate; and an amplification reaction tube, connected to the other side to realize the Cas12a-PB detection based on CRISPR reaction and RPA reaction ( Wu et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

Liu

Zhao

et al. 2022

Biosensors and Bioelectronics

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The pandemic due to the outbreak of 2019 coronavirus disease (COVID-19) caused by novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised significant public health concerns. Rapid, affordable, and accurate diagnostic testing not only paves the way for the effective treatment of diseases, but also plays a crucial role in preventing the spreading of infectious diseases. Herein, a one-pot CRISPR/Cas13a-based visual biosensor was proposed and developed for the rapid and low-cost nucleic acid detection. By combining Cas13a cleavage and Recombinase Polymerase Amplification (RPA) in a one-pot reaction in a disposable tube-in-tube vessel, amplicon contamination could be completely avoided. The RPA reaction is carried out in the inner tube containing two hydrophobic holes at the bottom. After the completion of amplification reaction, the reaction solution enters the outer tube containing pre-stored Cas13a reagent under the action of centrifugation or shaking. Inner and outer tubes are combined to form an independent reaction pot to complete the nucleic acid detection without opening the lid. This newly developed nucleic acid detection method not only meets the need of rapid nucleic acid detection at home without the need for any specialized equipment, but also fulfils the requirement of rapid on-site nucleic acid detection with the aid of small automated instruments. In this study, CRISPR/Cas13a and CRISPR/Cas12a were used to verify the reliability of the developed one-pot nucleic acid detection method. The performance of the system was verified by detecting the DNA virus, i.e., African swine fever virus (ASFV) and the RNA virus, i.e., SARS-Cov-2. The results indicate that the proposed method possesses a limit of detection of 3 copy/μL. The negative and positive test results are consistent with the results of real-time fluorescence quantitative polymerase chain reaction (PCR), but the time required is shorter and the cost is lower. Thus, this study makes this method available in resource-limited areas for the purpose of large-scale screening and in case of epidemic outbreak.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

Liu

Zhao

et al. 2022

Biosensors and Bioelectronics

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“…First, it is more specific than colorimetric methods. The biggest drawback of colorimetric methods is that most of them are lack of detection specificity because their detection objects are based on by-products of LAMP amplification including H + induced pH change ( Zhang et al, 2020a , Zhang et al, 2020b ; Huang et al, 2020 ; Lu et al, 2020a , Lu et al, 2020b ), double stranded DNA secondary structure ( Wang et al, 2020a , Wang et al, 2020b ; Park et al, 2020 ) and Magnesium pyrophosphate ( Lau et al, 2020 ; Yang et al, 2020 ). Our proposed method employed CRISPR, which even could achieve single base mutation (SNP) detection by crRNA specially targeting target sequence, greatly improving detection specificity.…”

Section: Discussionmentioning

confidence: 99%

“…Third, our method is more distinguishable with naked eye than colorimetric methods. Colorimetric methods have high background for naked eye observation because the color always changes from one color to an adjacent one ( Wang et al, 2020a , Wang et al, 2020b ; Lau et al, 2020 ; Park et al, 2020 ). Our proposed method is based on fluorescence detection, which is more distinguishable with naked eye than colorimetric methods.…”

Section: Discussionmentioning

confidence: 99%

“…Upon recognition of their RNA or DNA targets, activated CRISPR-Cas nucleases indiscriminately cleave nearby single-stranded non-targeted nucleic acids ( Chen et al, 2018 ). Combining with nucleic acid amplification, amplified products will trigger CRISPR-Cas nucleases for collateral cleavage of reporters to achieve additional sensitivity ( Kellner et al, 2019 ; Wang et al, 2020a , Wang et al, 2020b ). Researchers have developed CRISPR-based tests by integrating PCR or recombinase polymerase amplification (RPA) with CRISPR cleavage ( Ding et al, 2020 ; Guo et al, 2020 ; Tian et al, 2020 ; Wang et al, 2019 , Wang et al, 2020a , Wang et al, 2020b ; Zhang et al, 2020a , Zhang et al, 2020b ).…”

Section: Introductionmentioning

confidence: 99%

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opvCRISPR: One-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection

Wang

Qian

Pang

et al. 2021

Biosensors and Bioelectronics

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220

156

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The 2019 novel coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected all aspects of human life. Rapid, accurate, sensitive and user friendly detection method is urgently needed to facilitate early intervention and control the spread of SARS-CoV-2. Here, we propose a one-pot visual SARS-CoV-2 detection system named “opvCRISPR” by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a cleavage in a single reaction system. We demonstrate that the collateral activity against single-stranded DNA (ssDNA) reporters of activated Cas12a triggered by RT-LAMP amplicon increases detection sensitivity and makes detection results observable with naked eye. The opvCRISPR enables detection at nearly single molecule level in 45 min. We validate this method with 50 SARS-CoV-2 potentially infected clinical samples. The opvCRISPR diagnostic results provide 100% agreement with the Centers for Disease Control and Prevention (CDC)-approved quantitative RT-PCR assay. The opvCRISPR holds great potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics.

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A Simplified Amplification‐Free Strategy with Lyophilized CRISPR‐CcrRNA System for Drug‐Resistant Salmonella Detection

Bao

Sun

et al. 2023

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With the widespread abuse of antibiotics, the problem of bacterial resistance is becoming increasingly serious, resulting in more than 700 000 deaths annually, worldwide. [1] Salmonella, one of the most dangerous and infectious foodborne pathogens, ranks second among pathogens that endanger public food safety. [2] Salmonella accounts for 41% of global diarrhea-related deaths. [1,3] The emergence of drug-resistant Salmonella has increased the danger to public health. Early diagnosis and prompt detection of drug resistance require a rapid, sensitive, and cost-effective detection program from the perspective of prevention, screening for drug-resistant bacteria, and management of food-related accidents. [4,5] Traditional culture-based antibiotic sensitivity testing (AST) methods are complicated and often require 2 to 7 days. [6] In contrast, nucleic acid detection of drug-resistance genes has significant advantages in terms of sensitivity and specificity. [7] The gold standard for nucleic acid detection, quantitative polymerase chain reaction (qPCR), can detect 10 CFU mL −1 of resistant bacteria within a few hours. [8,9] However, it still relies heavily on specialized equipment and expertise and is difficult to use outside a laboratory setting. [4] Lateral flow assay (LFA) is the most promising point-of-care testing (POCT) platform owing to its minimal device dependence, speed, sensitivity, and user-friendliness. [10,11] A visual readout LFA based on antigen capture with a faster and more accurate reading within 15 min was developed. [12] Unfortunately, this immunology-based pathogenic bacterial detection via LFA generally has low sensitivity and cannot be used for AST. [10,13] Combining the CRISPR/Cas system with LFA allows for sensitive and specific detection of targets while maintaining its fast and user-friendly features. For instance. Gootenberg et al [14] proposed a method called SHERLOCK for SARS-CoV-2 detection, [15] Joung et al. [13] demonstrated the direct detection of SARS-CoV-2 samples and further developed the SHINE. [16] All of the LFA-CRISPR methods mentioned above enable Drug resistance in pathogenic bacteria has become a major threat to global health. The misuse of antibiotics has increased the number of resistant bacteria in the absence of rapid, accurate, and cost-effective diagnostic tools. Here, an amplification-free CRISPR-Cas12a time-resolved fluorescence immunochromatographic assay (AFC-TRFIA) is used to detect drugresistant Salmonella. Multi-locus targeting in combination crRNA (CcrRNA) is 27-fold more sensitive than a standalone crRNA system. The lyophilized CRISPR system further simplifies the operation and enables one-pot detection. Induction of nucleic acid fixation via differentially charged interactions reduced the time and cost required for flowmetric chromatography with enhanced stability. The induction of nucleic acid fixation via differentially charged interactions reduces the time and cost required for flowmetric chromatography with enhanced stability. The platform developed for th...

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Ultrafast visual nucleic acid detection with CRISPR/Cas12a and rapid PCR in single capillary

Cited by 31 publications

References 35 publications

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

opvCRISPR: One-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection

A Simplified Amplification‐Free Strategy with Lyophilized CRISPR‐CcrRNA System for Drug‐Resistant Salmonella Detection

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