scholarly journals CRISPR-Cas12b-assisted nucleic acid detection platform

2018 ◽  
Author(s):  
Linxian Li ◽  
Shiyuan Li ◽  
Jin Wang
Keyword(s):  
Nucleic Acid ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Rna Detection ◽  
Environmental Testing ◽  
Cleavage Activity ◽  
Target Nucleic Acid ◽  
Reverse Transcription Step ◽  
Diagnostic Technology ◽  
One Step

AbstractRapid molecular diagnostic technology is very useful in many areas, including public health, environmental testing and criminal investigation. We recently showed that Cas12a had trans-cleavage activity upon collateral single-stranded DNA (ssDNA), with which the HOLMES platform (one-HOur Low-cost Multipurpose highly Efficient System) was developed. Here, we combine the thermophilic Cas12b, which also has the ssDNA trans-cleavage activity, with Loop-Mediated Isothermal Amplification (LAMP), and create HOLMESv2. In HOLMESv2, LAMP amplification and Cas12b trans-cleavage can be integrated into a one-step system with a constant temperature, which therefore brings much convenience in nucleic acid detection. Moreover, we also simplify the RNA detection procedures in HOLMESv2, using an RNA-dependent DNA polymerase for amplification and therefore omitting an extra reverse transcription step.One Sentence SummaryWe combine LAMP and Cas12b to develop HOLMESv2 for conveniently detecting target nucleic acid in a one-step approach.

scholarly journals Scanning single-molecule counting system for Eprobe with highly simple and effective approach

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243319
Author(s):  
Takeshi Hanami ◽  
Tetsuya Tanabe ◽  
Takuya Hanashi ◽  
Mitsushiro Yamaguchi ◽  
Hidetaka Nakata ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Detection System ◽  
Signal To Noise Ratio ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Digital Measurement ◽  
Excitation Light ◽  
Target Nucleic Acid ◽  
Fluorescent Molecules

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

scholarly journals IsoPCR: An Analytically Sensitive, Nested, Multiplex Nucleic Acid Amplification Method

2013 ◽  
Vol 59 (2) ◽  
pp. 436-439 ◽  
Author(s):  
Martin Jensen Søe ◽  
Mikkel Rohde ◽  
Jens Mikkelsen ◽  
Peter Warthoe
Keyword(s):  
Nucleic Acid ◽  
Candida Glabrata ◽  
Simultaneous Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Amplification Method

BACKGROUND Nucleic acid tests that can simultaneously detect multiple targets with high sensitivity, specificity, and speed are highly desirable. To meet this need, we developed a new approach we call the isoPCR method. METHODS The isoPCR method is a 2-stage nested-like nucleic acid amplification method that combines a single multiplex preamplification PCR with subsequent distinct detection of specific targets by use of isothermal amplification. We compared isoPCR to nested quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and nested LAMP (PCR followed by LAMP), for detection of DNA from Candida glabrata. We evaluated the method's multiplex capability for detecting low copy numbers of pathogens commonly involved in sepsis. RESULTS IsoPCR provided detection of 1 copy of Candida glabrata, an LOD that was 5-fold lower than a nested qPCR assay (5 copies), while the amplification time was simultaneously halved. Similarly, the LOD for isoPCR was lower than that for a LAMP assay (1000 copies) and a nested LAMP assay (5 copies). IsoPCR required recognition of 6 regions for detection, thereby providing a theoretically higher specificity compared to nested qPCR (4 regions). The isoPCR multiplexing capability was demonstrated by simultaneous detection of 4 pathogens with individual LODs of 10 copies or fewer. Furthermore, the specificity of isoPCR was demonstrated by successful pathogen detection from samples with more than 1 pathogen present. CONCLUSIONS IsoPCR provides a molecular diagnostic tool for multiplex nucleic acid detection, with an LOD down to 1 copy, high theoretical specificity, and halving of the amplification time compared to a nested qPCR assay.

scholarly journals Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Long T. Nguyen ◽  
Brianna M. Smith ◽  
Piyush K. Jain
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Divalent Cations ◽  
Nucleic Acid Detection ◽  
Wild Type ◽  
Detailed Characterization ◽  
Cleavage Activity ◽  
Optimized Conditions ◽  
Higher Sensitivity

AbstractThe CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3′- or 5′-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40–60 min.

A catalytic DNA circuit-programmed and enzyme-powered autonomous DNA machine for nucleic acid detection

The Analyst ◽  
2019 ◽  
Vol 144 (20) ◽  
pp. 5923-5927 ◽  
Author(s):  
Shuang Liu ◽  
Chen Xin ◽  
Xiaoxiao Yu ◽  
Zhenbo Ding ◽  
Shufeng Liu
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Detection ◽  
One Step ◽  
Catalytic Dna ◽  
Dna Machine

A catalytic DNA circuit-programmed and enzyme-powered autonomous DNA machine was proposed for one-step, isothermal and dual-level amplified detection of nucleic acids.

scholarly journals A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): application for the diagnosis of COVID-19

Talanta ◽  
2021 ◽  
pp. 122591
Author(s):  
Shijun Li ◽  
Junfei Huang ◽  
Lijuan Ren ◽  
Weijia Jiang ◽  
Ming Wang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Detection ◽  
One Pot ◽  
One Step

scholarly journals Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

2020 ◽  
Author(s):  
Kenneth N. Hass ◽  
Mengdi Bao ◽  
Qian He ◽  
Myeongkee Park ◽  
Peiwu Qin ◽  
...  
Keyword(s):  
Point Of Care ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Viral Dna ◽  
Fully Integrated ◽  
Impact System ◽  
Target Dna ◽  
Reporter Probe ◽  
One Step ◽  
Labeled Probe

ABSTRACTA fully Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) system is developed for viral DNA detection. This powerful system is patterned with high-aspect ratio micropillars to enhance reporter probe binding. After surface modification and probe immobilization, CRISPR Cas12a/crRNA complex is injected into the fully enclosed system. With the presence of double-stranded DNA target, the CRISPR enzyme is activated and non-specifically cleaves the ssDNA reporters initially immobilized on the micropillars. This collateral cleavage releases fluorescence dyes into the assay, and the intensity is linearly proportional to the target DNA concentration ranging from 0.1 to 10 nM. Importantly, this system does not rely on traditional dye-quencher labeled probe thus eliminating the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed at isothermal conditions (37°C) without using complicated and time-consuming off-chip probe hybridization and denaturation. This miniaturized and fully packed IMPACT chip demonstrates rapid, sensitive, and simple nucleic acid detection and is an ideal candidate for the next generation molecular diagnostic platform for point-of-care (POC) applications, responding to emerging and deadly pathogen outbreaks.

scholarly journals Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

2020 ◽  
Author(s):  
Long T. Nguyen ◽  
Brianna M. Smith ◽  
Piyush K. Jain
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Nucleic Acid Detection ◽  
Wild Type ◽  
Catalytic Behavior ◽  
Methylated Dna ◽  
Cleavage Activity ◽  
Target Dna ◽  
Optimized Conditions ◽  
Higher Sensitivity

AbstractThe CRISPR/Cas12a RNA-guided complexes have a tremendous potential for nucleic acid detection due to its ability to indiscriminately cleave ssDNA once bound to a target DNA. However, the current CRISPR/Cas12a systems are limited to detecting DNA in a picomolar detection limit without an amplification step. Here, we developed a platform with engineered crRNAs and optimized conditions that enabled us to detect DNA, DNA/RNA heteroduplex and methylated DNA with higher sensitivity, achieving a limit of detection of in femtomolar range without any target pre-amplification step. By extending the 3’- or 5’-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discovered a new self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA. We applied this sensitive system to detect as low as 25 fM dsDNA from the PCA3 gene, an overexpressed biomarker in prostate cancer patients, in simulated urine over 6 hours. The same platform was used to detect as low as ~700 fM cDNA from HIV, 290 fM RNA from HCV, and 370 fM cDNA from SARS-CoV-2, all within 30 minutes without a need for target amplification. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs were incorporated in a paper-based lateral flow assay that could detect the target with up to 23-fold higher sensitivity within 40-60 minutes.

scholarly journals Recent Improvements in CRISPR-Based Amplification-Free Pathogen Detection

2021 ◽  
Vol 12 ◽  
Author(s):  
Jian Zhang ◽  
Hailong Lv ◽  
Linxian Li ◽  
Minjie Chen ◽  
Dayong Gu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Pathogen Detection ◽  
Direct Detection ◽  
Detection Sensitivity ◽  
Molecular Diagnostic ◽  
Target Nucleic Acid ◽  
Precise Diagnosis ◽  
Important Approach ◽  
Pathogen Diagnosis ◽  
Diagnostic Technologies

Molecular diagnostic (MDx) methods directly detect target nucleic acid sequences and are therefore an important approach for precise diagnosis of pathogen infection. In comparison with traditional MDx techniques such as PCR, the recently developed CRISPR-based diagnostic technologies, which employ the single-stranded nucleic acid trans-cleavage activities of either Cas12 or Cas13, show merits in both sensitivity and specificity and therefore have great potential in both pathogen detection and beyond. With more and more efforts in improving both the CRISPR trans-cleavage efficiencies and the signal detection sensitivities, CRISPR-based direct detection of target nucleic acids without preamplification can be a possibility. Here in this mini-review, we summarize recent research progresses of amplification-free CRISPR-Dx systems and explore the potential changes they will lead to pathogen diagnosis. In addition, discussion of the challenges for both detection sensitivity and cost of the amplification-free systems will also be covered.

scholarly journals Advanced CRISPR-Cas Effector Enzyme-Based Diagnostics for Infectious Diseases, Including COVID-19

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1356
Author(s):  
Sangha Kwon ◽  
Ha Youn Shin
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Pathogen Detection ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Time Accuracy

Rapid and precise diagnostic tests can prevent the spread of diseases, including worldwide pandemics. Current commonly used diagnostic methods include nucleic-acid-amplification-based detection methods and immunoassays. These techniques, however, have several drawbacks in diagnosis time, accuracy, and cost. Nucleic acid amplification methods are sensitive but time-consuming, whereas immunoassays are more rapid but relatively insensitive. Recently developed CRISPR-based nucleic acid detection methods have been found to compensate for these limitations. In particular, the unique collateral enzymatic activities of Cas12 and Cas13 have dramatically reduced the diagnosis times and costs, while improving diagnostic accuracy and sensitivity. This review provides a comprehensive description of the distinct enzymatic features of Cas12 and Cas13 and their applications in the development of molecular diagnostic platforms for pathogen detection. Moreover, it describes the current utilization of CRISPR-Cas-based diagnostic techniques to identify SARS-CoV-2 infection, as well as recent progress in the development of CRISPR-Cas-based detection strategies for various infectious diseases. These findings provide insights into designing effective molecular diagnostic platforms for potential pandemics.

scholarly journals Sensitive one-step isothermal detection of pathogen-derived RNAs

2020 ◽  
Author(s):  
Chang Ha Woo ◽  
Sungho Jang ◽  
Giyoung Shin ◽  
Gyoo Yeol Jung ◽  
Jeong Wook Lee
Keyword(s):  
Diagnostic Method ◽  
High Sensitivity ◽  
T7 Rna Polymerase ◽  
Molecular Diagnostic ◽  
Enzymatic Reactions ◽  
Rna Aptamer ◽  
Rna Detection ◽  
Highly Sensitive ◽  
One Step ◽  
Sensitivity Specificity

AbstractThe recent outbreaks of Ebola, Zika, MERS, and SARS-CoV-2 (2019-nCoV) require fast, simple, and sensitive onsite nucleic acid diagnostics that can be developed rapidly to prevent the spread of diseases. We have developed a SENsitive Splint-based one-step isothermal RNA detection (SENSR) method for rapid and straightforward onsite detection of pathogen RNAs with high sensitivity and specificity. SENSR consists of two simple enzymatic reactions: a ligation reaction by SplintR ligase and subsequent transcription by T7 RNA polymerase. The resulting transcript forms an RNA aptamer that induces fluorescence. Here, we demonstrate that SENSR is an effective and highly sensitive method for the detection of the current epidemic pathogen, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We also show that the platform can be extended to the detection of five other pathogens. Overall, SENSR is a molecular diagnostic method that can be developed rapidly for onsite uses requiring high sensitivity, specificity, and short assaying times.

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