scholarly journals Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription

2020 ◽  
Vol 4 (12) ◽  
pp. 1168-1179 ◽  
Author(s):  
Chang Ha Woo ◽  
Sungho Jang ◽  
Giyoung Shin ◽  
Gyoo Yeol Jung ◽  
Jeong Wook Lee
Keyword(s):  
Fluorescence Detection ◽  
Clinical Samples ◽  
One Pot

scholarly journals Sensitive quantitative detection of SARS-CoV-2 in clinical samples using digital warm-start CRISPR assay

2020 ◽  
Author(s):  
Xiong Ding ◽  
Kun Yin ◽  
Ziyue Li ◽  
Maroun M. Sfeir ◽  
Changchun Liu
Keyword(s):  
Room Temperature ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
High Tolerance ◽  
Rt Pcr ◽  
One Pot ◽  
Nucleoprotein Gene ◽  
Warm Start ◽  
Heat Treated

AbstractQuantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is crucial for assessing the infectivity of coronavirus disease 2019 and the efficacy of antiviral drugs. Here, we describe a digital warm-start CRISPR (WS-CRISPR) assay for sensitive quantitative detection of SARS-CoV-2 in clinical samples. The WS-CRISPR assay combines low-temperature reverse transcription dual-priming mediated isothermal amplification (RT-DAMP) and CRISPR-Cas12a-based detection in one-pot, attributed to the mediation role by pyrophosphatase and phosphorothioated primers. The WS-CRISPR assay is initiated at above 50 °C and overcomes undesired premature target amplification at room temperature, enabling accurate digital nucleic acid quantification. By targeting SARS-CoV-2’s nucleoprotein gene, digital WS-CRISPR assay is able to detect down to 5 copies/μl SARS-CoV-2 RNA in the chip within 90 minutes. It is clinically validated by quantitatively determining 32 clinical swab samples and three clinical saliva samples, showing 100% agreement with RT-PCR results. Moreover, the digital WS-CRISPR assay has been demonstrated to directly detect SARS-CoV-2 in heat-treated saliva samples without RNA extraction, showing high tolerance to inhibitors. Thus, the digital WS-CRISPR method, as a sensitive and reliable CRISPR assay, facilitates accurate SARS-CoV-2 detection toward digitized quantification.

scholarly journals A multi-analyte selective dansyl derivative for the fluorescence detection of Cu(ii) and cysteine

2019 ◽  
Vol 18 (6) ◽  
pp. 1533-1539 ◽  
Author(s):  
Anand Thangaraj ◽  
Vinita Bhardwaj ◽  
Suban K. Sahoo
Keyword(s):  
Fluorescence Detection ◽  
Dansyl Chloride ◽  
One Pot ◽  
Dansyl Derivative

A new multi-analyte selective fluorescence chemosensor DA was synthesized by a simple one pot reaction between dansyl chloride and 2-aminobenzohydrazide in the presence of a base.

One-pot synthesis of carbon dots co-doped with N and S: high quantum yield governed by molecular state and fluorescence detection of Ag+

2019 ◽  
Vol 117 (18) ◽  
pp. 2500-2510 ◽  
Author(s):  
Wei-jie Ren ◽  
Jing-jing Bai ◽  
Yan-liang Zhao ◽  
Yu-long Wang ◽  
Fei Liu ◽  
...  
Keyword(s):  
Quantum Yield ◽  
Fluorescence Detection ◽  
Carbon Dots ◽  
Molecular State ◽  
High Quantum Yield ◽  
One Pot ◽  
High Quantum ◽  
One Pot Synthesis ◽  
Co Doped

scholarly journals One-Seq: A Highly Scalable Sequencing-Based Diagnostic for SARS-CoV-2 and Other Single-Stranded Viruses

2021 ◽  
Author(s):  
Mingjie Dai ◽  
Wenzhe Ma ◽  
Hong Kang ◽  
Matthew Sonnett ◽  
George M. Church ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Pcr Amplification ◽  
High Dynamic Range ◽  
Clinical Samples ◽  
One Pot ◽  
Reagent Cost ◽  
Early Processing ◽  
Cost Scaling

The management of pandemics such as COVID-19 requires highly scalable and sensitive viral diagnostics, together with variant identification. Next-generation sequencing (NGS) has many attractive features for highly multiplexed testing, however current sequencing-based methods are limited in throughput by early processing steps on individual samples (e.g. RNA extraction and PCR amplification). Here we report a new method, "One-Seq", that eliminates the current bottlenecks in scalability by enabling early pooling of samples, before any extraction or amplification steps. To enable early pooling, we developed a one-pot reaction for efficient reverse transcription (RT) and upfront barcoding in extraction-free clinical samples, and a "protector" strategy in which carefully designed competing oligonucleotides prevent barcode crosstalk and preserve detection of the high dynamic range of viral load in clinical samples. This method is highly sensitive, achieving a limit of detection (LoD) down to 2.5 genome copy equivalent (gce) in contrived RT samples, 10 gce in multiplexed sequencing, and 2-5 gce with multi-primer detection, suggesting an LoD of 200-500 gce/ml for clinical testing. In clinical specimens, One-Seq showed quantitative viral detection against clinical Ct values with 6 logs of linear dynamic range and detection of SARS-CoV-2 positive samples down to ~360 gce/ml. In addition, One-Seq reports a number of hotspot viral mutations at equal scalability at no extra cost. Scaling up One-Seq would allow a throughput of 100,000-1,000,000 tests per day per single clinical lab, at an estimated amortized reagent cost of $1.5 per test and turn-around time of 7.5-15 hr.

scholarly journals A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern

2021 ◽  
Author(s):  
Long T. Nguyen ◽  
Nicolas C. Macaluso ◽  
Brianna L.M. Pizzano ◽  
Melanie N. Cash ◽  
Jan Spacek ◽  
...  
Keyword(s):  
Thermal Instability ◽  
Reaction System ◽  
High Viral Load ◽  
Detection Methods ◽  
Clinical Samples ◽  
One Pot ◽  
Efficient Manner ◽  
Guide Rna ◽  
Design Requirements ◽  
The Cost

Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has the potential to transform diagnostics due to its programmability. However, many of the CRISPR-based detection methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. A complete one-pot detection reaction using alternative Cas effector endonucleases has been proposed to overcome these challenges. Yet, current approaches using Alicyclobacillus acidiphilus Cas12b (AapCas12b) are limited by its thermal instability at optimum reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction temperatures. Herein, we demonstrate that a novel Cas12b from Brevibacillus sp. SYP-B805 (referred to as BrCas12b) has robust trans-cleavage activity at ideal RT-LAMP conditions. A competitive profiling study of BrCas12b against Cas12b homologs from other bacteria genera underscores the potential of BrCas12b in the development of new diagnostics. As a proof-of-concept, we incorporated BrCas12b into an RT-LAMP-mediated one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs) to enable rapid, differential detection of SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) in 205 clinical samples. Notably, a BrCas12b detection signal was observed within 1-3 minutes of amplification, achieving an overall 98.1% specificity, 91.2% accuracy, and 88.1% sensitivity within 30 minutes. Significantly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, we combined the lyophilized one-pot reagents with a portable multiplexing device capable of interpreting fluorescence signals at a fraction of the cost of a qPCR system. With relaxed design requirements, one-pot detection, and simple instrumentation, this assay has the capability to advance future diagnostics.

scholarly journals Cell-Free Synthetic Biosensors Based on CRISPR/Cas Mediated Cascade Signal Amplification for Precise RNA Detection

2021 ◽  
Author(s):  
Chao Zhang ◽  
Penghui Zhang ◽  
Hui Ren ◽  
Pengpeng Jia ◽  
Jingcheng Ji ◽  
...  
Keyword(s):  
Cancer Diagnosis ◽  
Enzyme Synthesis ◽  
Clinical Samples ◽  
One Pot ◽  
Rna Detection ◽  
Detection Technology ◽  
A Cell ◽  
Glucose Meter ◽  
Cascade Amplification ◽  
Diagnostic Technologies

Abstract Synthetic biology has been harnessed to create new diagnostic technologies. However, most synthetic biosensors involve error-prone amplification steps and limitations of accuracy in RNA detection. Here, we report a cell-free synthetic biosensing platform, termed as SHARK (Synthetic Enzyme Shift RNA Signal Amplifier Related Cas13a Knockdown Reaction), to efficiently and accurately amplify RNA signal by leveraging the collateral cleavage of activated Cas13a to regulate cell-free enzyme synthesis. Based on cascade amplification and customized enzyme output, SHARK behaves a broad compatibility in different scenarios. Using a personal glucose meter, we detected 50 copies/μl SARS-CoV-2 on a SHARK-loaded paper. In addition, when combined with machine learning, SHARK can perform bio-computations and thus provide miRNA patterns for cancer diagnosis and staging. SHARK shows characteristics of precise recognition, cascade amplification and customizable signal outputting in one pot comparisons with established assays based on 64 clinical samples, presenting great potential in developing next-generation RNA detection technology.

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