scholarly journals Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection

2022 ◽  
Vol 2 (1) ◽  
Author(s):  
Long T. Nguyen ◽  
Santosh R. Rananaware ◽  
Brianna L. M. Pizzano ◽  
Brandon T. Stone ◽  
Piyush K. Jain
Keyword(s):  
High Sensitivity ◽  
High Specificity ◽  
Detection Methods ◽  
Clinical Validation ◽  
Respiratory Pathogens ◽  
Detection Capability ◽  
Guide Rna ◽  
Assay Optimization ◽  
Dual Reporter ◽  
Wide Range

Abstract Background The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies have emerged as a rapid and affordable platform that can shape the future of diagnostics. Methods We developed ENHANCEv2 that is composed of a chimeric guide RNA, a modified LbCas12a enzyme, and a dual reporter construct to improve the previously reported ENHANCE system. We validated both ENHANCE and ENHANCEv2 using 62 nasopharyngeal swabs and compared the results to RT-qPCR. We created a lyophilized version of ENHANCEv2 and characterized its detection capability and stability. Results Here we demonstrate that when coupled with an RT-LAMP step, ENHANCE detects COVID-19 samples down to a few copies with 95% accuracy while maintaining a high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. ENHANCE works robustly in a wide range of magnesium concentrations (3 mM-13 mM), allowing for further assay optimization. Our clinical validation results for both ENHANCE and ENHANCEv2 show 60/62 (96.7%) sample agreement with RT-qPCR results while only using 5 µL of sample and 20 minutes of CRISPR reaction. We show that the lateral flow assay using paper-based strips displays 100% agreement with the fluorescence-based reporter assay during clinical validation. Finally, we demonstrate that a lyophilized version of ENHANCEv2 shows high sensitivity and specificity for SARS-CoV-2 detection while reducing the CRISPR reaction time to as low as 3 minutes while maintaining its detection capability for several weeks upon storage at room temperature. Conclusions CRISPR-based diagnostic platforms offer many advantages as compared to conventional qPCR-based detection methods. Our work here provides clinical validation of ENHANCE and its improved form ENHANCEv2 for the detection of COVID-19.

scholarly journals Engineered CRISPR/Cas12a Enables Rapid SARS-CoV-2 Detection

2020 ◽  
Author(s):  
Long T. Nguyen ◽  
Santosh R. Rananaware ◽  
Brianna L.M. Pizzano ◽  
Brandon T. Stone ◽  
Piyush K. Jain
Keyword(s):  
High Sensitivity ◽  
High Specificity ◽  
Single Copy ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Magnesium Concentration ◽  
Clinical Validation ◽  
Respiratory Pathogens ◽  
Reporter Assay ◽  
Wide Range

ABSTRACTThe coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies such as DETECTR, SHERLOCK, and STOPCovid have emerged as a rapid and affordable platform that can shape the future of diagnostics. Recently, we reported engineered crRNAs for Cas12a, called ENHANCE, that enables enhanced detection of nucleic acids. Here we report development, clinical validation, and advancement of ENHANCE platform for detecting SARS-CoV-2. With an RT-LAMP pre-amplification step, ENHANCE detects samples down to a single copy with 95% accuracy and shows high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. Utilizing LbCas12a-mediated trans-cleavage activity, ENHANCE works robustly in a wide range of magnesium concentration (3 mM-13 mM), allowing for further assay optimization. Additionally, ENHANCEv2 is developed to further improve the previously reported ENHANCE. ENHANCEv2 employs mutated LbCas12aD156R, engineered chimeric DNA-extended crRNA, and a dual reporter for both fluorescence-based reporter assay and lateral flow assay. Both ENHANCE and ENHANCEv2 are validated in 62 clinical nasopharyngeal swabs, showing 60/62 (96.7%) agreement with RT-qPCR results, and using only 5 μL of sample and 20 minutes of CRISPR reaction. Lateral flow assay on paper strips displays 100% agreement with fluorescence-based reporter assay in the clinical validation. Following a 30-minute pre-amplification RT-LAMP step, the lyophilized ENHANCEv2 is shown to achieve high sensitivity and specificity while reducing CRISPR reaction time to as low as 3 minutes and maintaining its detection capability upon storage at room temperature for several weeks.

scholarly journals Engineered CRISPR/Cas12a Enables Rapid SARS-CoV-2 Detection

2021 ◽  
Author(s):  
Long Nguyen ◽  
Santosh Rananaware ◽  
Brianna Pizzano ◽  
Brandon Stone ◽  
Piyush Jain
Keyword(s):  
High Sensitivity ◽  
High Specificity ◽  
Single Copy ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Magnesium Concentration ◽  
Clinical Validation ◽  
Respiratory Pathogens ◽  
Reporter Assay ◽  
Wide Range

Abstract The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies such as DETECTR, SHERLOCK, and others have emerged as a rapid and affordable platform that can shape the future of diagnostics. Recently, we reported engineered crRNAs for Cas12a, called ENHANCE, that enables enhanced detection of nucleic acids. Here we report development, clinical validation, and advancement of ENHANCE platform for detecting SARS-CoV-2. With an RT-LAMP pre-amplification step, ENHANCE detects samples down to a single copy with 95% accuracy and shows high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. Utilizing LbCas12a-mediated trans-cleavage activity, ENHANCE works robustly in a wide range of magnesium concentration (3 mM-13 mM), allowing for further assay optimization. Additionally, ENHANCEv2 is developed to further improve the previously reported ENHANCE. ENHANCEv2 employs mutated LbCas12aD156R, engineered chimeric DNA-extended crRNA, and a dual reporter for both fluorescence-based reporter assay and lateral flow assay. Both ENHANCE and ENHANCEv2 are validated in 62 clinical nasopharyngeal swabs, showing 60/62 (96.7%) agreement with RT-qPCR results, and using only 5 µL of sample and 20 minutes of CRISPR reaction. Lateral flow assay on paper strips displays 100% agreement with fluorescence-based reporter assay in the clinical validation. Following a 30-minute pre-amplification RT-LAMP step, the lyophilized ENHANCEv2 is shown to achieve high sensitivity and specificity while reducing CRISPR reaction time to as low as 3 minutes and maintaining its detection capability upon storage at room temperature for several weeks.

scholarly journals DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

2015 ◽  
Vol 8 ◽  
pp. MBI.S29736 ◽  
Author(s):  
Kenjiro Nagamine ◽  
Guo-Chiuan Hung ◽  
Bingjie Li ◽  
Shyh-Ching Lo
Keyword(s):  
Safety Concern ◽  
Rapid Development ◽  
High Sensitivity ◽  
High Specificity ◽  
Clostridium Tetani ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Pcr Assays ◽  
Primer Sets ◽  
Species Specific

Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

scholarly journals Long Noncoding RNAs as Novel Biomarkers Have a Promising Future in Cancer Diagnostics

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Ting Shi ◽  
Ge Gao ◽  
Yingli Cao
Keyword(s):  
Noncoding Rnas ◽  
High Sensitivity ◽  
High Specificity ◽  
Body Fluids ◽  
Cancer Diagnostics ◽  
Long Noncoding Rnas ◽  
Detection Methods ◽  
Tumor Biomarkers ◽  
Biological Functions ◽  
Sample Collection

Cancers have a high mortality rate due to lack of suitable specific early diagnosis tumor biomarkers. Emerging evidence is accumulating that lncRNAs (long noncoding RNAs) are involved in tumorigenesis, tumor cells proliferation, invasion, migration, apoptosis, and angiogenesis. Furthermore, extracellular lncRNAs can circulate in body fluids; they can be detected and strongly resist RNases. Many researchers have found that lncRNAs could be good candidates for tumor biomarkers and possessed high specificity, high sensitivity, and noninvasive characteristics. In this review, we summarize the detection methods and possible sources of circulating lncRNAs and outline the biological functions and expression level of the most significant lncRNAs in tissues, cell lines, and body fluids (whole blood, plasma, urine, gastric juice, and saliva) of different kinds of tumors. We evaluate the diagnostic performance of lncRNAs as tumor biomarkers. However, the biological functions and the mechanisms of circulating lncRNAs secretion have not been fully understood. The uniform normalization protocol of sample collection, lncRNAs extraction, endogenous control selection, quality assessment, and quantitative data analysis has not been established. Therefore, we put forward some recommendations that might be investigated in the future if we want to adopt lncRNAs in clinical practice.

scholarly journals Recent advances of fluorescent biosensors based on cyclic signal amplification technology in biomedical detection

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Hongke Qu ◽  
Chunmei Fan ◽  
Mingjian Chen ◽  
Xiangyan Zhang ◽  
Qijia Yan ◽  
...  
Keyword(s):  
Signal Amplification ◽  
Low Cost ◽  
Rolling Circle Amplification ◽  
High Sensitivity ◽  
High Specificity ◽  
Detection Methods ◽  
Research Progress ◽  
Rolling Circle ◽  
Displacement Reactions ◽  
Short Time

AbstractThe cyclic signal amplification technology has been widely applied for the ultrasensitive detection of many important biomolecules, such as nucleic acids, proteins, enzymes, adenosine triphosphate (ATP), metal ions, exosome, etc. Due to their low content in the complex biological samples, traditional detection methods are insufficient to satisfy the requirements for monitoring those biomolecules. Therefore, effective and sensitive biosensors based on cyclic signal amplification technology are of great significance for the quick and simple diagnosis and treatment of diseases. Fluorescent biosensor based on cyclic signal amplification technology has become a research hotspot due to its simple operation, low cost, short time, high sensitivity and high specificity. This paper introduces several cyclic amplification methods, such as rolling circle amplification (RCA), strand displacement reactions (SDR) and enzyme-assisted amplification (EAA), and summarizes the research progress of using this technology in the detection of different biomolecules in recent years, in order to provide help for the research of more efficient and sensitive detection methods. Graphical Abstract

Microarray Analysis Reveals the Actual Specificity of Enrichment Media Used for Food Safety Assessment

2011 ◽  
Vol 74 (6) ◽  
pp. 1030-1034 ◽  
Author(s):  
TANJA KOSTIĆ ◽  
BEATRIX STESSL ◽  
MARTIN WAGNER ◽  
ANGELA SESSITSCH
Keyword(s):  
Food Safety ◽  
Food Analysis ◽  
Safety Assessment ◽  
Marker Gene ◽  
Simultaneous Detection ◽  
High Sensitivity ◽  
High Specificity ◽  
Microarray Platform ◽  
Detection Methods ◽  
Indicator Organisms

Microbial diagnostic microarrays are tools for simultaneous detection and identification of microorganisms in food, clinical, and environmental samples. In comparison to classic methods, microarray-based systems have the potential for high throughput, parallelism, and miniaturization. High specificity and high sensitivity of detection have been demonstrated. A microbial diagnostic microarray for the detection of the most relevant bacterial food- and waterborne pathogens and indicator organisms was developed and thoroughly validated. The microarray platform based on sequence-specific end labeling of oligonucleotides and the phylogenetically robust gyrB marker gene allowed a highly specific (resolution on genus and/or species level) and sensitive (0.1% relative and 104 CFU absolute sensitivity) detection of the target pathogens. In initial challenge studies of the applicability of microarray-based food analysis, we obtained results demonstrating the questionable specificity of standardized culture-dependent microbiological detection methods. Taking into consideration the importance of reliable food safety assessment methods, comprehensive performance assessment is essential. Results demonstrate the potential of this new pathogen diagnostic microarray to evaluate culture-based standard methods in microbiological food analysis.

scholarly journals Recent Advances and Perspectives of Molecularly Imprinted Polymer-Based Fluorescent Sensors in Food and Environment Analysis

Nanomaterials ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 1030 ◽  
Author(s):  
Guangyang Liu ◽  
Xiaodong Huang ◽  
Lingyun Li ◽  
Xiaomin Xu ◽  
Yanguo Zhang ◽  
...  
Keyword(s):  
Molecularly Imprinted Polymer ◽  
New Technology ◽  
Three Dimensional ◽  
High Sensitivity ◽  
Detection Methods ◽  
Fluorescent Nanoparticles ◽  
Imprinted Polymer ◽  
Recognition Mechanism ◽  
Molecularly Imprinted ◽  
Wide Range

Molecular imprinting technology (MIT), also known as molecular template technology, is a new technology involving material chemistry, polymer chemistry, biochemistry, and other multi-disciplinary approaches. This technology is used to realize the unique recognition ability of three-dimensional crosslinked polymers, called the molecularly imprinted polymers (MIPs). MIPs demonstrate a wide range of applicability, good plasticity, stability, and high selectivity, and their internal recognition sites can be selectively combined with template molecules to achieve selective recognition. A molecularly imprinted fluorescence sensor (MIFs) incorporates fluorescent materials (fluorescein or fluorescent nanoparticles) into a molecularly imprinted polymer synthesis system and transforms the binding sites between target molecules and molecularly imprinted materials into readable fluorescence signals. This sensor demonstrates the advantages of high sensitivity and selectivity of fluorescence detection. Molecularly imprinted materials demonstrate considerable research significance and broad application prospects. They are a research hotspot in the field of food and environment safety sensing analysis. In this study, the progress in the construction and application of MIFs was reviewed with emphasis on the preparation principle, detection methods, and molecular recognition mechanism. The applications of MIFs in food and environment safety detection in recent years were summarized, and the research trends and development prospects of MIFs were discussed.

scholarly journals High specificity antibodies and detection methods for quantifying phosphorylated tau from clinical samples

2021 ◽  
Author(s):  
Monika Arbaciauskaite ◽  
Yu Lei ◽  
Yong Ku Cho
Keyword(s):  
Neurodegenerative Diseases ◽  
Memory Loss ◽  
High Sensitivity ◽  
High Specificity ◽  
Detection Methods ◽  
Clinical Samples ◽  
Phosphorylated Tau ◽  
Comprehensive Survey ◽  
Low Levels ◽  
Development Engineering

Abstract The ability to measure total and phosphorylated tau levels in clinical samples is transforming the detection of Alzheimer’s disease (AD) and other neurodegenerative diseases. In particular, recent reports indicate that accurate detection of low levels of phosphorylated tau (p-tau) in plasma provides a reliable biomarker of AD long before sensing memory loss. Therefore, the diagnosis and monitoring of neurodegenerative diseases progression using blood samples is becoming a reality. These major advances were achieved by using antibodies specific to p-tau as well as sophisticated high sensitivity immunoassay platforms. This review focuses on these enabling advances in high-specificity antibody development, engineering, and novel signal detection methods. We will draw insights from structural studies on p-tau antibodies, engineering efforts to improve their binding properties, and efforts to validate their specificity. A comprehensive survey of high-sensitivity p-tau immunoassay platforms along with sensitivity limits will be provided. We conclude that although robust approaches for detecting certain p-tau species have been established, systematic efforts to validate antibodies for assay development is still needed for the recognition of biomarkers for AD and other neurodegenerative diseases.

scholarly journals Quantum dot biosensor combined with antibody and aptamer for tracing food-borne pathogens

2021 ◽  
Vol 5 ◽  
Author(s):  
Feifei Sun ◽  
Jing Zhang ◽  
Qingli Yang ◽  
Wei Wu
Keyword(s):  
Quantum Dots ◽  
Pathogen Detection ◽  
High Sensitivity ◽  
High Specificity ◽  
Detection Methods ◽  
Food Borne Pathogens ◽  
Food Borne ◽  
Recognition Elements ◽  
Different Characteristics ◽  
And Control

Abstract Due to the increasing number of food-borne diseases, more attention is being paid to food safety. Food-borne pathogens are the main cause of food-borne diseases, which seriously endanger human health, so it is necessary to detect and control them. Traditional detection methods cannot meet the requirements of rapid detection of food due to many shortcomings, such as being time-consuming, laborious or requiring expensive instrumentation. Quantum dots have become a promising nanotechnology in pathogens tracking and detection because of their excellent optical properties. New biosensor detection methods based on quantum dots are have been gradually developed due to their high sensitivity and high specificity. In this review, we summarize the different characteristics of quantum dots synthesized by carbon, heavy metals and composite materials firstly. Then, attention is paid to the principles, advantages and limitations of the quantum dots biosensor with antibodies and aptamers as recognition elements for recognition and capture of food-borne pathogens. Finally, the great potential of quantum dots in pathogen detection is summarized.

scholarly journals The molecular basis for recognition of 5′-NNNCC-3′ PAM and its methylation state by Acidothermus cellulolyticus Cas9

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Anuska Das ◽  
Travis H. Hand ◽  
Chardasia L. Smith ◽  
Ethan Wickline ◽  
Michael Zawrotny ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Methylation Status ◽  
Extreme Environments ◽  
High Sensitivity ◽  
High Specificity ◽  
Acidothermus Cellulolyticus ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Bond Network ◽  
Rna And Dna

AbstractAcidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5′-NNNCC-3′ Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.

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