scholarly journals Highly Specific and Sensitive Detection of Yersinia pestis by Portable Cas12a-UPTLFA Platform

2021 ◽  
Vol 12 ◽  
Author(s):  
Yang You ◽  
Pingping Zhang ◽  
Gengshan Wu ◽  
Yafang Tan ◽  
Yong Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Yersinia Pestis ◽  
Rapid Detection ◽  
Detection Method ◽  
Detection System ◽  
Point Of Care ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Great Promise ◽  
High Background

The recent discovery of collateral cleavage activity of class-II clustered regularly interspaced short palindromic repeats–CRISPR-associated protein (CRISPR-Cas) makes CRISPR-based diagnosis a potential high-accuracy nucleic acid detection method. Colloidal gold-based lateral flow immunochromatographic assay (LFA), which has been combined with CRISPR/Cas-based nucleic detection, usually associates with drawbacks of relative high background and the subjectivity in naked-eye read-out of the results. Here, we developed a novel system composed of Cas12a-based nucleic acid detection and up-converting phosphor technology (UPT)-based LFA (UPT–LFA), termed Cas12a-UPTLFA. We further demonstrated the utility of this platform in highly sensitive and specific detection of Yersinia pestis, the causative agent of the deadly plague. Due to high infectivity and mortality, as well as the potential to be misused as bioterrorism agent, a culture-free, ultrasensitive, specific, and rapid detection method for Y. pestis has long been desired. By incorporating isothermal recombinase polymerase amplification, the Cas12a-UPTLFA we established can successfully detect genomic DNA of Y. pestis as low as 3 attomolar (aM) and exhibited high sensitivity (93.75%) and specificity (90.63%) for detection of spiked blood samples with a detection limit of 102 colony-forming unit per 100 μl of mouse blood. With a portable biosensor, Cas12a-UPTLFA assay can be operated easily by non-professional personnel. Taken together, we have developed a novel Cas12a-UPTLFA platform for rapid detection of Y. pestis with high sensitivity and specificity, which is portable, not expensive, and easy to operate as a point-of-care method. This detection system can easily be extended to detect other pathogens and holds great promise for on-site detection of emerging infectious pathogens.

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 Virus detection via programmable Type III-A CRISPR-Cas systems

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sagar Sridhara ◽  
Hemant N. Goswami ◽  
Charlisa Whyms ◽  
Jonathan H. Dennis ◽  
Hong Li
Keyword(s):  
Nucleic Acid ◽  
Detection Method ◽  
Virus Detection ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Immune Effector ◽  
Type Iii ◽  
System A ◽  
Acid Cleavage

AbstractAmong the currently available virus detection assays, those based on the programmable CRISPR-Cas enzymes have the advantage of rapid reporting and high sensitivity without the requirement of thermocyclers. Type III-A CRISPR-Cas system is a multi-component and multipronged immune effector, activated by viral RNA that previously has not been repurposed for disease detection owing in part to the complex enzyme reconstitution process and functionality. Here, we describe the construction and application of a virus detection method, based on an in vivo-reconstituted Type III-A CRISPR-Cas system. This system harnesses both RNA- and transcription-activated dual nucleic acid cleavage activities as well as internal signal amplification that allow virus detection with high sensitivity and at multiple settings. We demonstrate the use of the Type III-A system-based method in detection of SARS-CoV-2 that reached 2000 copies/μl sensitivity in amplification-free and 60 copies/μl sensitivity via isothermal amplification within 30 min and diagnosed SARS-CoV-2-infected patients in both settings. The high sensitivity, flexible reaction conditions, and the small molecular-driven amplification make the Type III-A system a potentially unique nucleic acid detection method with broad applications.

scholarly journals A Chemical-Enhanced System for CRISPR-Based Nucleic Acid Detection

2021 ◽  
Author(s):  
Zihan Li ◽  
Wenchang Zhao ◽  
Shixin Ma ◽  
Zexu Li ◽  
Yingjia Yao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Detection System ◽  
Point Of Care ◽  
Detection Sensitivity ◽  
Nucleic Acid Detection ◽  
Chemical Additives ◽  
Lateral Flow Strip ◽  
Viral Pathogens ◽  
Detection Systems ◽  
System Robustness

The CRISPR-based nucleic acid detection systems such as SHERLOCK, DETECTR and HOLMES have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness towards practical application of CRISPR-based diagnostics.

scholarly journals Rapidemic, a versatile and label-free DNAzyme-based platform for visual nucleic acid detection

2020 ◽  
Author(s):  
Marijn van den Brink ◽  
Sebastian T. Tandar ◽  
Tim A. P. van den Akker ◽  
Sinisha Jovikj ◽  
Violette Defourt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Detection Method ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Nucleic Acid Detection ◽  
Label Free ◽  
Strand Displacement Amplification ◽  
Sequence Detection ◽  
Accessible Point

AbstractIn the last three decades, there have been recurring outbreaks of infectious diseases, brought to light with the recent outbreak of coronavirus disease 2019 (COVID-19). Attempts to effectively contain the spread of infectious diseases have been hampered by the lack of rapidly adaptable, accurate, and accessible point-of-care diagnostic testing. In this study, we present a novel design of a label-free DNAzyme-based detection method called Rapidemic. This assay combines recombinase polymerase amplification (RPA) with linear strand-displacement amplification (LSDA) and guanine-quadruplex (GQ) DNAzyme-catalysed colour-changing reaction. The colorimetry basis of the signal readout omits the need for extensive instrumentation. Moreover, the primer-based sequence detection of RPA gives Rapidemic a potential to be rapidly adapted to target a new sequence. As a proof of concept, we developed the assay to detect isolated genomic DNA of Saccharomyces cerevisiae. The use of low-pH buffers and the optimization of the dilution rates from each preceding reaction to the next showed to be successful strategies to enable visible detection with this method. These findings demonstrate for the first time that a label-free DNAzyme-based detection method can be coupled to RPA and LSDA for nucleic acid detection.

scholarly journals Rapid detection of SARS-CoV-2 with CRISPR-Cas12a

PLoS Biology ◽  
2020 ◽  
Vol 18 (12) ◽  
pp. e3000978
Author(s):  
Dan Xiong ◽  
Wenjun Dai ◽  
Jiaojiao Gong ◽  
Guande Li ◽  
Nansong Liu ◽  
...  
Keyword(s):  
Detection Method ◽  
Point Of Care ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Global Pandemic ◽  
Portable Detection ◽  
Recent Outbreak ◽  
Associated Proteins ◽  
Specialized Equipment ◽  
Viral Diagnosis

The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1–10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.

scholarly journals A Novel Amplification-free SARS-CoV-2 Point-of-care Nucleic Acid Detection System based on Hybrid Capture Fluorescence Immunoassay

2020 ◽  
Author(s):  
Daming Wang ◽  
Shaogui He ◽  
Xiaohui Wang ◽  
Min Chen ◽  
Li Li ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Detection System ◽  
Point Of Care ◽  
Quantitative Polymerase Chain Reaction ◽  
Nucleic Acid Detection ◽  
Kappa Statistics ◽  
Fluorescence Immunoassay ◽  
Time Saving ◽  
Hybrid Capture ◽  
Test Kit

Abstract Herein we presented a novel, rapid and amplification-free SARS-CoV-2 nucleic acid detection system based on hybrid capture fluorescence immunoassay (HC-FIA) technology. The usage of the monoclonal antibody S9.6 in recognizing DNA-RNA double-stranded hybrids enabled the conversion of nucleic acid testing into immunofluorescence carrying on a simple lateral flow dipstick. The established HC-FIA also exhibited satisfactory sensitivity, specificity and great robustness. The clinical evaluation of HC-FIA kit and fluorescence reading device are further processed in three hospitals independently. The results of 734 samples from 670 subjects indicated high consistency between our HC-FIA and quantitative polymerase chain reaction based commercially available kit or clinical diagnosis according to Kappa statistics. Altogether, HC-FIA related method and commercial test kit show unparalleled advantages as time saving, amplification-free, high throughput and portable POCT molecular diagnosis, which facilitates its application as on-site Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection in epidemic prevention and control worldwide, especially during the outbreak.

Design and Implementation of Experimental Consumable Management Device for the Automatic Nucleic Acid Detection System

2016 ◽  
Vol 16 (7) ◽  
pp. 7069-7076
Author(s):  
Jun Yu ◽  
Zhu Chen ◽  
Chao Wang ◽  
Yana Hu ◽  
Hongming Dong ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Detection System ◽  
Nucleic Acid Detection ◽  
Design And Implementation

The potential impact of new generation molecular point-of-care tests on gonorrhoea and chlamydia in a setting of high endemic prevalence

Sexual Health ◽  
2013 ◽  
Vol 10 (4) ◽  
pp. 348 ◽  
Author(s):  
Ben B. Hui ◽  
David P. Wilson ◽  
James S. Ward ◽  
Rebecca J. Guy ◽  
John M. Kaldor ◽  
...  
Keyword(s):  
Point Of Care ◽  
Treatment Strategies ◽  
High Sensitivity ◽  
Indigenous Communities ◽  
Great Promise ◽  
Screening Methods ◽  
Sexually Transmissible Infections ◽  
Screening Coverage ◽  
New Generation ◽  
The Impact

Background Despite the availability of testing and treatment, bacterial sexually transmissible infections (STIs) continue to occur at endemic levels in many remote Indigenous communities in Australia. New generation molecular point-of-care (POC) tests have high sensitivity, comparable with conventional diagnostic tests, and have the potential to increase the impact of STI screening. Methods: We developed mathematical models of gonorrhoea (Neisseria gonorrhoeae) and chlamydia (Chlamydia trachomatis) transmission in remote Indigenous communities in Australia to evaluate screening and treatment strategies that utilise POC tests. Results: The introduction of POC testing with 95% sensitivity could reduce the prevalence of gonorrhoea and chlamydia from 7.1% and 11.9% to 5.7% and 8.9%, respectively, under baseline screening coverage of 44% per year. If screening coverage is increased to 60% per year, prevalence is predicted to be reduced to 3.6% and 6.7%, respectively, under conventional testing, and further reduced to 1.8% and 3.1% with the introduction of POC testing. Increasing screening coverage to 80% per year will result in a reduction in the prevalence of gonorrhoea and chlamydia to 0.6% and 1.5%, respectively, and the virtual elimination of both STIs if POC testing is introduced. Conclusions: Modelling suggests that molecular POC tests of high sensitivity have great promise as a public health strategy for controlling chlamydia and gonorrhoea. However, evaluation of the cost-effectiveness of POC testing needs to be made before widespread implementation of this technology can be considered.

scholarly journals Ultrasensitive CRISPR-based diagnostic for field-applicable detection ofPlasmodiumspecies in symptomatic and asymptomatic malaria

2020 ◽  
Vol 117 (41) ◽  
pp. 25722-25731 ◽  
Author(s):  
Rose A. Lee ◽  
Helena De Puig ◽  
Peter Q. Nguyen ◽  
Nicolaas M. Angenent-Mari ◽  
Nina M. Donghia ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Sensitivity And Specificity ◽  
Parasite Density ◽  
Point Of Care ◽  
High Sensitivity ◽  
World Health ◽  
Ultrasensitive Detection ◽  
Asymptomatic Malaria ◽  
Radical Cure ◽  
Asymptomatic Carriers

Asymptomatic carriers ofPlasmodiumparasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care diagnostics are also lacking for nonfalciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods, such as PCR, have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS. Here we describe a CRISPR-based diagnostic for ultrasensitive detection and differentiation ofPlasmodium falciparum,Plasmodium vivax,Plasmodium ovale, andPlasmodium malariae, using the nucleic acid detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking). We present a streamlined, field-applicable, diagnostic comprised of a 10-min SHERLOCK parasite rapid extraction protocol, followed by SHERLOCK for 60 min forPlasmodiumspecies-specific detection via fluorescent or lateral flow strip readout. We optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction, and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. OurP. falciparumandP. vivaxassays exhibited 100% sensitivity and specificity on clinical samples (5P. falciparumand 10P. vivaxsamples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid point-of-care clinical diagnostic for nonfalciparum malaria species and low parasite densityP. falciparuminfections.

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