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 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 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 new method for quantitative detection of Lactobacillus casei based on casx gene and its application

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xiaoyang Pang ◽  
Ziyang Jia ◽  
Jing Lu ◽  
Shuwen Zhang ◽  
Cai Zhang ◽  
...  
Keyword(s):  
16S Rrna ◽  
Detection Method ◽  
High Sensitivity ◽  
Accurate Method ◽  
Quantitative Detection ◽  
Bacterial Identification ◽  
Associated Proteins ◽  
16S Sequence ◽  
Species Specific ◽  
16 S Rrna

Abstract Background The traditional method of bacterial identification based on 16S rRNA is a widely used and very effective detection method, but this method still has some deficiencies, especially in the identification of closely related strains. A high homology with little differences is mostly observed in the 16S sequence of closely related bacteria, which results in difficulty to distinguish them by 16S rRNA-based detection method. In order to develop a rapid and accurate method of bacterial identification, we studied the possibility of identifying bacteria with other characteristic fragments without the use of 16S rRNA as detection targets. Results We analyzed the potential of using cas (CRISPR-associated proteins) gene as a target for bacteria detection. We found that certain fragment located in the casx gene was species-specific and could be used as a specific target gene. Based on these fragments, we established a TaqMan MGB Real-time PCR method for detecting bacteria. We found that the method used in this study had the advantages of high sensitivity and good specificity. Conclusions The casx gene-based method of bacterial identification could be used as a supplement to the conventional 16 s rRNA-based detection method. This method has an advantage over the 16 s rRNA-based detection method in distinguishing the genetic relationship between closely-related bacteria, such as subgroup bacteria, and can be used as a supplement to the 16 s rRNA-based detection method.

scholarly journals Portable tumor biosensing of serum by plasmonic biochips in combination with nanoimprint and microfluidics

Nanophotonics ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 307-316 ◽  
Author(s):  
Jianyang Zhou ◽  
Feng Tao ◽  
Jinfeng Zhu ◽  
Shaowei Lin ◽  
Zhengying Wang ◽  
...  
Keyword(s):  
Nanoimprint Lithography ◽  
Clinical Medicine ◽  
Point Of Care ◽  
Low Cost ◽  
Human Tumor ◽  
High Sensitivity ◽  
Visible Range ◽  
Label Free ◽  
Portable Detection ◽  
Clinical Experiments

AbstractPlasmonic sensing has a great potential in the portable detection of human tumor markers, among which the carcinoembryonic antigen (CEA) is one of the most widely used in clinical medicine. Traditional plasmonic and non-plasmonic methods for CEA biosensing are still not suitable for the fast developing era of Internet of things. In this study, we build up a cost-effective plasmonic immunochip platform for rapid portable detection of CEA by combining soft nanoimprint lithography, microfluidics, antibody functionalization, and mobile fiber spectrometry. The plasmonic gold nanocave array enables stable surface functionality, high sensitivity, and simple reflective measuring configuration in the visible range. The rapid quantitative CEA sensing is implemented by a label-free scheme, and the detection capability for the concentration of less than 5 ng/ml is achieved in clinical experiments, which is much lower than the CEA cancer diagnosis threshold of 20 ng/ml and absolutely sufficient for medical applications. Clinical tests of the chip on detecting human serums demonstrate good agreement with conventional medical examinations and great advantages on simultaneous multichannel detections for high-throughput and multi-marker biosensing. Our platform provides promising opportunities on low-cost and compact medical devices and systems with rapid and sensitive tumor detection for point-of-care diagnosis and mobile healthcare.

scholarly journals CRISPR/Cas13-Based Approaches for Ultrasensitive and Specific Detection of microRNAs

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1655
Author(s):  
Javier T. Granados-Riveron ◽  
Guillermo Aquino-Jarquin
Keyword(s):  
Cell Biology ◽  
Low Cost ◽  
High Sensitivity ◽  
Mature Mirnas ◽  
Diagnostic Tools ◽  
Nucleic Acid Detection ◽  
Target Sequence ◽  
Associated Proteins ◽  
Mirna Detection ◽  
Mirna Expression Profiling

MicroRNAs (miRNAs) have a prominent role in virtually every aspect of cell biology. Due to the small size of mature miRNAs, the high degree of similarity between miRNA family members, and the low abundance of miRNAs in body fluids, miRNA expression profiling is technically challenging. Biosensors based on electrochemical detection for nucleic acids are a novel category of inexpensive and very sensitive diagnostic tools. On the other hand, after recognizing the target sequence, specific CRISPR-associated proteins, including orthologues of Cas12, Cas13, and Cas14, exhibit collateral nonspecific catalytic activities that can be employed for specific and ultrasensitive nucleic acid detection from clinically relevant samples. Recently, several platforms have been developed, connecting the benefits of enzyme-assisted signal amplification and enzyme-free amplification biosensing technologies with CRISPR-based approaches for miRNA detection. Together, they provide high sensitivity, precision, and fewer limitations in diagnosis through efficient sensors at a low cost and a simple miniaturized readout. This review provides an overview of several CRISPR-based biosensing platforms that have been developed and successfully applied for ultrasensitive and specific miRNA detection.

scholarly journals A Simple Colorimetric Molecular Detection of Novel Coronavirus (COVID-19), an Essential Diagnostic Tool for Pandemic Screening

2020 ◽  
Author(s):  
Pazhanimuthu Annamalai ◽  
Madhu Kanta ◽  
Pazhanivel Ramu ◽  
Baskar Ravi ◽  
Kokilavani Veerapandian ◽  
...  
Keyword(s):  
Molecular Detection ◽  
Point Of Care ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Cost Effective ◽  
Cost Effective Method ◽  
Recent Outbreak ◽  
Pcr Method ◽  
Novel Coronavirus ◽  
Virus Isolates

AbstractThe recent outbreak of the newly emerged novel coronavirus (SARS-CoV-2) presents a big challenge for public health laboratories as virus isolates are not available while there is an increasing evidence that the epidemic is more widespread than initially thought, as well as spreading internationally across borders through travellers does already happen warranting a methodology for the rapid detection of the infection to control SARS-CoV-2. Aim: We intended to develop and deploy a robust and rapid diagnostic methodology using LAMP assay for use in point of care settings to detect SARS-COV-2 infection. Methodology: In the present study, we have developed a validated rapid diagnostic procedure to detect SARS-CoV-2 using LAMP assay, its design relying on isothermal amplification of the nucleic acids of the SARS-CoV-2. Results: The LAMP assay developed detects SARS-CoV-2 infection rapidly with high sensitivity and reliability. The data generated by LAMP assay were comparable and at par with the data generated by real-time PCR method. Conclusion: The present study demonstrates that the LAMP assay developed was a rapid, reliable, sensitive and cost effective method to detect SARS-CoV-2 infection in a point of care as well as in laboratory settings.

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 Instrument-Free and Visual Detection of Salmonella Based on Magnetic Nanoparticles and an Antibody Probe Immunosensor

2019 ◽  
Vol 20 (18) ◽  
pp. 4645 ◽  
Author(s):  
Liding Zhang ◽  
Xuewei Du ◽  
Zhixin Chen ◽  
Congjie Chen ◽  
Nanxin Gong ◽  
...  
Keyword(s):  
Point Of Care ◽  
Foodborne Pathogen ◽  
Diagnostic System ◽  
Detection Systems ◽  
Technical Requirements ◽  
Salmonella Infections ◽  
Portable Detection ◽  
Specialized Equipment ◽  
Microbial Strains ◽  
Point Of Care Detection

Salmonella, a common foodborne pathogen, causes many cases of foodborne illness and poses a threat to public health worldwide. Immunological detection systems can be combined with nanoparticles to develop sensitive and portable detection technologies for timely screening of Salmonella infections. Here, we developed an antibody-probe-based immuno-N-hydroxysuccinimide (NHS) bead (AIB) system to detect Salmonella. After adding the antibody probe, Salmonella accumulated in the samples on the surfaces of the immuno-NHS beads (INBs), forming a sandwich structure (INB–Salmonella–probes). We demonstrated the utility of our AIB diagnostic system for detecting Salmonella in water, milk, and eggs, with a sensitivity of 9 CFU mL−1 in less than 50 min. The AIB diagnostic system exhibits highly specific detection and no cross-reaction with other similar microbial strains. With no specialized equipment or technical requirements, the AIB diagnostic method can be used for visual, rapid, and point-of-care detection of Salmonella.

Study on Field Demonstration of High Sensitivity SF6 Leakage Detection Method for Gas Insulated Switchgear

2020 ◽  
Vol 140 (5) ◽  
pp. 409-414
Author(s):  
Masaru Tatemi ◽  
Hisao Inami ◽  
Toshiaki Rokunohe ◽  
Makoto Hirose
Keyword(s):  
Detection Method ◽  
High Sensitivity ◽  
Leakage Detection ◽  
Gas Insulated Switchgear

Novel High Sensitivity Tuberculosis Point-of-Care Test for People Living with HIV

2018 ◽  
Author(s):  
Tobias Broger ◽  
Bianca Sossen ◽  
Elloise du Toit ◽  
Andrew D. Kerkhoff ◽  
Charlotte Schutz ◽  
...  
Keyword(s):  
Point Of Care ◽  
High Sensitivity ◽  
People Living With Hiv ◽  
Point Of Care Test ◽  
Living With Hiv
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