scholarly journals Detection of Coronaviruses Using RNA Toehold Switch Sensors

2021 ◽  
Vol 22 (4) ◽  
pp. 1772
Author(s):  
Soan Park ◽  
Jeong Wook Lee
Keyword(s):  
Nucleic Acid ◽  
Diagnostic Test ◽  
Pathogen Detection ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
Cross Reactivity ◽  
Amplification Method ◽  
Reactivity Test ◽  
Target Rna ◽  
Microbiome Profiling

A rapid, sensitive and simple point-of-care (POC) nucleic acid diagnostic test is needed to prevent spread of infectious diseases. Paper-based toehold reaction, a recently emerged colorimetric POC nucleic acid diagnostic test, has been widely used for pathogen detection and microbiome profiling. Here, we introduce an amplification method called reverse transcription loop-mediated amplification (RT-LAMP) prior to the toehold reaction and modify it to enable more sensitive and faster colorimetric detection of RNA viruses. We show that incorporating the modified RT-LAMP to the toehold reaction detects as few as 120 copies of coronavirus RNA in 70 min. Cross-reactivity test against other coronaviruses indicates this toehold reaction with the modified RT-LAMP is highly specific to the target RNA. Overall, the paper-based toehold switch sensors with the modified RT-LAMP allow fast, sensitive, specific and colorimetric coronavirus detection.

scholarly journals A robust, hand-powered, instrument-free sample preparation system for point-of-care pathogen detection

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Fei Zhao ◽  
Eun Yeong Lee ◽  
Geun Su Noh ◽  
Jaehyup Shin ◽  
Huifang Liu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Sample Preparation ◽  
Pathogen Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Sample Pretreatment ◽  
Sample Volume ◽  
Nucleic Acid Isolation ◽  
Point Of Care Diagnostics ◽  
Reaction Matrix

Abstract Here, we describe a simple, universal protocol for use in nucleic acid testing-based pathogen diagnostics, which requires only hand-powered sample preparation, including the processes of pathogen enrichment and nucleic acid isolation. The protocol uses low-cost amine-functionalized diatomaceous earth with a 1-μm Teflon filter as a reaction matrix in both stages of the process, using homobifunctional imidoesters. Using a simple syringe as a pump, the capture efficiency for a large sample volume (<50 mL) was enhanced by up to 98.3%, and the detection limit was 1 CFU/mL, 100-fold better than that of common commercial nucleic acid isolation kit. This protocol can also be combined with commercialized 96-well filter plates for robust sample preparation. Our proposed system is robust, simple, low-cost, universal, and rapid (taking <20 min), and it works regardless of the ambient environment and sample pretreatment, requiring no electricity or instruments. Its benefits include the simplicity of producing its components and its ease of operation, and it can be readily integrated with other assays for point-of-care diagnostics.

scholarly journals Nucleic Acid-Based Sensing Techniques for Diagnostics and Surveillance of Influenza

Biosensors ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 47 ◽  
Author(s):  
Samantha J. Courtney ◽  
Zachary R. Stromberg ◽  
Jessica Z. Kubicek-Sutherland
Keyword(s):  
Influenza Virus ◽  
Nucleic Acid ◽  
Diagnostic Test ◽  
Influenza A ◽  
Direct Detection ◽  
Point Of Care ◽  
Influenza Viruses ◽  
Nucleic Acid Amplification ◽  
High Genetic Diversity ◽  
Mild Disease

Influenza virus poses a threat to global health by causing seasonal outbreaks as well as three pandemics in the 20th century. In humans, disease is primarily caused by influenza A and B viruses, while influenza C virus causes mild disease mostly in children. Influenza D is an emerging virus found in cattle and pigs. To mitigate the morbidity and mortality associated with influenza, rapid and accurate diagnostic tests need to be deployed. However, the high genetic diversity displayed by influenza viruses presents a challenge to the development of a robust diagnostic test. Nucleic acid-based tests are more accurate than rapid antigen tests for influenza and are therefore better candidates to be used in both diagnostic and surveillance applications. Here, we review various nucleic acid-based techniques that have been applied towards the detection of influenza viruses in order to evaluate their utility as both diagnostic and surveillance tools. We discuss both traditional as well as novel methods to detect influenza viruses by covering techniques that require nucleic acid amplification or direct detection of viral RNA as well as comparing advantages and limitations for each method. There has been substantial progress in the development of nucleic acid-based sensing techniques for the detection of influenza virus. However, there is still an urgent need for a rapid and reliable influenza diagnostic test that can be used at point-of-care in order to enhance responsiveness to both seasonal and pandemic influenza outbreaks.

scholarly journals A Low-Cost, 3D-Printed Biosensor for Rapid Detection of Escherichia coli

2022 ◽  
Author(s):  
Samir Malhotra ◽  
Dang Song Pham ◽  
Michael P.H. Lau ◽  
Anh H. Nguyen ◽  
Hung Cao
Keyword(s):  
Escherichia Coli ◽  
Rapid Detection ◽  
Pathogen Detection ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Bacterial Pathogens ◽  
Cross Reactivity ◽  
Potential Candidate ◽  
Limit Of Quantification ◽  
3D Printed

Detection of bacterial pathogens is significant in the fields of food safety, medicine, public health, etc. If bacterial pathogens are not treated promptly, antimicrobial resistance is possible and can lead to morbidity and mortality. Current bacterial detection methodologies rely on laboratory-based techniques that pose limitations such as long turnaround detection times, expensive costs, in-adequate accuracy, and required trained specialists. Here, we describe a cost-effective and port-able 3D-printed electrochemical biosensor that facilitates rapid detection of certain Escherichia coli (E. coli) strains (DH5α, BL21, TOP10, and JM109) within 15 minutes using 500 μL of sample and costs $2.50 per test. The sensor displayed an excellent limit of detection (LOD) of 53 CFU, limit of quantification (LOQ) of 270 CFU, and showed cross-reactivity with strains BL21 and JM109 due to shared epitopes. This advantageous diagnostic device is a potential candidate for high-frequency testing at the point of care as well as applicable to various fields where pathogen detection is of interest.

scholarly journals An evaluation of the diagnostic performance characteristics of the Yellow Fever IgM immunochromatographic rapid diagnostic test kit from SD Biosensor in Ghana

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0262312
Author(s):  
Lawrence Henry Ofosu-Appiah ◽  
Dodzi Kofi Amelor ◽  
Bright Ayensu ◽  
Ernest Akyereko ◽  
Nafisah Issah Rabiwu ◽  
...  
Keyword(s):  
Diagnostic Test ◽  
Rapid Diagnostic Test ◽  
Yellow Fever ◽  
Diagnostic Performance ◽  
Yellow Fever Virus ◽  
Point Of Care ◽  
Clinical Signs ◽  
Cross Reactivity ◽  
Performance Characteristics ◽  
Test Kit

Yellow fever is endemic in Ghana and outbreaks occur periodically. The prodromal signs due to Yellow Fever Virus (YFV) infection are non-specific, making clinical signs unreliable as the sole criteria for diagnosis. Accurate laboratory confirmation of suspected yellow fever cases is therefore vital in surveillance programs. Reporting of ELISA IgM testing results by laboratories can delay due to late arrival of samples from the collection sites as well as limited availability of ELISA kits. In this study, the diagnostic performance characteristics of a rapid immunochromatographic Standard Q Yellow Fever IgM test kit (SD Biosensor) was evaluated for the rapid diagnosis of Yellow Fever infection in Ghana. A panel of 275 sera, comprising 81 confirmed YFV positives and 194 negatives were re-tested in this study using the Standard Q Yellow Fever IgM test kit. Using the CDC/WHO Yellow Fever IgM capture ELISA as a benchmark, the sensitivity, specificity and accuracy of the Standard Q Yellow Fever test kit were 96.3%, 97.9% and 97.5%, respectively. The false positivity rate was 5.1% and there was no cross-reactivity when the Standard Q Yellow Fever test kit was tested against dengue, malaria and hepatitis B and C positive samples. In addition, inter-reader variability and invalid rate were both zero. The results indicate that the diagnostic performance of the Standard Q Yellow Fever IgM test kit on serum or plasma is comparable to the serum IgM detection by ELISA and can be used as a point of care rapid diagnostic test kit for YFV infection in endemic areas.

scholarly journals LabDisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza A H3N2 virus

Lab on a Chip ◽  
2016 ◽  
Vol 16 (1) ◽  
pp. 199-207 ◽  
Author(s):  
F. Stumpf ◽  
F. Schwemmer ◽  
T. Hutzenlaub ◽  
D. Baumann ◽  
O. Strohmeier ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Influenza A ◽  
Pathogen Detection ◽  
Point Of Care ◽  
H3n2 Virus ◽  
Respiratory Pathogens ◽  
Manual Handling

Sample supply remains the only manual handling step for pathogen detection at the point-of-care.

scholarly journals Cleavable hairpin beacon-enhanced fluorescence detection of nucleic acid isothermal amplification and smartphone-based readout

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xiong Ding ◽  
Kun Yin ◽  
Ziyue Li ◽  
Vikram Pandian ◽  
Joan A. Smyth ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Fluorescence Detection ◽  
Pathogen Detection ◽  
Point Of Care ◽  
Lamp Assay ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
Specific Method ◽  
Enhanced Fluorescence ◽  
Resource Limited

Abstract Fluorescence detection of nucleic acid isothermal amplification utilizing energy-transfer-tagged oligonucleotide probes provides a highly sensitive and specific method for pathogen detection. However, currently available probes suffer from relatively weak fluorescence signals and are not suitable for simple, affordable smartphone-based detection at the point of care. Here, we present a cleavable hairpin beacon (CHB)-enhanced fluorescence detection for isothermal amplification assay. The CHB probe is a single fluorophore-tagged hairpin oligonucleotide with five continuous ribonucleotides which can be cleaved by the ribonuclease to specifically initiate DNA amplification and generate strong fluorescence signals. By coupling with loop-mediated isothermal amplification (LAMP), the CHB probe could detect Borrelia burgdorferi (B. burgdorferi) recA gene with a sensitivity of 100 copies within 25 min and generated stronger specific fluorescence signals which were easily read and analysed by our programmed smartphone. Also, this CHB-enhanced LAMP (CHB-LAMP) assay was successfully demonstrated to detect B. burgdorferi DNA extracted from tick species, showing comparable results to real-time PCR assay. In addition, our CHB probe was compatible with other isothermal amplifications, such as isothermal multiple-self-matching-initiated amplification (IMSA). Therefore, CHB-enhanced fluorescence detection is anticipated to facilitate the development of simple, sensitive smartphone-based point-of-care pathogen diagnostics in resource-limited settings.

scholarly journals Toward a CRISPR-based point-of-care test for tomato brown rugose fruit virus detection

2021 ◽  
Author(s):  
Joan Miquel Bernabé-Orts ◽  
Yolanda Hernando ◽  
Miguel A. Aranda
Keyword(s):  
Nucleic Acid ◽  
Plant Pathogens ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Genetic Resistance ◽  
Virus Detection ◽  
Cross Reactivity ◽  
World Population ◽  
Acid Extraction ◽  
Point Of Care Test

Implementing effective monitoring strategies is fundamental to protect crops from pathogens and to ensure the food supply as the world population continues to grow. This is especially important for emergent plant pathogens such as tomato brown rugose fruit virus (ToBRFV), which overcomes the genetic resistance resources used in tomato breeding against tobamoviruses and has become pandemic in less than a decade. Here we report the development of a CRISPR/Cas12a-based test to detect ToBRFV in the laboratory and potentially in a field setting. Using different tobamoviruses to assess specificity, our test showed a clear positive signal for ToBRFV-infected samples, while no cross-reactivity was observed for closely related viruses. Next, we compared the limit of detection of our CRISPR-based test with a reference real-time quantitative PCR test widely used, revealing similar sensitivities for both tests. Finally, to reduce complexity and achieve field-applicability, we used a fast nucleic acid purification step and compared its results side-to-side with those of a commonly used column-mediated protocol. The rapid protocol saved time and resources but at the expense of sensitivity. However, it still may be useful to confirm ToBRFV detection in samples with incipient symptoms of infection. Although there is room for improvement, to our knowledge this is the first field-compatible CRISPR-based test to detect ToBRFV which combines isothermal amplification with a simplified nucleic acid extraction protocol.

Development of Ball-Enabled Miniaturized Valves for Sample Preparation and Microheaters for Pathogen Detection

2020 ◽  
Author(s):  
Carlos Manzanas ◽  
Xiao Jiang ◽  
John A. Lednicky ◽  
Z. Hugh Fan
Keyword(s):  
Zika Virus ◽  
Pathogen Detection ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
Public Health Problem ◽  
Cost Effective ◽  
Nucleic Acid Amplification ◽  
Rna Amplification ◽  
Testing Platform ◽  
The Past

Abstract There have been numerous Zika virus (ZIKV) outbreaks in the past few years, representing a public health problem. The recommended tests for the diagnosis of Zika infections are performed in a laboratory setting. However, diagnostics platforms at the point-of-care (POC) are highly desirable for understanding and preventing ZIKV transmission. To address this need, we have developed a testing platform that (1) can be operated in the field for pathogen detection, (2) is rapid, cost-effective, and reliable, and (3) does not require a power supply. To realize the platform, we have developed (1) a series of ball-based valves for the storage and sequential delivery of reagents and (2) microheater-enabled RNA amplification, both of which are integral components of this POC device. The multiple reagents are needed for virus lysis, RNA enrichment and purification. These ball-based are employed for fluid-control and they are actuated manually by sliding the unit and a pole under it, which can lift the balls. Nucleic acid amplification is then performed by a smart coffee mug that provides a constant temperature for reverse transcription loop mediated isothermal amplification (RT-LAMP), followed by colorimetric detection. We have demonstrated the detection of Zika virus in human urine and saliva samples using this testing platform.

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