scholarly journals Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2

2020 ◽  
Vol 117 (47) ◽  
pp. 29518-29525 ◽  
Author(s):  
Ashwin Ramachandran ◽  
Diego A. Huyke ◽  
Eesha Sharma ◽  
Malaya K. Sahoo ◽  
ChunHong Huang ◽  
...  
Keyword(s):  
Electric Field ◽  
Microfluidic Chip ◽  
Molecular Diagnostic ◽  
Nasopharyngeal Swab ◽  
Guide Rna ◽  
Diagnostic Assays ◽  
Field Gradients ◽  
Target Dna ◽  
Rapid Spread ◽  
Diagnostic Approaches

The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR–Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore−quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12–gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.

scholarly journals Electric-field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2

2020 ◽  
Author(s):  
Ashwin Ramachandran ◽  
Diego A. Huyke ◽  
Eesha Sharma ◽  
Malaya K. Sahoo ◽  
Niaz Banaei ◽  
...  
Keyword(s):  
Electric Field ◽  
Early Stage ◽  
Diagnostic Methods ◽  
Disease Spread ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Diagnostic Assays ◽  
Field Control

AbstractThe rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In a basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex is activated when it highly specifically binds to target DNA, and the activated complex non-specifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We recently discovered that electric field gradients can be used to control and accelerate this CRISPR assay by co-focusing Cas12-gRNA, reporters, and target. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab sample. We here combine this ITP purification with loop-mediated isothermal amplification, and the ITP-enhanced CRISPR assay to achieve detection of SARS-CoV-2 RNA (from raw sample to result) in 30 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables a new modality for a suite of microfluidic CRISPR-based diagnostic assays.Significance statementRapid, early-stage screening is especially crucial during pandemics for early identification of infected patients and control of disease spread. CRISPR biology offers new methods for rapid and accurate pathogen detection. Despite their versatility and specificity, existing CRISPR-diagnostic methods suffer from the requirements of up-front nucleic acid extraction, large reagent volumes, and several manual steps—factors which prolong the process and impede use in low resource settings. We here combine on-chip electric-field control in combination with CRIPSR biology to directly address these limitations of current CRISPR-diagnostic methods. We apply our method to the rapid detection of SARS-CoV-2 RNA in clinical samples. Our method takes 30 min from raw sample to result, a significant improvement over existing diagnostic methods for COVID-19.

scholarly journals Enhancing Colorimetric LAMP Amplification Speed and Sensitivity with Guanidine Chloride

2020 ◽  
Author(s):  
Yinhua Zhang ◽  
Guoping Ren ◽  
Jackson Buss ◽  
Andrew J. Barry ◽  
Gregory C. Patton ◽  
...  
Keyword(s):  
Guanidine Hydrochloride ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Simple Method ◽  
Diagnostic Assays ◽  
Dna And Rna ◽  
Target Dna ◽  
Versatile Technique ◽  
Guanidine Chloride ◽  
Primer Sets

AbstractLoop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated technique and enabling accurate and high-throughput diagnostics.

scholarly journals Microfluidic Chip with Two-Stage Isothermal Amplification Method for Highly Sensitive Parallel Detection of SARS-CoV-2 and Measles Virus

Micromachines ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1582
Author(s):  
Qin Huang ◽  
Xiaohui Shan ◽  
Ranran Cao ◽  
Xiangyu Jin ◽  
Xue Lin ◽  
...  
Keyword(s):  
Microfluidic Chip ◽  
Measles Virus ◽  
Point Of Care ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Nasopharyngeal Swab ◽  
Two Stage ◽  
Parallel Detection ◽  
Amplification Method ◽  
Specificity And Sensitivity

A two-stage isothermal amplification method, which consists of a first-stage basic recombinase polymerase amplification (RPA) and a second-stage fluorescence loop-mediated isothermal amplification (LAMP), as well as a microfluidic-chip-based portable system, were developed in this study; these enabled parallel detection of multiplex targets in real time in around one hour, with high sensitivity and specificity, without cross-contamination. The consumption of the sample and the reagent was 2.1 μL and 10.6 μL per reaction for RPA and LAMP, respectively. The lowest detection limit (LOD) was about 10 copies. The clinical amplification of about 40 nasopharyngeal swab samples, containing 17 SARS-CoV-2 (severe acute respiratory syndrome coronavirus) and 23 measles viruses (MV), were parallel tested by using the microfluidic chip. Both clinical specificity and sensitivity were 100% for MV, and the clinical specificity and sensitivity were 94.12% and 95.83% for SARS-CoV-2, respectively. This two-stage isothermal amplification method based on the microfluidic chip format offers a convenient, clinically parallel molecular diagnostic method, which can identify different nucleic acid samples simultaneously and in a timely manner, and with a low cost of the reaction reagent. It is especially suitable for resource-limited areas and point-of-care testing (POCT).

scholarly journals Comparative study of four SARS-CoV-2 Nucleic Acid Amplification Test (NAAT) platforms demonstrates that ID NOW performance is impaired substantially by patient and specimen type

2020 ◽  
Author(s):  
Paul R. Lephart ◽  
Michael Bachman ◽  
William LeBar ◽  
Scott McClellan ◽  
Karen Barron ◽  
...  
Keyword(s):  
Nasal Swab ◽  
The United States ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Nasopharyngeal Swab ◽  
Diagnostic Assays ◽  
Comparable Performance ◽  
Indications For Use ◽  
Significant Factors

AbstractThe advent of the COVID-19 pandemic in the United States created a unique situation where multiple molecular diagnostic assays with various indications for use in the detection of SARS-CoV-2 rapidly received Emergency Use Authorization by the FDA, were validated by laboratories and utilized clinically, all within a period of a few weeks. We compared the performance of four of these assays that were being evaluated for use at our institution: Abbott RealTime m2000 SARS-CoV-2 Assay, DiaSorin Simplexa COVID-19 Direct, Cepheid Xpert Xpress SARS-CoV-2 and Abbott ID NOW COVID-19. Nasopharyngeal and nasal specimens were collected from 88 ED and hospital-admitted patients and tested by the four methods in parallel to compare performance. ID NOW performance stood out as significantly worse than the other three assays despite demonstrating comparable analytic sensitivity. Further study determined that the use of a foam nasal swab compared to a nylon flocked nasopharyngeal swab, as well as use in a population chronically vs. acutely positive for SARS-CoV-2, were significant factors in the poor comparable performance.

scholarly journals Comparison of SARS-CoV-2 RT-PCR on a high-throughput molecular diagnostic platform and the cobas SARS-CoV-2 test for the diagnostic of COVID-19 on various clinical samples

2020 ◽  
Vol 78 (8) ◽  
Author(s):  
Onya Opota ◽  
René Brouillet ◽  
Gilbert Greub ◽  
Katia Jaton
Keyword(s):  
High Throughput ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Test Results ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Rapid Spread ◽  
Bronchial Aspirate ◽  
Very High

ABSTRACT Objectives:In order to cope with the rapid spread of the COVID-19 pandemic, we introduced on our in-house high-throughput molecular diagnostic platform (MDx Platform) a real-time reverse transcriptase PCR (RT-PCR) to detect the SARS-CoV-2 from any clinical specimens. The aim of this study was to compare the RT-PCR results obtain with the MDx Platform and the commercial assay cobas SARS-CoV-2 (Roche) on nasopharyngeal swab and other clinical specimens including sputum, bronchial aspirate, bronchoalveolar lavage and anal swabs. Methods: Samples received in our laboratory from patients suspected of COVID-19 (n = 262) were tested in parallel with our MDx platform SARS-CoV-2 PCR and with the cobas SARS-CoV-2 test. Results: The overall agreement between the two tests for all samples tested was 99.24% (260/262), which corresponded to agreements of 100% (178/178) on nasopharyngeal swabs, 95.45% (42/44) on lower respiratory tract specimen with discordant resultS obtained for very high cycle threshold (Ct) value and 100% (40/40) on anorectal swabs. The Ct values for nasopharyngeal swabs displayed an excellent correlation (R2 > 96%) between both tests. Conclusions: The high agreements between the cobas SARS-CoV-2 test and the MDx platform supports the use of both methods for the diagnostic of COVID-19 on various clinical samples. Very few discrepant results may occur at very low viral load.

Effects of non-axial electric field gradients on the ferromagnetic state of amorphous rare-earth alloys

1980 ◽  
Vol 58 (5) ◽  
pp. 629-632 ◽  
Author(s):  
H. Hernandez ◽  
R. Ferrer ◽  
M. J. Zuckermann
Keyword(s):  
Rare Earth ◽  
Electric Field ◽  
Ferromagnetic State ◽  
Rare Earth Alloys ◽  
Electric Field Gradients ◽  
Axial Electric Field ◽  
Ferromagnetic Alloys ◽  
Field Gradients ◽  
Ordered State

We discuss the influence of non-axial electric field gradients on the ordered state of amorphous ferromagnetic alloys containing rare-earth atoms.

scholarly journals CRISPR as a Diagnostic Tool

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1162
Author(s):  
Seohyun Kim ◽  
Sangmin Ji ◽  
Hye Ran Koh
Keyword(s):  
Diagnostic Tool ◽  
Specific Binding ◽  
Diagnostic Methods ◽  
Specific Gene ◽  
Guide Rna ◽  
Engineering Technique ◽  
Target Dna ◽  
Target Binding ◽  
Genetic Engineering Technique ◽  
Disease Related Gene

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.

Synergistic use of electroosmotic flow and magnetic forces for nucleic acid extraction

The Analyst ◽  
2020 ◽  
Vol 145 (6) ◽  
pp. 2412-2419 ◽  
Author(s):  
Rachel N. Deraney ◽  
Lindsay Schneider ◽  
Anubhav Tripathi
Keyword(s):  
Nucleic Acid ◽  
Electric Field ◽  
Microfluidic Chip ◽  
Applied Electric Field ◽  
Electroosmotic Flow ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Magnetic Forces ◽  
Extraction And Purification

NA extraction and purification utilitzing a microfluidic chip with applied electric field to induce electroosmotic flow opposite the magnetic NA-bound bead mix.

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