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 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.

A point-of-care diagnostic for differentiating Ebola from endemic febrile diseases

2018 ◽  
Vol 10 (471) ◽  
pp. eaat0944 ◽  
Author(s):  
David Sebba ◽  
Alexander G. Lastovich ◽  
Melody Kuroda ◽  
Eric Fallows ◽  
Joshua Johnson ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Clinical Symptoms ◽  
Point Of Care ◽  
Hemorrhagic Fever ◽  
Diagnostic Methods ◽  
Outbreak Detection ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Live Virus ◽  
And Control

Hemorrhagic fever outbreaks such as Ebola are difficult to detect and control because of the lack of low-cost, easily deployable diagnostics and because initial clinical symptoms mimic other endemic diseases such as malaria. Current molecular diagnostic methods such as polymerase chain reaction require trained personnel and laboratory infrastructure, hindering diagnostics at the point of need. Although rapid tests such as lateral flow can be broadly deployed, they are typically not well-suited for differentiating among multiple diseases presenting with similar symptoms. Early detection and control of Ebola outbreaks require simple, easy-to-use assays that can detect and differentiate infection with Ebola virus from other more common febrile diseases. Here, we developed and tested an immunoassay technology that uses surface-enhanced Raman scattering (SERS) tags to simultaneously detect antigens from Ebola, Lassa, and malaria within a single blood sample. Results are provided in <30 min for individual or batched samples. Using 190 clinical samples collected from the 2014 West African Ebola outbreak, along with 163 malaria positives and 233 negative controls, we demonstrated Ebola detection with 90.0% sensitivity and 97.9% specificity and malaria detection with 100.0% sensitivity and 99.6% specificity. These results, along with corresponding live virus and nonhuman primate testing of an Ebola, Lassa, and malaria 3-plex assay, indicate the potential of the SERS technology as an important tool for outbreak detection and clinical triage in low-resource settings.

scholarly journals Detection of LytA Genes in Streptococcus pneumoniae Isolated from sputum pneumonia patients

Biomedika ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 23-30
Author(s):  
Mustika Sari Hutabarat ◽  
Firdaus Hamid ◽  
Irawaty Djaharuddin ◽  
Alfian Zainuddin ◽  
Rossana Agus ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
False Negative ◽  
Pneumococcal Infection ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Biochemical Tests ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

Streptococcus pneumoniae (pneumococcus) is a Gram-positive facultative anaerobic bacterium that is a major cause of morbidity and mortality worldwide. But the lack of reporting of disease by this bacterium in Indonesia, one of the causes is because the diagnosis of pneumococcal infection is often clinically not typical and conventional methods which are still the standard gold method often give false-negative results. So the purpose of this study was to evaluate the performance of culture and molecular diagnostic methods using the Polymerase Chain Reaction (PCR) technique in detecting Streptococcus pneumoniae in sputum clinical samples using the Autolysin (LytA) gene which is a virulence factor of this bacterium. 57 isolates from 60 samples were confirmed as Streptococcus sp through microscopic identification, culture, and biochemical tests. Then the sensitivity test with an optochin test of 9 (9%) compared the results descriptively with the PCR technique using the Autolysin A (LytA) gene which was obtained more sensitive by 15 (25%).

scholarly journals Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride

BioTechniques ◽  
2020 ◽  
Vol 69 (3) ◽  
pp. 178-185 ◽  
Author(s):  
Yinhua Zhang ◽  
Guoping Ren ◽  
Jackson Buss ◽  
Andrew J Barry ◽  
Gregory C Patton ◽  
...  
Keyword(s):  
Guanidine Hydrochloride ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Simple Method ◽  
Diagnostic Assays ◽  
Loop Mediated Isothermal Amplification ◽  
Dna And Rna ◽  
Versatile Technique ◽  
Primer Sets

Loop-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 techniques and enabling accurate, high-throughput diagnostics.

scholarly journals Comparison Between a Standard and SalivaDirect RNA Extraction Protocol for Molecular Diagnosis of SARS-CoV-2 Using Nasopharyngeal Swab and Saliva Clinical Samples

2021 ◽  
Vol 9 ◽  
Author(s):  
Sofía N. Rodríguez Flores ◽  
Luis Mario Rodríguez-Martínez ◽  
Bernardita L. Reyes-Berrones ◽  
Nadia A. Fernández-Santos ◽  
Elthon J. Sierra-Moncada ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Proteinase K ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Sample Collection ◽  
Extraction Protocol ◽  
Assay Performance ◽  
Two Samples ◽  
Oropharyngeal Swab

During the COVID-19 pandemic, a certified laboratory of Tamaulipas, Mexico has processed over 100,000 samples of COVID-19 suspected patients, working a minimum of 100 tests daily. Thus, it would be beneficial for such certified laboratories nationwide to reduce the time and cost involved in performing the diagnosis of COVID-19, from sample collection, transportation to local lab, processing of samples, and data acquisition. Here, 30 nasopharyngeal swab and saliva samples from the same COVID-19 individuals were assessed by a standard nucleic acid extraction protocol, including protein lysis with proteinase K followed by binding to column, washing, and elution, and by the SalivaDirect protocol based on protein lysis, skipping the other steps to reduce processing time and costs. The genomic RNA was amplified using a SARS-CoV-2 Real-Time PCR kit. A variation (P &gt; 0.05) in the 95% CIs = 72.6%–96.7% was noted by using the SalivaDirect protocol and saliva samples (sensitivity of 88.2%) in comparison to those of standard protocol with oropharyngeal swab samples (95% CIs = 97.5%–100%; sensitivity of 100%) as reported elsewhere. However, when using nasopharyngeal swab samples in the SalivaDirect protocol (sensitivity of 93.6%; 95% CIs = 79.2%–99.2%), it was in concordance (P &lt; 0.05) with those of the standard one. The logical explanation to this was that two samples with Ct values of 38, and 40 cycles for gene E produced two false negatives in the SalivaDirect protocol in relation to the standard one; thus, there was a reduction of the sensitivity of 6.4% in the overall assay performance.

scholarly journals Application of the amplification-free SERS-based CRISPR/Cas12a platform in the identification of SARS-CoV-2 from clinical samples

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jiajie Liang ◽  
Peijun Teng ◽  
Wei Xiao ◽  
Guanbo He ◽  
Qifang Song ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Point Of Care ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Enhancement Effect ◽  
Diagnostic Assays ◽  
Point Of Care Test ◽  
Raman Enhancement ◽  
Potential Strategy ◽  
Refractory Diseases

AbstractThe control of contagious or refractory diseases requires early, rapid diagnostic assays that are simple, fast, and easy-to-use. Here, easy-to-implement CRISPR/Cas12a-based diagnostic platform through Raman transducer generated by Raman enhancement effect, term as SERS-CRISPR (S-CRISPR), are described. The S-CRISPR uses high-activity noble metallic nanoscopic materials to increase the sensitivity in the detection of nucleic acids, without amplification. This amplification-free platform, which can be performed within 30–40 min of incubation time, is then used for detection of SARS-CoV-2 derived nucleic acids in RNA extracts obtained from nasopharyngeal swab specimens (n  =  112). Compared with the quantitative reverse transcription polymerase chain reaction (RT-qPCR), the sensitivity and specificity of S-CRISPR reaches 87.50% and 100%, respectively. In general, the S-CRISPR can rapidly identify the RNA of SARS-CoV-2 RNA without amplification and is a potential strategy for nucleic acid point of care test (POCT).

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 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.

Laboratory Methods for the Diagnosis of SARS-Cov-2

2022 ◽  
pp. 58-77
Author(s):  
Mohamed Echchakery ◽  
Samia Boussaa ◽  
Souad El Mouahid ◽  
Maryam Mountassir ◽  
Said El Hizazi ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Diagnostic Tests ◽  
Epidemiological Studies ◽  
Virus Antigen ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Laboratory Methods ◽  
Virus Surface ◽  
Diagnostic Assays ◽  
Human Endeavor

The coronavirus disease 2019 (COVID-19) which has become the pandemic par excellence of our time places pressure on various aspects of human endeavor and as such requires detailed study to better combat it. However, diagnostic tests were used to provide data on the incidence of COVID-19 and to assess the immune status of infected individuals. The objective of this chapter is to describe the diagnostic methods currently used to identify SARS-CoV-2 infection. Obtaining the first SARS-CoV-2 genome sequence was decisive for the development of molecular diagnostic assays that currently make it possible to diagnose and screen for the Sars-CoV-2 infection. Their uses depend on the target to be detected. Antigenic tests detect the presence of a virus antigen, which usually makes a proteinaceous part of the virus surface. The serology tests detect the presence of antibodies generated against SARS-CoV-2 and are also a relevant tool for epidemiological studies.

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 &gt; 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.

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