scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2020 ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Nucleic Acid Amplification ◽  
Nasopharyngeal Swab ◽  
Viral Transport ◽  
Extraction Step ◽  
Highly Sensitive

AbstractThe COVID-19 pandemic has resulted in an urgent global need for rapid, point-of-care diagnostic testing. Existing methods for nucleic acid amplification testing (NAAT) require an RNA extraction step prior to amplification of the viral RNA. This step necessitates the use of a centralized laboratory or complex and costly proprietary cartridges and equipment, and thereby prevents low-cost, scalable, point-of-care testing. We report the development of a highly sensitive and robust, easy-to-implement, SARS-CoV-2 test that utilizes isothermal amplification and can be run directly on viral transport media following a nasopharyngeal swab without the need for prior RNA extraction. Our assay provides visual results in 30 min with 85% sensitivity, 100% specificity, and a limit of detection (LoD) of 2.5 copies/μl, and can be run using a simple heat block.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals Highly performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

2020 ◽  
Author(s):  
Pierre Garneret ◽  
Etienne Coz ◽  
Elian Martin ◽  
Jean-Claude Manuguerra ◽  
Elodie Brient-Litzler ◽  
...  
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Rna Extraction ◽  
Dna Amplification ◽  
Developed Countries ◽  
Temperature Cycling ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Molecular Testing

In order to respond to the urgent request of massive testing, developed countries perform nucleic acid amplification tests (NAAT) of SARS-CoV-2 in centralized laboratories. Real-time RT - PCR (Reverse transcription - Polymerase Chain Reaction) is used to amplify the viral RNA and enable its detection. Although PCR is 37 years old, it is still considered, without dispute, as the gold standard. PCR is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings. In the present work, by harnessing progress made in the past two decades in DNA amplification, microfluidics and membrane technologies, we succeeded to create a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT - LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or highly fluorescent probes. Depending on the viral load, the detection takes between twenty minutes and one hour. Using pools of naso-pharyngal clinical samples, we estimated a sensitivity comparable to RT-qPCR (up to a Cycle threshold of 39, equivalent to <0.1 TCID50 per mL) and a 100% specificity, for other human coronaviruses and eight respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called COVIDISC to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment paves the way towards a large dissemination of this device. The perspective of a reliable SARS-CoV-2 point of care detection, highly performing, that would deliver on-site results in less than one hour opens up a new efficient approach to manage the pandemics.

scholarly journals LATE-PCR for LoC Molecular Diagnostics Devices and Its Application to the Sensitive Detection of SARS-CoV-2

2021 ◽  
Vol 6 (1) ◽  
pp. 43
Author(s):  
Dimitrios Karadimas ◽  
George Tsekenis
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Diagnostic Testing ◽  
Heat Denaturation ◽  
Nasopharyngeal Swab ◽  
The Novel ◽  
Rt Pcr ◽  
Qualitative Detection ◽  
Novel Coronavirus ◽  
Real Patients

The emergence of the novel coronavirus, SARS-CoV-2, has highlighted the need for rapid, accurate, and point-of-care diagnostic testing. Lab-on-a-Chip (LoC) devices offer the possibility to run such tests at a low cost, while at the same time permitting the multiplexed detection of several viruses when coupled with microarray detection of the amplified products. Herein, we report the development of a protocol for the qualitative detection of SARS-CoV-2, through the design of appropriate primers that target the evolutionary conserved regions of the virus. The proposed protocol relies on an improved version of asymmetric RT-PCR, the linear-after-the-exponential (LATE)-PCR that uses primers that are deliberately designed for use at unequal concentrations. As a result, LATE-PCR exhibits similar efficiency to symmetric PCR, while promoting accumulation of single-stranded products that can subsequently hybridize to a single-strand DNA probe-spotted microarray. The performance of the developed LATE-PCR protocol was compared to that of symmetric RT-PCR, and validated with the use of artificial viral RNA and nasopharyngeal swab samples from real patients. Furthermore, and in order to illustrate its potential for integration into a biosensor platform, the amplicons were allowed to hybridize with probes that were covalently immobilized onto commercially available functionalized glass, without the need for heat denaturation.

scholarly journals Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0243712
Author(s):  
Pierre Garneret ◽  
Etienne Coz ◽  
Elian Martin ◽  
Jean-Claude Manuguerra ◽  
Elodie Brient-Litzler ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Clinical Sample ◽  
Point Of Care ◽  
Rna Extraction ◽  
Temperature Cycling ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Molecular Testing ◽  
Rt Pcr

To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT—PCR (Reverse transcription—Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT—LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross‐reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called “COVIDISC” to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.

scholarly journals Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared to endpoint assessment

2021 ◽  
Author(s):  
Lena M. Diaz ◽  
Brandon E. Johnson ◽  
Daniel M. Jenkins
Keyword(s):  
Nucleic Acid ◽  
Quantitative Method ◽  
Limit Of Detection ◽  
Detection System ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Ph Indicator

AbstractControlling the course of the COVID-19 pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel coronavirus SARS-CoV-2. Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current FDA-approved RT-qPCR-based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than two hours to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS- CoV-2 at either 1X (50 genomes/reaction) or 2X (100 genomes/reaction) of the LOD. Importantly the quantitative method was based on dynamic optical changes during the reaction so was able to correctly classify samples that were misclassified by endpoint observation of color.

Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints

2020 ◽  
Vol 15 (15) ◽  
pp. 1483-1487
Author(s):  
Nikhil S Sahajpal ◽  
Ashis K Mondal ◽  
Allan Njau ◽  
Sudha Ananth ◽  
Kimya Jones ◽  
...  
Keyword(s):  
Supply Chain ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Sample Collection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Short Supply ◽  
Viral Transport ◽  
3D Printed ◽  
Bridging Studies

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

scholarly journals A rapid, accurate, scalable, and portable testing system for COVID-19 diagnosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Guanhua Xun ◽  
Stephan Thomas Lane ◽  
Vassily Andrew Petrov ◽  
Brandon Elliott Pepa ◽  
Huimin Zhao
Keyword(s):  
Limit Of Detection ◽  
Low Cost ◽  
High Volume ◽  
N Gene ◽  
Testing System ◽  
Target Sequence ◽  
One Pot ◽  
Sequence Detection ◽  
Highly Sensitive ◽  
Speed Accuracy

AbstractThe need for rapid, accurate, and scalable testing systems for COVID-19 diagnosis is clear and urgent. Here, we report a rapid Scalable and Portable Testing (SPOT) system consisting of a rapid, highly sensitive, and accurate assay and a battery-powered portable device for COVID-19 diagnosis. The SPOT assay comprises a one-pot reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) followed by PfAgo-based target sequence detection. It is capable of detecting the N gene and E gene in a multiplexed reaction with the limit of detection (LoD) of 0.44 copies/μL and 1.09 copies/μL, respectively, in SARS-CoV-2 virus-spiked saliva samples within 30 min. Moreover, the SPOT system is used to analyze 104 clinical saliva samples and identified 28/30 (93.3% sensitivity) SARS-CoV-2 positive samples (100% sensitivity if LoD is considered) and 73/74 (98.6% specificity) SARS-CoV-2 negative samples. This combination of speed, accuracy, sensitivity, and portability will enable high-volume, low-cost access to areas in need of urgent COVID-19 testing capabilities.

scholarly journals Rapid and Sensitive Detection of miRNA Based on AC Electrokinetic Capacitive Sensing for Point-of-Care Applications

Sensors ◽  
2021 ◽  
Vol 21 (12) ◽  
pp. 3985
Author(s):  
Nan Wan ◽  
Yu Jiang ◽  
Jiamei Huang ◽  
Rania Oueslati ◽  
Shigetoshi Eda ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Clinical Diagnostics ◽  
Detection Methods ◽  
Capacitive Sensing ◽  
Application Potential ◽  
Mirna Detection ◽  
Mirna 25 ◽  
Low Sensitivity

A sensitive and efficient method for microRNAs (miRNAs) detection is strongly desired by clinicians and, in recent years, the search for such a method has drawn much attention. There has been significant interest in using miRNA as biomarkers for multiple diseases and conditions in clinical diagnostics. Presently, most miRNA detection methods suffer from drawbacks, e.g., low sensitivity, long assay time, expensive equipment, trained personnel, or unsuitability for point-of-care. New methodologies are needed to overcome these limitations to allow rapid, sensitive, low-cost, easy-to-use, and portable methods for miRNA detection at the point of care. In this work, to overcome these shortcomings, we integrated capacitive sensing and alternating current electrokinetic effects to detect specific miRNA-16b molecules, as a model, with the limit of detection reaching 1.0 femto molar (fM) levels. The specificity of the sensor was verified by testing miRNA-25, which has the same length as miRNA-16b. The sensor we developed demonstrated significant improvements in sensitivity, response time and cost over other miRNA detection methods, and has application potential at point-of-care.

scholarly journals Demonstration of a Label-Free and Low-Cost Optical Cavity-Based Biosensor Using Streptavidin and C-Reactive Protein

Biosensors ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 4
Author(s):  
Donggee Rho ◽  
Seunghyun Kim
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Optical Cavity ◽  
Sample Volume ◽  
Small Sample ◽  
Label Free ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Cavity Structure

An optical cavity-based biosensor (OCB) has been developed for point-of-care (POC) applications. This label-free biosensor employs low-cost components and simple fabrication processes to lower the overall cost while achieving high sensitivity using a differential detection method. To experimentally demonstrate its limit of detection (LOD), we conducted biosensing experiments with streptavidin and C-reactive protein (CRP). The optical cavity structure was optimized further for better sensitivity and easier fluid control. We utilized the polymer swelling property to fine-tune the optical cavity width, which significantly improved the success rate to produce measurable samples. Four different concentrations of streptavidin were tested in triplicate, and the LOD of the OCB was determined to be 1.35 nM. The OCB also successfully detected three different concentrations of human CRP using biotinylated CRP antibody. The LOD for CRP detection was 377 pM. All measurements were done using a small sample volume of 15 µL within 30 min. By reducing the sensing area, improving the functionalization and passivation processes, and increasing the sample volume, the LOD of the OCB are estimated to be reduced further to the femto-molar range. Overall, the demonstrated capability of the OCB in the present work shows great potential to be used as a promising POC biosensor.

scholarly journals Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6617
Author(s):  
Eva Rajh ◽  
Tina Šket ◽  
Arne Praznik ◽  
Petra Sušjan ◽  
Alenka Šmid ◽  
...  
Keyword(s):  
Oral Cavity ◽  
Reverse Transcription ◽  
Screening Program ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Diagnostic Sensitivity ◽  
Nucleic Acid Amplification ◽  
Nasopharyngeal Swab ◽  
Viral Spread ◽  
One Step

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

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