scholarly journals Integration of RT-LAMP and Microfluidic Technology for Detection of SARS-CoV-2 in Wastewater as an Advanced Point-of-care Platform

2021 ◽  
Author(s):  
Ahmed Donia ◽  
Muhammad Furqan Shahid ◽  
Aftab Ahmad ◽  
Aneela Javed ◽  
Muhammad Nawaz ◽  
...  
Keyword(s):  
Point Of Care ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Surrogate Marker ◽  
Lamp Assay ◽  
N Gene ◽  
Microfluidic Chips ◽  
Microfluidic Technology ◽  
Wastewater Samples ◽  
The One

Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of 7 wastewater samples. RT-LAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best surrogate marker for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be used as a surrogate marker for detection of SARS-CoV-2. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except the one which subjected to RT-LAMP for targeting N region after RNA extraction (yellowish/orange color). This study shows for the first time that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips.

scholarly journals Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Udeni B. R. Balasuriya ◽  
Lindomar Pena
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Serum Samples ◽  
Low Resource ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

scholarly journals Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Matthew A Lalli ◽  
Joshua S Langmade ◽  
Xuhua Chen ◽  
Catrina C Fronick ◽  
Christopher S Sawyer ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Viral Detection ◽  
Loop Mediated Isothermal Amplification

Abstract Background Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. Methods To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. Results The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44–104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Conclusions Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

scholarly journals Rapid, reliable, and cheap point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP (Cap-iLAMP)

2020 ◽  
Author(s):  
Lukas Bokelmann ◽  
Olaf Nickel ◽  
Tomislav Maricic ◽  
Svante Paabo ◽  
Matthias Meyer ◽  
...  
Keyword(s):  
Point Of Care ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Cost Effective ◽  
Diagnostic Methods ◽  
Current Standard ◽  
Laboratory Equipment ◽  
Hybridization Capture ◽  
Level Of Automation ◽  
High Level

Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can easily be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation, sophisticated laboratory equipment and trained personnel to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification). This method combines a hybridization capture-based RNA extraction of non-invasive gargle lavage samples to concentrate samples and remove inhibitors with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP does not result in false positives and single infected samples can be detected in a pool among 25 uninfected samples, thus reducing the technical cost per test to ~1 Euro per individual.

Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y, and del156-157/R158G) in settled solids using digital RT-PCR v2

2021 ◽  
Author(s):  
Bridgette Hughes ◽  
Bradley J. White ◽  
Marlene K. Wolfe ◽  
Krista Wigginton ◽  
Alexandria B Boehm
Keyword(s):  
Quantitative Analysis ◽  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Population Level ◽  
Digital Pcr ◽  
N Gene ◽  
Rt Pcr ◽  
Pcr Inhibitor ◽  
Wastewater Samples

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one for HV69-70, one for E484K/N501Y, and one for del156-157/R158G) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y,del156-157/R158G, and Del143-145) in settled solids using digital RT-PCR v2

2021 ◽  
Author(s):  
Bridgette Hughes ◽  
Bradley J. White ◽  
Marlene K. Wolfe ◽  
Krista Wigginton ◽  
Alexandria B Boehm
Keyword(s):  
Quantitative Analysis ◽  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Population Level ◽  
Digital Pcr ◽  
N Gene ◽  
Rt Pcr ◽  
Pcr Inhibitor ◽  
Wastewater Samples

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one each for HV69-70, E484K/N501Y, del156-157/R158G, and Del143-145) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

scholarly journals Point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lukas Bokelmann ◽  
Olaf Nickel ◽  
Tomislav Maricic ◽  
Svante Pääbo ◽  
Matthias Meyer ◽  
...  
Keyword(s):  
Point Of Care ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Cost Effective ◽  
Diagnostic Methods ◽  
Current Standard ◽  
Laboratory Equipment ◽  
Hybridization Capture ◽  
Single Positive ◽  
High Level

AbstractEfforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation and sophisticated laboratory equipment to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification) which combines a hybridization capture-based RNA extraction of gargle lavage samples with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP prevents false positives and allows single positive samples to be detected in pools of 25 negative samples, reducing the reagent cost per test to ~1 Euro per individual.

scholarly journals Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification

2020 ◽  
Vol 14 (11) ◽  
pp. e0008855
Author(s):  
Rokusuke Yoshikawa ◽  
Haruka Abe ◽  
Yui Igasaki ◽  
Saeki Negishi ◽  
Hiroaki Goto ◽  
...  
Keyword(s):  
Clinical Evaluation ◽  
Rural Areas ◽  
Point Of Care ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Diagnostic Assay ◽  
Loop Mediated Isothermal Amplification ◽  
Efficient Detection ◽  
Novel Coronavirus

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic novel coronavirus that has caused a worldwide outbreak. Here we describe a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that uses a portable device for efficient detection of SARS-CoV-2. This RT-LAMP assay specifically detected SARS-CoV-2 without cross-reacting with the most closely related human coronavirus, SARS-CoV. Clinical evaluation of nasal swab samples from suspected SARS-CoV-2 pneumonia (COVID-19) patients showed that the assay could detect over 23.7 copies within 15 min with a 100% probability. Since the RT-LAMP assay can be performed with a portable battery-supported device, it is a rapid, simple, and sensitive diagnostic assay for COVID-19 that can be available at point-of-care. We also developed the RT-LAMP assay without the RNA extraction step–Direct RT-LAMP, which could detect more than 1.43 x 103 copies within 15 min with a 100% probability in clinical evaluation test. Although the Direct RT-LAMP assay was less sensitive than the standard RT-LAMP, the Direct RT-LAMP assay can be available as the rapid first screening of COVID-19 in poorly equipped areas, such as rural areas in developing countries.

scholarly journals One-pot Detection of COVID-19 with Real-time Reverse-transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assay and Visual RT-LAMP Assay

2020 ◽  
Author(s):  
Deguo Wang
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Prevention And Control ◽  
Gene Sequence ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
N Gene ◽  
One Pot ◽  
The One ◽  
And Control

AbstractBackgroundRapid and reliable diagnostic assays were critical for prevention and control of the coronavirus pneumonia caused by COVID-19.ObjectiveThis study was to establish one-pot real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay and one-pot visual RT-LAMP assay for the detection of COVID-19.MethodsSix specific LAMP primers targeting the N gene of COVID-19 were designed, the RT-LAMP reaction system was optimized with plasmid pUC57 containing N gene sequence, the detection limit was determined with a serial dilution of the plasmid pUC57 containing N gene sequence, and the one-pot real-time RT-LAMP assay and one-pot visual RT-LAMP assay for the detection of COVID-19 were established.ResultsOur results showed that the one-pot RT-LAMP assays can detect COVID-19 with a limit of ≥ 6 copies per μl−1 of pUC57 containing N gene sequence.ConclusionThis study provides rapid, reliable and sensitive tools for facilitating preliminary and cost-effective prevention and control of COVID-19.

High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR v2

2021 ◽  
Author(s):  
Aaron Topol (Verily Life Sciences) ◽  
marlene.wolfe not provided ◽  
Brad White (Verily Life Sciences) ◽  
Krista Wigginton ◽  
Alexandria B Boehm
Keyword(s):  
Quantitative Analysis ◽  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Digital Pcr ◽  
N Gene ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Pcr Inhibitor ◽  
Wastewater Samples

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and ORF1a and a duplex assay targeting Bovine Coronavirus Vaccine (BCoV) and Pepper Mild mottle virus (PMMoV) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. This protocol describes 2 separate PCR reactions, one containing primer/probe mixtures targeting the three SARS-CoV-2 targets and one containing primer/probe mixtures targeting BCoV and PMMoV. BCoV is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. PMMoV is an enveloped virus which is abundant in human fecal waste and serves as an endogenous control for data normalization. PMMoV RNA is abundant at such high levels in wastewater samples that the samples must be diluted by a factor of 100 before quantification. The readout of this assay is a concentration of each target in the extracted RNA samples (copies/uL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

scholarly journals Detection of SARS-CoV-2 in Saliva by RT-LAMP During a Screening of Workers in Brazil, Including Pre-Symptomatic Carriers

2021 ◽  
Author(s):  
Carlos dos Santos ◽  
Kézia de Oliveira ◽  
Geovana Mendes ◽  
Lívia Silva ◽  
Marcio de Souza Jr. ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Asymptomatic Carrier ◽  
Point Of Care ◽  
Quantitative Polymerase Chain Reaction ◽  
Lamp Assay ◽  
Diagnostic Methods ◽  
Public And Private ◽  
Polymerase Chain ◽  
Increasing Demand

The coronavirus pandemic has been causing damage to many nations, as public and private health systems deteriorate by the increasing demand. Some infected patients have culturable severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) even though not presenting any symptoms, and therefore, are probably able to transmit it. Correctly diagnosing and isolating infected patients is an important step towards preventing new infections. Current diagnostic methods rely mainly on reverse transcription quantitative polymerase chain reaction (RT-qPCR). Methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) have risen as viable alternatives, as they are cheaper and require less infrastructure, they have the potential to be applied in low-resource scenarios and even at point-of-care. Here we report a colorimetric RT‑LAMP assay capable of detecting SARS-CoV-2 in ribonucleic acid (RNA) from saliva. In some cases, the test was able to detect viral RNA before symptom onset and even in a self-reported asymptomatic carrier. It had a limit of detection of 300 copies per reaction and showed a sensitivity of 80%, a specificity of 100%, a general accuracy of 99.59%, and a Cohen’s kappa of 0.887. The possibility of detecting positive cases even before the clinical manifestation shows great potential and can contribute to controlling the pandemic.

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