scholarly journals Development and validation of direct RT-LAMP for SARS-CoV-2

2020 ◽  
Author(s):  
Abu Naser Mohon ◽  
Jana Hundt ◽  
Guido van Marle ◽  
Kanti Pabbaraju ◽  
Byron Berenger ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Extraction Procedure ◽  
Lamp Assay ◽  
Cold Chain ◽  
Clinical Samples ◽  
Lamp Method ◽  
Rt Pcr ◽  
Percent Agreement

AbstractWe have developed a reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2. The LAMP assay achieves comparable limit of detection as commonly used RT-PCR protocols based on artificial targets, recombinant Sindbis virus, and clinical samples. Clinical validation of single-target (S gene) LAMP (N=120) showed a positive percent agreement (PPA) of 41/42 (97.62%) and negative percent agreement (NPA) of 77/78 (98.72%) compared to reference RT-PCR. Dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 44/48 (91.97%) and NPA 72/72 (100%) when including discrepant samples. The assay can be performed without a formal extraction procedure, with lyophilized reagents which do need cold chain, and is amenable to point-of-care application with visual detection.

scholarly journals Rapid isothermal amplification and portable detection system for SARS-CoV-2

2020 ◽  
Vol 117 (37) ◽  
pp. 22727-22735 ◽  
Author(s):  
Anurup Ganguli ◽  
Ariana Mostafa ◽  
Jacob Berger ◽  
Mehmet Y. Aydin ◽  
Fu Sun ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Alternative Pathway ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Complete Agreement ◽  
Rt Pcr ◽  
Viral Transport Medium

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

scholarly journals Optimization of Lyophilized LAMP and RT-PCR Reaction Mixes for Detection of Tuberculosis

2020 ◽  
Vol 4 (4) ◽  
pp. 230-236
Author(s):  
Esra Agel ◽  
Hasan Sagcan
Keyword(s):  
Rural Areas ◽  
Point Of Care ◽  
Culture Method ◽  
Molecular Techniques ◽  
Cold Chain ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Gold Standard Method ◽  
Tuberculosis Diagnosis ◽  
Peg 8000

AbstractUndoubtedly, one of the most infectious diseases in the world is tuberculosis. Key factor for tuberculosis control is to prevent possible contagion with rapid diagnosis and effective treatment. The culture method, which it takes several weeks to obtain results, is the gold standard method for laboratory diagnosis of tuberculosis. In order to prevent possible contagion of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. Normally, clinical samples are studied in advanced laboratories designed for this purpose. However, especially after the screening in rural areas, the transmission of the samples to the centers has many negative effects on the clinical material. Therefore, the latest trend molecular techniques in microbiological diagnosis are developing into point of care systems that can be applied in the field without laboratory infrastructure. The major challenge for molecular-based point-of-care tests is the need to store polymerase enzymes and some of the ingredients used in the cold chain. The aim of this study is to increase the resistance of the amplification reaction mixtures by lyophilizing the tuberculosis diagnosis. Lyophilization was performed on Loop-mediated isothermal amplification (LAMP) and Real-time PCR mixtures. For the lyophilization of LAMP and RT-PCR mixtures, two different experimental setups were tried from the literature except for the developed content. Chemicals such as stachyose, trehalose, glycerol and PEG 8000 are widely using as cryoprotectants. As a result, the developed content (0.5% PEG 8000, 2.0 % Stachyose) was determined the best cryoprotectant mixture. Accordingly, amplification mixtures can be produced with the developed lyophilization method and point of care kits can be developed.

scholarly journals A handheld point-of-care system for rapid detection of SARS-CoV-2 in under 20 minutes

2020 ◽  
Author(s):  
Jesus Rodriguez-Manzano ◽  
Kenny Malpartida-Cardenas ◽  
Nicolas Moser ◽  
Ivana Pennisi ◽  
Matthew Cavuto ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Lamp Assay ◽  
High Rate ◽  
Epidemiological Surveillance ◽  
Clinical Samples ◽  
Lab On Chip ◽  
Point Of Care System ◽  
On Chip

AbstractThe COVID-19 pandemic is a global health emergency characterized by the high rate of transmission and ongoing increase of cases globally. Rapid point-of-care (PoC) diagnostics to detect the causative virus, SARS-CoV-2, are urgently needed to identify and isolate patients, contain its spread and guide clinical management. In this work, we report the development of a rapid PoC diagnostic test (< 20 min) based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and semiconductor technology for the detection of SARS-CoV-2 from extracted RNA samples. The developed LAMP assay was tested on a real-time benchtop instrument (RT-qLAMP) showing a lower limit of detection of 10 RNA copies per reaction. It was validated against 183 clinical samples including 127 positive samples (screened by the CDC RT-qPCR assay). Results showed 90.55% sensitivity and 100% specificity when compared to RT-qPCR and average positive detection times of 15.45 ± 4.43 min. For validating the incorporation of the RT-LAMP assay onto our PoC platform (RT-eLAMP), a subset of samples was tested (n=40), showing average detection times of 12.89 ± 2.59 min for positive samples (n=34), demonstrating a comparable performance to a benchtop commercial instrument. Paired with a smartphone for results visualization and geo-localization, this portable diagnostic platform with secure cloud connectivity will enable real-time case identification and epidemiological surveillance.One Sentence SummaryWe demonstrate isothermal detection of SARS-CoV-2 in under 20 minutes from extracted RNA samples with a handheld Lab-on-Chip platform.

scholarly journals Evaluation of the QIAstat-Dx Respiratory SARS-CoV-2 Panel, the First Rapid Multiplex PCR Commercial Assay for SARS-CoV-2 Detection

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Benoit Visseaux ◽  
Quentin Le Hingrat ◽  
Gilles Collin ◽  
Donia Bouzid ◽  
Samuel Lebourgeois ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Limit Of Detection ◽  
Clinical Sample ◽  
Point Of Care ◽  
Sample Pretreatment ◽  
Tracheal Aspirate ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Viral Neutralization ◽  
Efficient Detection

ABSTRACT In the race to contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), efficient detection and triage of infected patients must rely on rapid and reliable testing. In this work, we performed the first evaluation of the QIAstat-Dx respiratory SARS-CoV-2 panel (QIAstat-SARS) for SARS-CoV-2 detection. This assay is the first rapid multiplex PCR (mPCR) assay, including SARS-CoV-2 detection, and is fully compatible with a non-PCR-trained laboratory or point-of-care (PoC) testing. This evaluation was performed using 69 primary clinical samples (66 nasopharyngeal swabs [NPS], 1 bronchoalveolar lavage fluid sample [BAL], 1 tracheal aspirate sample, and 1 bronchial aspirate sample) comparing SARS-CoV-2 detection with the currently WHO-recommended reverse transcription-PCR (RT-PCR) (WHO-RT-PCR) workflow. Additionally, a comparative limit of detection (LoD) assessment was performed for QIAstat-SARS and WHO-RT-PCR using a quantified clinical sample. Compatibility of sample pretreatment for viral neutralization or viscous samples with the QIAstat-SARS system were also tested. The QIAstat-Dx respiratory SARS-CoV-2 panel demonstrated a sensitivity comparable to that of the WHO-recommended assay with a limit of detection at 1,000 copies/ml. The overall percent agreement between QIAstat-Dx SARS and WHO-RT-PCR on 69 clinical samples was 97% with a sensitivity of 100% (40/40) and specificity at 93% (27/29). No cross-reaction was encountered for any other respiratory viruses or bacteria included in the panel. The QIAstat-SARS rapid multiplex PCR panel provides a highly sensitive, robust, and accurate assay for rapid detection of SARS-CoV-2. This assay allows rapid decisions even in non-PCR-trained laboratory or point-of-care testing, allowing innovative organization.

scholarly journals Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Andreas Hober ◽  
Khue Hua Tran-Minh ◽  
Dominic Foley ◽  
Thomas McDonald ◽  
Johannes PC Vissers ◽  
...  
Keyword(s):  
Large Scale ◽  
Limit Of Detection ◽  
Virus Detection ◽  
Viral Proteins ◽  
Clinical Samples ◽  
Molecular Testing ◽  
Rt Pcr ◽  
Percent Agreement ◽  
Peptide Enrichment ◽  
Asymptomatic Individuals

Reliable, robust, large-scale molecular testing for SARS-CoV-2 is essential for monitoring the ongoing Covid-19 pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immunoaffinity enrichment combined with liquid chromatography - mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in PBS swab media from combined throat/nasopharynx/saliva samples.<br />The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their corresponding RT-PCR readout (r=0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative readout of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

scholarly journals Lumipulse G SARS-CoV-2 Ag assay evaluation using clinical samples from different testing groups

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Giulia Menchinelli ◽  
Licia Bordi ◽  
Flora Marzia Liotti ◽  
Ivana Palucci ◽  
Maria Rosaria Capobianchi ◽  
...  
Keyword(s):  
Positive Result ◽  
Limit Of Detection ◽  
False Negative ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Repeated Testing ◽  
Rt Pcr ◽  
Subgenomic Rna ◽  
Negative Results ◽  
Percent Agreement

Abstract Objectives Compared to RT-PCR, lower performance of antigen detection assays, including the Lumipulse G SARS-CoV-2 Ag assay, may depend on specific testing scenarios. Methods We tested 594 nasopharyngeal swab samples from individuals with COVID-19 (RT-PCR cycle threshold [Ct] values ≤ 40) or non-COVID-19 (Ct values > 40) diagnoses. RT-PCR positive samples were assigned to diagnostic, screening, or monitoring groups of testing. Results With a limit of detection of 1.2 × 104 SARS-CoV-2 RNA copies/ml, Lumipulse showed positive percent agreement (PPA) of 79.9% (155/194) and negative percent agreement of 99.3% (397/400), whereas PPAs were 100% for samples with Ct values of <18 or 18–<25 and 92.5% for samples with Ct values of 25–<30. By three groups, Lumipulse showed PPA of 87.0% (60/69), 81.1% (43/53), or 72.2% (52/72), respectively, whereas PPA was 100% for samples with Ct values of <18 or 18–<25, and was 94.4, 80.0, or 100% for samples with Ct values of 25–<30, respectively. Additional testing of RT-PCR positive samples for SARS-CoV-2 subgenomic RNA showed that, by three groups, PPA was 63.8% (44/69), 62.3% (33/53), or 33.3% (24/72), respectively. PPAs dropped to 55.6, 20.0, or 41.7% for samples with Ct values of 25–<30, respectively. All 101 samples with a subgenomic RNA positive result had a Lumipulse assay’s antigen positive result, whereas only 54 (58.1%) of remaining 93 samples had a Lumipulse assay’s antigen positive result. Conclusions Lumipulse assay was highly sensitive in samples with low RT-PCR Ct values, implying repeated testing to reduce consequences of false-negative results.

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 Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252687
Author(s):  
Sukalyani Banik ◽  
Kaheerman Saibire ◽  
Shraddha Suryavanshi ◽  
Glenn Johns ◽  
Soumitesh Chakravorty ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Viral Rna ◽  
Clinical Samples ◽  
Sample Collection ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Guanidinium Thiocyanate ◽  
Upper Respiratory ◽  
Polymerase Chain ◽  
The Stability

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

scholarly journals Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

2020 ◽  
Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Virus Disease ◽  
Lamp Assay ◽  
Nucleic Acid Amplification ◽  
N Gene ◽  
Nucleic Acid Detection ◽  
Lamp Method ◽  
Rt Pcr ◽  
Rapid Amplification

AbstractCorona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 °C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

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.

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