scholarly journals Diagnosis of SARS-CoV-2 infection with LamPORE, a high-throughput platform combining loop-mediated isothermal amplification and nanopore sequencing

2021 ◽  
Author(s):  
Leon Peto ◽  
Gillian Rodger ◽  
Daniel P Carter ◽  
Karen L Osman ◽  
Mehmet Yavuz ◽  
...  
Keyword(s):  
High Throughput ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Nanopore Sequencing ◽  
Rt Pcr ◽  
Symptomatic Infection ◽  
Clinical Specimens ◽  
Loop Mediated Isothermal Amplification

Background LamPORE is a novel diagnostic platform for the detection of SARS-CoV-2 RNA combining loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyse thousands of samples per day on a single instrument. Methods We evaluated the performance of LamPORE against RT-PCR using RNA extracted from spiked respiratory samples and stored nose and throat swabs collected at two UK hospitals. Findings The limit of detection of LamPORE was ten genome copies/μl of extracted RNA, which is above the limit achievable by RT-PCR but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among these LamPORE had a diagnostic sensitivity of 99.1% (226/228 [95% CI 96.9–99.9%]). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279 [98.0–100.0%]). Overall, 1.4% (7/514 [0.5–2.9%]) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494 [94.8–98.1%]). Interpretation LamPORE has a similar performance to RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients, and offers a promising approach to high-throughput testing.

scholarly journals Diagnosis of SARS-CoV-2 infection with LamPORE, a high-throughput platform combining loop-mediated isothermal amplification and nanopore sequencing

2020 ◽  
Author(s):  
Leon Peto ◽  
Gillian Rodger ◽  
Daniel P Carter ◽  
Karen L Osman ◽  
Mehmet Yavuz ◽  
...  
Keyword(s):  
High Throughput ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Nanopore Sequencing ◽  
Rt Pcr ◽  
Symptomatic Infection ◽  
Clinical Specimens ◽  
Loop Mediated Isothermal Amplification

LamPORE is a novel diagnostic platform for the detection of SARS-CoV-2 RNA that combines loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyse thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against RT-PCR using RNA extracted from spiked respiratory samples and from stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 7-10 genome copies/microlitre of extracted RNA. This is above the limit achievable by RT-PCR but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among these LamPORE had a diagnostic sensitivity of 99.1% (226/228 [95% CI 96.9-99.9%]). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279 [98.0-100.0%]). Overall, 1.4% (7/514 [0.5-2.9]) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494 [94.8-98.1]). This indicates that LamPORE has a similar performance to RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients, and offers a promising approach to high-throughput testing.

scholarly journals A quite sensitive fluorescent loop-mediated isothermal amplification for rapid detection of respiratory syncytial virus

2019 ◽  
Vol 13 (12) ◽  
pp. 1135-1141 ◽  
Author(s):  
Yihong Hu ◽  
Zhenzhou Wan ◽  
Yonglin Mu ◽  
Yi Zhou ◽  
Jia Liu ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
High Morbidity ◽  
Rt Pcr ◽  
Loop Mediated Isothermal Amplification ◽  
Syncytial Virus ◽  
Syto 9

Introduction: Human respiratory syncytial virus (hRSV) is a common respiratory virus closely related to respiratory tract infection (RTI). Rapid and accurate detection of hRSV is urgently needed to reduce the high morbidity and mortality due to hRSV infection. Methodology: Here, we established a highly sensitive and specific reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of A and B group hRSV simultaneously. The specific primer sets for hRSV A and B groups were designed in the M and M2-2 gene, respectively. SYTO 9 was used as the fluorescent dye for real-time monitoring of the amplification of hRSV RNA without cross reaction between hRSV A and B. Results: The limit of detection (LOD) of our new method was 281.17 50% tissue culture infective doses (TCID50)/mL for hRSV A and 1.58 TCID50/mL for hRSV B. Using 90 clinical samples, a comparison to traditional RT-PCR was performed to validate this assay. The positivity rate of RT-LAMP and RT-PCR were 67.8% and 55.6%, respectively, and the positivity rate of RT-LAMP was significantly higher than RT-PCR (χ2 test, P < 0.01). Conclusions: Compared with traditional RT-PCR method, the newly developed fluorescent RT-LAMP combined with well-designed primers and SYTO 9 is quite sensitive, specific, rapid and well applicable to hRSV clinical diagnosis.

scholarly journals A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jonas Schmidt ◽  
Sandro Berghaus ◽  
Frithjof Blessing ◽  
Folker Wenzel ◽  
Holger Herbeck ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Mass Screening ◽  
High Throughput ◽  
Clinical Laboratory ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Rt Pcr ◽  
Loop Mediated Isothermal Amplification ◽  
Clinical Diagnostic

AbstractShortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen’s kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar’s test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.

scholarly journals SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

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 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 Reverse Transcription Loop-Mediated Isothermal Amplification for the Detection of Porcine Reproductive and Respiratory Syndrome Virus

2009 ◽  
Vol 21 (3) ◽  
pp. 350-354 ◽  
Author(s):  
Albert Rovira ◽  
Juan Abrahante ◽  
Michael Murtaugh ◽  
Muñoz-Zanzi Claudia
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Restriction Analysis ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Serum Samples ◽  
Loop Mediated Isothermal Amplification

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine. The objective of the current study is to investigate the feasibility of using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of PRRSV. The RT-LAMP is a recently described DNA amplification technique reported to be simple, inexpensive, fast, and accurate. The RT-LAMP reaction was set up using 2 sets of primers that were designed to detect North American and European strains of PRRSV and performed successfully in a simple heat block. The specificity of the amplified product was demonstrated by restriction analysis. The RT-LAMP was able to detect 5 different PRRSV isolates. However, the limit of detection ranged between 10 2 and 10 4 50% tissue culture infective dose/ml. The RT-LAMP was further evaluated using serum samples from animals of known infection status. The ability of RT-LAMP to detect PRRSV in serum from acutely infected animals was evaluated with 114 serum samples from 18 experimentally inoculated boars. Forty-nine of these samples tested positive by RT-LAMP, while 94 were positive by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic specificity, evaluated with 100 known negative serum samples, was estimated as 99%. The feasibility of RT-LAMP to detect PRRSV was demonstrated in the current study. The RT-LAMP reaction could be performed in just 1 hr with a simple and inexpensive heat block. However, the sensitivity of this technique was significantly lower than that of RT-PCR.

scholarly journals A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

2020 ◽  
Vol 21 (8) ◽  
pp. 2826 ◽  
Author(s):  
Renfei Lu ◽  
Xiuming Wu ◽  
Zhenzhou Wan ◽  
Yingxue Li ◽  
Xia Jin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
N Gene ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

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.

scholarly journals Evaluation of RT-qPCR and Loop-Mediated Isothermal Amplification (LAMP) Assays for the Detection of SARS-CoV-2 in Argentina

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 659
Author(s):  
María Dolores Fellner ◽  
Romina Bonaventura ◽  
Jorge Basiletti ◽  
Martín Avaro ◽  
Estefanía Benedetti ◽  
...  
Keyword(s):  
World Health Organization ◽  
Reverse Transcription ◽  
Diagnostic Performance ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Isothermal Amplification ◽  
World Health ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Health Organization

Our aim was to evaluate the analytical and clinical performance of the SARS-CoV-2 molecular detection kits used in Argentina. Nine real-time reverse-transcription polymerase chain reaction (RT-qPCR) and three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were evaluated using the World Health Organization (WHO) recommended test as reference method. A secondary standard calibrated for the E, N and RdRp genes against the Pan American Health Organization—World Health Organization—International Standard was used to calculate the limit of detection (LoD). A panel of artificial clinical samples, 32 positive and 30 negative for SARS-CoV-2, were analyzed to estimate the kappa concordance (κ) and the diagnostic performance. Differences among the LoD values for the target genes amplified by each kit were >1 log copies/reaction. The κ for the RT-qPCR kits was greater than 0.9, whereas that for the RT-LAMP assays ranged from 0.75 to 0.93. The clinical performance of RT-qPCR kits showed 100% specificity and high sensitivity, although with variations according to the gene analyzed. The E and N genes provided greater clinical sensitivity, whereas the RdRp gene increased the clinical specificity. The RT-LAMP assays revealed a variable diagnostic performance. The information provided can be useful to choose the most appropriate diagnostic test and may contribute to the establishment of a consensus in the diagnosis of SARS-CoV-2 in Argentina and the region.

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