scholarly journals Colorimetric Detection of Dengue by Single Tube Reverse-Transcription-Loop-Mediated Isothermal Amplification

PLoS ONE ◽  
2015 ◽  
Vol 10 (9) ◽  
pp. e0138694 ◽  
Author(s):  
Yee-Ling Lau ◽  
Meng-Yee Lai ◽  
Boon-Teong Teoh ◽  
Juraina Abd-Jamil ◽  
Jefree Johari ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Colorimetric Detection ◽  
Isothermal Amplification ◽  
Single Tube ◽  
Loop Mediated Isothermal Amplification

scholarly journals Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Bruna de Oliveira Coelho ◽  
Heloisa Bruna Soligo Sanchuki ◽  
Dalila Luciola Zanette ◽  
Jeanine Marie Nardin ◽  
Hugo Manuel Paz Morales ◽  
...  
Keyword(s):  
Viral Load ◽  
Internal Control ◽  
Reverse Transcription ◽  
Color Change ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
False Negative ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

scholarly journals Validation of a single-step, single-tube reverse transcription-loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA

2020 ◽  
Author(s):  
Jean Y. H. Lee ◽  
Nickala Best ◽  
Julie McAuley ◽  
Jessica L. Porter ◽  
Torsten Seemann ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Rapid Detection ◽  
Single Step ◽  
Isothermal Amplification ◽  
N Gene ◽  
Assay Sensitivity ◽  
Single Tube ◽  
Clinical Specimens ◽  
Loop Mediated Isothermal Amplification

2.AbstractIntroductionThe SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.AimTo establish and validate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.MethodologyWe used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby as little as 1 μL of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65°C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals.ResultsAssay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 minutes (SD ±7 minutes). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 mL−1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.ConclusionWith a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.3.Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

scholarly journals Development of Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assays Targeting SARS-CoV-2

2020 ◽  
Author(s):  
Gun-Soo Park ◽  
Keunbon Ku ◽  
Seung-Hwa Baek ◽  
Seong Jun Kim ◽  
Seung Il Kim ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Detection Method ◽  
Colorimetric Detection ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Genomic Rna ◽  
Loop Mediated Isothermal Amplification ◽  
Human Coronaviruses ◽  
High Level

AbstractEpidemics of Coronavirus Disease 2019 (COVID-19) now have more than 100,000 confirmed cases worldwide. Diagnosis of COVID-19 is currently performed by RT-qPCR methods, but the capacity of RT-qPCR methods is limited by its requirement of high-level facilities and instruments. Here, we developed and evaluated RT-LAMP assays to detect genomic RNA of SARS-CoV-2, the causative virus of COVID-19. RT-LAMP assays in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human Coronaviruses was not observed. We also adapted a colorimetric detection method for our RT-LAMP assay so that the tests potentially performed in higher throughput.

scholarly journals Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis

2021 ◽  
Vol 22 (11) ◽  
pp. 5743
Author(s):  
Igor P. Oscorbin ◽  
Georgiy Yu. Shevelev ◽  
Ksenia A. Pronyaeva ◽  
Andrey A. Stepanov ◽  
Darya V. Shamovskaya ◽  
...  
Keyword(s):  
Internal Control ◽  
Reverse Transcription ◽  
Control Sample ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Single Tube ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Process

Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including an internal control sample, which provides the option of controlling all analytical steps. We developed a method of SARS-CoV-2 viral RNA detection based on multiplex reverse-transcription LAMP, with single-tube qualitative analysis of SARS-CoV-2 RNA and MS2 phage used as a control RNA. The multiplexing is based on the differences in characteristic melting peaks generated during the amplification process. The developed technique detects at least 20 copies of SARS-CoV-2 RNA per reaction on a background of 12,000 MS2 RNA copies. The total time of analysis does not exceed 40 min. The method validation, performed on 125 clinical samples of patients’ nasal swabs, showed a 97.6% concordance rate with the results of real-time (RT)-PCR assays. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time.

scholarly journals Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA

2020 ◽  
Vol 69 (9) ◽  
pp. 1169-1178 ◽  
Author(s):  
Jean Y. H. Lee ◽  
Nickala Best ◽  
Julie McAuley ◽  
Jessica L. Porter ◽  
Torsten Seemann ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Single Step ◽  
Isothermal Amplification ◽  
Assay Sensitivity ◽  
Single Tube ◽  
Clinical Specimens ◽  
Loop Mediated Isothermal Amplification

Introduction. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. Aim. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. Methodology. We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals. Results. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min (sd±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 ml−1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay. Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.

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