مورد إلكتروني
Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA.
| العنوان: | Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA. |
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| بيانات النشر: | Microbiology Society United Kingdom 2020-11-16 |
| تفاصيل مُضافة: | Mercoulia K. Luttick A. McDonald S. Greenhalgh A. Kwong J.C. Sherry N.L. Graham M. Hoang T. Herisse M. Pidot S.J. Williamson D.A. Howden B.P. Monk I.R. Stinear T.P. Lee J.Y.H. Best N. McAuley J. Porter J.L. Seemann T. Schultz M.B. Sait M. Orlando N. Ballard S.A. Druce J. Tran T. Catton M.G. Pryor M.J. Cui H.L. |
| نوع الوثيقة: | Electronic Resource |
| مستخلص: | 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 microl 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 degreeC 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 r |
| مصطلحات الفهرس: | OptiGene, article, comparative study, controlled study, coronavirus disease 2019/di [Diagnosis], diagnostic test accuracy study, dilution, fluorescence analysis, human, infectious dose, limit of detection, nonhuman, priority journal, real time reverse transcription polymerase chain reaction, reverse transcription loop mediated isothermal amplification, RNA extraction, RNA purification, sensitivity and specificity, Severe acute respiratory syndrome coronavirus 2, throat culture, tissue culture, validation study, virus detection, virus spike, nucleocapsid protein/ec [Endogenous Compound], virus RNA/ec [Endogenous Compound], RT-LAMP system, transport medium, Article |
| URL: | Journal of Medical Microbiology LibKey Link |
| الإتاحة: | Open access content. Open access content Copyright 2020 Elsevier B.V., All rights reserved. |
| أرقام أخرى: | AUSHL oai:repository.monashhealth.org:1/28956 Journal of Medical Microbiology. 69 (9) (pp 1169-1178), 2020. Date of Publication: 2020. 0022-2615 https://repository.monashhealth.org/monashhealthjspui/handle/1/28956Test 32755529 [http://www.ncbi.nlm.nih.gov/pubmed/?term=32755529Test] 2008018621 (Lee, McAuley, Porter, Seemann, Kwong, Hoang, Herisse, Pidot, Williamson, Howden, Monk, Stinear) Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia (Lee) Department of Infectious Diseases, Monash Health, Clayton, VIC, Australia (Best, McDonald, Greenhalgh) GenWorks Pty Ltd, Thebarton, SA, Australia (Seemann, Schultz, Sait, Orlando, Mercoulia, Ballard, Sherry, Hoang, Williamson, Howden) Microbiological Diagnostic Unit Public Health Laboratory, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia (Druce, Tran, Catton) Victorian Infectious Diseases Reference Laboratory, Melbourne Health at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia (Pryor, Cui, Luttick) 360Biolabs, Melbourne, VIC, Australia (Kwong, Sherry, Howden) Department of Infectious Diseases, Austin Health, Heidelberg, VIC, Australia (Graham) Department of Microbiology, Monash Health, Clayton, VIC, Australia (Williamson) Melbourne Health, Melbourne, VIC, Australia Stinear T.P.; tstinear@unimelb.edu.au 1305115890 |
| المصدر المساهم: | MONASH HEALTH LIBRS From OAIster®, provided by the OCLC Cooperative. |
| رقم الانضمام: | edsoai.on1305115890 |
| قاعدة البيانات: | OAIster |
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