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Copyright © 2025 Hadi Mirzaei et al. Canadian Journal of Infectious Diseases and Medical Microbiology published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License (the “License”), which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. https://creativecommons.org/licenses/by/4.0/

Abstract

Background and Aims: Coronavirus disease 2019 (COVID-19), an emerging life-threatening viral disease, has rapidly spread worldwide, exerting a detrimental impact on public health. We aimed to devise an innovative platform based on the loop-mediated isothermal amplification (LAMP) method, having priorities over real-time PCR (RT–PCR) in terms of sensitivity, specificity, and low running costs.

Methods: To develop a novel assay, a new primer set plus four primer sets were designed targeting the N gene of the COVID-19 agent, resulting in the sensitivity reinforcement. The limit of detection (LOD) of the developed approach was determined and compared to those of the standard RT–LAMP and RT–PCR. Two hundred confirmed positive and negative samples initially tested by RT–PCR were recruited to assess the nested–RT–LAMP assay. Furthermore, for the one-step nested–RT–LAMP assay, positive samples were tested directly without the need for RNA extraction.

Results: The LOD of nested–RT–LAMP, LAMP, and RT–PCR were 5, 15, and 15 copies/μL, respectively. The findings of the investigation illustrated 100% sensitivity and 98% specificity for both LAMP assays. Moreover, respectively, 94% and 97% sensitivity and specificity were determined regarding the one-step nested–RT–LAMP assay.

Conclusion: We offered a novel approach with more sensitivity compared to RT–PCR and common RT–LAMP, not only being a simple, accurate, cost-effective alternative diagnostic tool for RT–PCR but also being able to detect asymptomatic or mildly symptomatic patients more accurately in 2 h by naked eyes.

Details

Title
Development of a Novel Nested–RT–LAMP Assay for the Rapid and Accurate Coronavirus Disease-2019 Diagnosis
Author
Mirzaei, Hadi 1 ; Sepahi, Neda 2 ; Ghasemian, Abdolmajid 2   VIAFID ORCID Logo  ; Ranjbar, Razie 2 ; Samsami, Sahar 3 ; Mansoori, Yaser 2   VIAFID ORCID Logo  ; Chenari, Maryam 4 ; Montaseri, Zahra 5 ; Namavari, Negin 6 ; Namavari, Sahar 7 ; Ghanbariasad, Ali 7   VIAFID ORCID Logo 

 Department of Medical Genetics School of Medicine Zabol University of Medical Sciences Zabol Iran 
 Noncommunicable Diseases Research Center Fasa University of Medical Sciences Fasa Iran 
 Student Research Committee Fasa University of Medical Sciences Fasa Iran 
 Department of Virology School of Public Health Tehran University of Medical Sciences Tehran Iran 
 Department of Infectious Diseases School of Medicine Fasa University of Medical Sciences Fasa Iran 
 School of Medicine Grenada St. George’s University St. George’s Grenada 
 Department of Medical Biotechnology School of Advanced Technologies in Medicine Fasa University of Medical Sciences Fasa Iran 
Editor
Amit Singh
Publication year
2025
Publication date
2025
Publisher
John Wiley & Sons, Inc.
ISSN
17129532
e-ISSN
19181493
Source type
Scholarly Journal
Language of publication
French; English
ProQuest document ID
3189545939
Copyright
Copyright © 2025 Hadi Mirzaei et al. Canadian Journal of Infectious Diseases and Medical Microbiology published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License (the “License”), which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. https://creativecommons.org/licenses/by/4.0/