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Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses
BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Influenza A, and Influenza B viruses is essential for rapid differential diagnosis in patients with similar symptoms, especially during...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10640757/ https://www.ncbi.nlm.nih.gov/pubmed/37951883 http://dx.doi.org/10.1186/s12866-023-03048-9 |
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author | Abbasi, Hamidreza Nikoo, Hadi Razavi Fotouhi, Fatemeh Khosravi, Ayyoob |
author_facet | Abbasi, Hamidreza Nikoo, Hadi Razavi Fotouhi, Fatemeh Khosravi, Ayyoob |
author_sort | Abbasi, Hamidreza |
collection | PubMed |
description | BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Influenza A, and Influenza B viruses is essential for rapid differential diagnosis in patients with similar symptoms, especially during “flu season” in the post-pandemic era. So far, several multiplex methods have been approved for the simultaneous detection of SARS-CoV-2, Influenza A, and Influenza B. However, due to the rapid mutation rate of the SARS-CoV-2 genome and the emergence of new variants, existing methods must be improved and updated. METHODS: To identify a highly conserved region in the SARS-CoV-2 N-gene, a genomic survey was performed to increase the sensitivity and specificity of primer and probe sets targeting the SARS-CoV-2 genome. The 95% LLOD (95% lower limits of detection) were calculated by probit analysis. A total of 70 predetermined clinical samples using singleplex RT-qPCR assays, were included. The clinical performance of the multiplex RT-qPCR assay was determined and compared with a commercial multiplex kit. The Cohen’s kappa coefficient, P-value (McNemar’s test), Passing-Bablok regression, and Bland Altman agreement analysis were determined to monitor the agreement of the assays. RESULTS: The novel SARS-CoV-2 primer and probe set designed in this assay was able to detect all variants of concern (VOCs) and variants of interest (VOIs) with high analytical and clinical performance. The 95% LLOD for the multiplex RT-qPCR was 20 copies per reaction for the N gene of SARS-CoV-2, 2 copies per reaction for M1 gene of Influenza A and NS1 gene of Influenza B. The diagnostic sensitivity of the multiplex RT-qPCR was 94.4%, 93.7%, and 100% for the detection of SARS-CoV-2, Influenza A, and Influenza B genomes, respectively. Moreover, the specificity was identical (100%) in both assays. According to the agreement analysis results, there was no statistical difference between our multiplex assay and the commercial kit. CONCLUSIONS: In this study, we developed a novel in-house made multiplex RT-qPCR assay, with high sensitivity, specificity, and reliability for the diagnosis of SARS-CoV-2 infection in clinical samples. This is valuable during Influenza seasons when influenza co-circulates with SARS-CoV-2, as it saves costs, time, and thus specific and timely treatment of patients. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12866-023-03048-9. |
format | Online Article Text |
id | pubmed-10640757 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-106407572023-11-11 Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses Abbasi, Hamidreza Nikoo, Hadi Razavi Fotouhi, Fatemeh Khosravi, Ayyoob BMC Microbiol Research BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Influenza A, and Influenza B viruses is essential for rapid differential diagnosis in patients with similar symptoms, especially during “flu season” in the post-pandemic era. So far, several multiplex methods have been approved for the simultaneous detection of SARS-CoV-2, Influenza A, and Influenza B. However, due to the rapid mutation rate of the SARS-CoV-2 genome and the emergence of new variants, existing methods must be improved and updated. METHODS: To identify a highly conserved region in the SARS-CoV-2 N-gene, a genomic survey was performed to increase the sensitivity and specificity of primer and probe sets targeting the SARS-CoV-2 genome. The 95% LLOD (95% lower limits of detection) were calculated by probit analysis. A total of 70 predetermined clinical samples using singleplex RT-qPCR assays, were included. The clinical performance of the multiplex RT-qPCR assay was determined and compared with a commercial multiplex kit. The Cohen’s kappa coefficient, P-value (McNemar’s test), Passing-Bablok regression, and Bland Altman agreement analysis were determined to monitor the agreement of the assays. RESULTS: The novel SARS-CoV-2 primer and probe set designed in this assay was able to detect all variants of concern (VOCs) and variants of interest (VOIs) with high analytical and clinical performance. The 95% LLOD for the multiplex RT-qPCR was 20 copies per reaction for the N gene of SARS-CoV-2, 2 copies per reaction for M1 gene of Influenza A and NS1 gene of Influenza B. The diagnostic sensitivity of the multiplex RT-qPCR was 94.4%, 93.7%, and 100% for the detection of SARS-CoV-2, Influenza A, and Influenza B genomes, respectively. Moreover, the specificity was identical (100%) in both assays. According to the agreement analysis results, there was no statistical difference between our multiplex assay and the commercial kit. CONCLUSIONS: In this study, we developed a novel in-house made multiplex RT-qPCR assay, with high sensitivity, specificity, and reliability for the diagnosis of SARS-CoV-2 infection in clinical samples. This is valuable during Influenza seasons when influenza co-circulates with SARS-CoV-2, as it saves costs, time, and thus specific and timely treatment of patients. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12866-023-03048-9. BioMed Central 2023-11-11 /pmc/articles/PMC10640757/ /pubmed/37951883 http://dx.doi.org/10.1186/s12866-023-03048-9 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Abbasi, Hamidreza Nikoo, Hadi Razavi Fotouhi, Fatemeh Khosravi, Ayyoob Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses |
title | Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses |
title_full | Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses |
title_fullStr | Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses |
title_full_unstemmed | Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses |
title_short | Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses |
title_sort | development of a robust taqman probe-based one-step multiplex rt-qpcr for simultaneous detection of sars-cov-2 and influenza a/b viruses |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10640757/ https://www.ncbi.nlm.nih.gov/pubmed/37951883 http://dx.doi.org/10.1186/s12866-023-03048-9 |
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