Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses
Abstract 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...
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| Format: | Article |
| Language: | English |
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BMC
2023-11-01
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| Series: | BMC Microbiology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12866-023-03048-9 |
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| author | Hamidreza Abbasi Hadi Razavi Nikoo Fatemeh Fotouhi Ayyoob Khosravi |
| author_facet | Hamidreza Abbasi Hadi Razavi Nikoo Fatemeh Fotouhi Ayyoob Khosravi |
| author_sort | Hamidreza Abbasi |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-03-11T11:07:29Z |
| format | Article |
| id | doaj.art-6a38249dc85e458588b4e97da0aac890 |
| institution | Directory Open Access Journal |
| issn | 1471-2180 |
| language | English |
| last_indexed | 2024-03-11T11:07:29Z |
| publishDate | 2023-11-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Microbiology |
| spelling | doaj.art-6a38249dc85e458588b4e97da0aac8902023-11-12T12:08:20ZengBMCBMC Microbiology1471-21802023-11-0123111310.1186/s12866-023-03048-9Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B virusesHamidreza Abbasi0Hadi Razavi Nikoo1Fatemeh Fotouhi2Ayyoob Khosravi3Department of Medical Biotechnology, Faculty of Advanced Medical Technologies, Golestan University of Medical SciencesDepartment of Microbiology, Faculty of Medicine, Golestan University of Medical SciencesDepartment of Influenza and other Respiratory Viruses, Pasteur Institute of IranStem Cell Research Center, Golestan University of Medical SciencesAbstract 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.https://doi.org/10.1186/s12866-023-03048-9SARS-CoV-2Influenza a virusInfluenza B virusMultiplex RT-qPCRCo-infection |
| spellingShingle | Hamidreza Abbasi Hadi Razavi Nikoo Fatemeh Fotouhi Ayyoob Khosravi Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses BMC Microbiology SARS-CoV-2 Influenza a virus Influenza B virus Multiplex RT-qPCR Co-infection |
| 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 | SARS-CoV-2 Influenza a virus Influenza B virus Multiplex RT-qPCR Co-infection |
| url | https://doi.org/10.1186/s12866-023-03048-9 |
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