A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples
Introduction: the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of acute respiratory disease (COVID-19). SARS-CoV-2 is a positive-strand RNA virus and its genomic characterization has played a vital role in the design of appropriate diagnostic...
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| Format: | Article |
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MDPI AG
2021-01-01
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| Series: | Healthcare |
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| Online Access: | https://www.mdpi.com/2227-9032/9/1/37 |
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| author | Sherif A. El-Kafrawy Mai M. El-Daly Ahmed M. Hassan Reham M. Kaki Adel M. Abuzenadah Mohammad A. Kamal Esam I. Azhar |
| author_facet | Sherif A. El-Kafrawy Mai M. El-Daly Ahmed M. Hassan Reham M. Kaki Adel M. Abuzenadah Mohammad A. Kamal Esam I. Azhar |
| author_sort | Sherif A. El-Kafrawy |
| collection | DOAJ |
| description | Introduction: the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of acute respiratory disease (COVID-19). SARS-CoV-2 is a positive-strand RNA virus and its genomic characterization has played a vital role in the design of appropriate diagnostics tests. The current RT-PCR protocol for SARS-CoV-2 detects two regions of the viral genome, requiring RNA extraction and several hours. There is a need for fast, simple, and cost-effective detection strategies. Methods: we optimized a protocol for direct RT-PCR detection of SARS-CoV-2 without the need for nucleic acid extraction. Nasopharyngeal samples were diluted to 1:3 using diethyl pyrocarbonate (DEPC)-treated water. The diluted samples were incubated at 95 °C for 5 min in a thermal cycler, followed by a cooling step at 4 °C for 5 min. Samples then underwent reverse transcription real-time RT-PCR in the E and RdRp genes. Results: our direct detection protocol showed 100% concordance with the standard protocol with an average Ct value difference of 4.38 for the E region and 3.85 for the RdRp region. Conclusion: the direct PCR technique was found to be a reliable and sensitive method that can be used to reduce the time and cost of the assay by removing the need for RNA extraction. It enables the use of the assay in research, diagnostics, and screening for COVID-19 in regions with fewer economic resources, where supplies are more limited allowing for wider use for screening. |
| first_indexed | 2024-03-10T13:31:20Z |
| format | Article |
| id | doaj.art-bbe951a12ded45dd86138bd7b336b30d |
| institution | Directory Open Access Journal |
| issn | 2227-9032 |
| language | English |
| last_indexed | 2024-03-10T13:31:20Z |
| publishDate | 2021-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Healthcare |
| spelling | doaj.art-bbe951a12ded45dd86138bd7b336b30d2023-11-21T07:59:40ZengMDPI AGHealthcare2227-90322021-01-01913710.3390/healthcare9010037A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical SamplesSherif A. El-Kafrawy0Mai M. El-Daly1Ahmed M. Hassan2Reham M. Kaki3Adel M. Abuzenadah4Mohammad A. Kamal5Esam I. Azhar6Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi ArabiaSpecial Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi ArabiaSpecial Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi ArabiaDepartment of Medicine, Department of Infectious Disease and Department of Infection Control and Environmental Health King Abdulaziz University Hospital, King Abdulaziz University, Jeddah 21589, Saudi ArabiaSpecial Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi ArabiaKing Fahd Medical Research Center, King Abdulaziz University, P. O. Box 80216, Jeddah 21589, Saudi ArabiaSpecial Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi ArabiaIntroduction: the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of acute respiratory disease (COVID-19). SARS-CoV-2 is a positive-strand RNA virus and its genomic characterization has played a vital role in the design of appropriate diagnostics tests. The current RT-PCR protocol for SARS-CoV-2 detects two regions of the viral genome, requiring RNA extraction and several hours. There is a need for fast, simple, and cost-effective detection strategies. Methods: we optimized a protocol for direct RT-PCR detection of SARS-CoV-2 without the need for nucleic acid extraction. Nasopharyngeal samples were diluted to 1:3 using diethyl pyrocarbonate (DEPC)-treated water. The diluted samples were incubated at 95 °C for 5 min in a thermal cycler, followed by a cooling step at 4 °C for 5 min. Samples then underwent reverse transcription real-time RT-PCR in the E and RdRp genes. Results: our direct detection protocol showed 100% concordance with the standard protocol with an average Ct value difference of 4.38 for the E region and 3.85 for the RdRp region. Conclusion: the direct PCR technique was found to be a reliable and sensitive method that can be used to reduce the time and cost of the assay by removing the need for RNA extraction. It enables the use of the assay in research, diagnostics, and screening for COVID-19 in regions with fewer economic resources, where supplies are more limited allowing for wider use for screening.https://www.mdpi.com/2227-9032/9/1/37SARS-CoV-2direct RT-PCRCOVID-19molecular detection |
| spellingShingle | Sherif A. El-Kafrawy Mai M. El-Daly Ahmed M. Hassan Reham M. Kaki Adel M. Abuzenadah Mohammad A. Kamal Esam I. Azhar A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples Healthcare SARS-CoV-2 direct RT-PCR COVID-19 molecular detection |
| title | A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples |
| title_full | A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples |
| title_fullStr | A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples |
| title_full_unstemmed | A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples |
| title_short | A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples |
| title_sort | direct method for rt pcr detection of sars cov 2 in clinical samples |
| topic | SARS-CoV-2 direct RT-PCR COVID-19 molecular detection |
| url | https://www.mdpi.com/2227-9032/9/1/37 |
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