Development and validation of an in-house, low-cost SARS-CoV-2 detection assay
Background: One major challenge for detecting the virus that causes COVID-19 is commercial SARS-CoV-2 testing kit or reagent availability. To allow every laboratory or hospital access to an in-house assay, we developed a low-cost SARS-CoV-2 detection assay protocol using in-house primers and reagent...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-09-01
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| Series: | Journal of Infection and Public Health |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1876034121001969 |
| _version_ | 1819091340544180224 |
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| author | Fatimah S. Alhamlan Ahmed A. Al-Qahtani Dana M. Bakheet Marie F. Bohol Sahar I. Althawadi Maysoon S. Mutabagani Reem S. Almaghrabi Dalia A. Obeid |
| author_facet | Fatimah S. Alhamlan Ahmed A. Al-Qahtani Dana M. Bakheet Marie F. Bohol Sahar I. Althawadi Maysoon S. Mutabagani Reem S. Almaghrabi Dalia A. Obeid |
| author_sort | Fatimah S. Alhamlan |
| collection | DOAJ |
| description | Background: One major challenge for detecting the virus that causes COVID-19 is commercial SARS-CoV-2 testing kit or reagent availability. To allow every laboratory or hospital access to an in-house assay, we developed a low-cost SARS-CoV-2 detection assay protocol using in-house primers and reagents/equipment on hand in most biology or diagnostic laboratories: a SYBR Green-based RT-PCR. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated an alternative RNA extraction protocol. Methods: We designed and synthesized in-house primers according to SARS-CoV-2 genome sequences retrieved from GISAID database. One hundred and ninety patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocol. RNA extraction was performed using TRI reagent-based RNA protocol to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory. Results: The sensitivity and specificity of the primers were evaluated using 190 patient samples previously tested for SARS-CoV-2. The positive amplicons were sequenced to confirm the results. The assay protocol was developed, and the specificity of each RT-PCR product was confirmed using melting curve analyses. Of 190 samples, the SYBR Green-based RT-PCR assay detected SARS-CoV-2 target genes in 88 samples, with no false-positive results. These findings indicate that the sensitivity of our assay was 97.7% and specificity of 100% with those of the diagnostic laboratory that tested the same samples using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit (R2 = 0.89). Conclusions: These approaches are reliable, repeatable, specific, sensitive, simple, and low-cost tools for the detection of SARS-CoV-2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or not affordable. |
| first_indexed | 2024-12-21T22:38:10Z |
| format | Article |
| id | doaj.art-9b7acde373814104aaad75b496e73b01 |
| institution | Directory Open Access Journal |
| issn | 1876-0341 |
| language | English |
| last_indexed | 2024-12-21T22:38:10Z |
| publishDate | 2021-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Infection and Public Health |
| spelling | doaj.art-9b7acde373814104aaad75b496e73b012022-12-21T18:47:55ZengElsevierJournal of Infection and Public Health1876-03412021-09-0114911391143Development and validation of an in-house, low-cost SARS-CoV-2 detection assayFatimah S. Alhamlan0Ahmed A. Al-Qahtani1Dana M. Bakheet2Marie F. Bohol3Sahar I. Althawadi4Maysoon S. Mutabagani5Reem S. Almaghrabi6Dalia A. Obeid7Department of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia; College of Medicine, Alfaisal University, Riyadh, Saudi Arabia; Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia; Corresponding author at: Department of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.Department of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia; College of Medicine, Alfaisal University, Riyadh, Saudi ArabiaCollege of Medicine, Alfaisal University, Riyadh, Saudi Arabia; Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi ArabiaDepartment of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi ArabiaDepartment of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi ArabiaDepartment of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi ArabiaDepartment of Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi ArabiaDepartment of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi ArabiaBackground: One major challenge for detecting the virus that causes COVID-19 is commercial SARS-CoV-2 testing kit or reagent availability. To allow every laboratory or hospital access to an in-house assay, we developed a low-cost SARS-CoV-2 detection assay protocol using in-house primers and reagents/equipment on hand in most biology or diagnostic laboratories: a SYBR Green-based RT-PCR. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated an alternative RNA extraction protocol. Methods: We designed and synthesized in-house primers according to SARS-CoV-2 genome sequences retrieved from GISAID database. One hundred and ninety patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocol. RNA extraction was performed using TRI reagent-based RNA protocol to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory. Results: The sensitivity and specificity of the primers were evaluated using 190 patient samples previously tested for SARS-CoV-2. The positive amplicons were sequenced to confirm the results. The assay protocol was developed, and the specificity of each RT-PCR product was confirmed using melting curve analyses. Of 190 samples, the SYBR Green-based RT-PCR assay detected SARS-CoV-2 target genes in 88 samples, with no false-positive results. These findings indicate that the sensitivity of our assay was 97.7% and specificity of 100% with those of the diagnostic laboratory that tested the same samples using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit (R2 = 0.89). Conclusions: These approaches are reliable, repeatable, specific, sensitive, simple, and low-cost tools for the detection of SARS-CoV-2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or not affordable.http://www.sciencedirect.com/science/article/pii/S1876034121001969SARS-CoV-2COVID19Diagnostic testsRT-PCR |
| spellingShingle | Fatimah S. Alhamlan Ahmed A. Al-Qahtani Dana M. Bakheet Marie F. Bohol Sahar I. Althawadi Maysoon S. Mutabagani Reem S. Almaghrabi Dalia A. Obeid Development and validation of an in-house, low-cost SARS-CoV-2 detection assay Journal of Infection and Public Health SARS-CoV-2 COVID19 Diagnostic tests RT-PCR |
| title | Development and validation of an in-house, low-cost SARS-CoV-2 detection assay |
| title_full | Development and validation of an in-house, low-cost SARS-CoV-2 detection assay |
| title_fullStr | Development and validation of an in-house, low-cost SARS-CoV-2 detection assay |
| title_full_unstemmed | Development and validation of an in-house, low-cost SARS-CoV-2 detection assay |
| title_short | Development and validation of an in-house, low-cost SARS-CoV-2 detection assay |
| title_sort | development and validation of an in house low cost sars cov 2 detection assay |
| topic | SARS-CoV-2 COVID19 Diagnostic tests RT-PCR |
| url | http://www.sciencedirect.com/science/article/pii/S1876034121001969 |
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