Background: Sever acute respiratory syndrome Coronavirus-2 (SARS-COV-2) spread prompted mass testing. The main method for testing is by any FDA approved kits for RNA extraction followed by One-Step RT-qPCR based on primer-probe assays. Yet, the high demand for these kits created a global bottleneck in the testing capacity. Methods: We developed a Full-In-House Method (FinHM) suitable for automated viral RNA extraction using full in-house solutions utilizing the MagMaxTM beads followed by an In-House RT-qPCR based on the CDC/WHO recommended ‘primer-probe’ assay targeting the following genes; E, RdRp2, and RdRp4. FinHM was validated by an FDA approved kit that targets S, N, and ORF1b genes made by Thermo Fisher Scientific (TF). Results: The sensitivity and specificity of the automated RNA extraction were evaluated on serial dilutions of in-laboratory propagated SARS-COV-2 with a successful detection down to 46 copies in both assays (P>0.05). Moreover, automated FinHM was successful in extraction of SARS-COV-2 RNA in 266 clinical samples, in which the test results replicated the FDA approved test results (>99% similarity, P>0.05). The In-House RT-qPCR assay had low limit of detection (5 RNA templates), with significant negative correlation between the Ct values and RNA titrations as shown by Pearson correlation (-0.8, -0.8 and -0.7 for E, RdRp2 and RdRp4, respectively). Finally, FinHM was also successful in extraction of SARS-COV-2-spiked plasma and patient plasma samples. Conclusion: We report a reliable, reproducible, specific, sensitive and low-cost platform for automated RNA extraction and detection from SARS-COV-2 and other viruses which is suitable for clinical and mass testing.
| Published in | American Journal of Internal Medicine (Volume 9, Issue 1) |
| DOI | 10.11648/j.ajim.20210901.11 |
| Page(s) | 1-10 |
| Creative Commons |
This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited. |
| Copyright |
Copyright © The Author(s), 2021. Published by Science Publishing Group |
SARS-COV-2, High Throughput Testing, One Step RT-qPCR, Primer-Probe, Diagnostic Testing
| [1] | Su S, Wong G, Shi W, Liu J, Lai ACK, Zhou J, et al. Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses. Trends Microbiol. 2016; 24 (6): 490-502. |
| [2] | Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020; 382 (8): 727-33. |
| [3] | Chu DKW, Pan Y, Cheng SMS, Hui KPY, Krishnan P, Liu Y, et al. Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia. Clin Chem. 2020; 66 (4): 549-55. |
| [4] | Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro surveillance: bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin. 2020; 25 (3): 2000045. |
| [5] | Cheng MP, Papenburg J, Desjardins M, Kanjilal S, Quach C, Libman M, et al. Diagnostic Testing for Severe Acute Respiratory Syndrome-Related Coronavirus-2: A Narrative Review. Annals of internal medicine. 2020: M20-1301. |
| [6] | Wells CR, Sah P, Moghadas SM, Pandey A, Shoukat A, Wang Y, et al. Impact of international travel and border control measures on the global spread of the novel 2019 coronavirus outbreak. Proceedings of the National Academy of Sciences of the United States of America. 2020; 117 (13): 7504-9. |
| [7] | Pasteur I. COVID-19 Disease (Novel Coronavirus): Institute Pasteur 2020 [Available from: https: //www.pasteur.fr/en/medical-center/disease-sheets/covid-19-disease-novel-coronavirus. |
| [8] | Bai Y, Yao L, Wei T, Tian F, Jin D-Y, Chen L, et al. Presumed Asymptomatic Carrier Transmission of COVID-19. JAMA. 2020; 323 (14): 1406-7. |
| [9] | Dong E, Du H, Gardner L. An interactive web-based dashboard to track COVID-19 in real time. Lancet Infect Dis. 2020; 20 (5): 533-4. |
| [10] | Liu R, Han H, Liu F, Lv Z, Wu K, Liu Y, et al. Positive rate of RT-PCR detection of SARS-CoV-2 infection in 4880 cases from one hospital in Wuhan, China, from Jan to Feb 2020. Clin Chim Acta. 2020; 505: 172-5. |
| [11] | Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395 (10223): 497-506. |
| [12] | Nalla AK, Casto AM, Huang MW, Perchetti GA, Sampoleo R, Shrestha L, et al. Comparative Performance of SARS-CoV-2 Detection Assays using Seven Different Primer/Probe Sets and One Assay Kit. J Clin Microbiol. 2020. |
| [13] | Vogels CBF, Brito AF, Wyllie AL, Fauver JR, Ott IM, Kalinich CC, et al. Analytical sensitivity and efficiency comparisons of SARS-COV-2 qRT-PCR primer-probe sets. medRxiv. 2020: 2020.03.30.20048108. |
| [14] | Young BE, Ong SWX, Kalimuddin S, Low JG, Tan SY, Loh J, et al. Epidemiologic Features and Clinical Course of Patients Infected With SARS-CoV-2 in Singapore. Jama. 2020; 323 (15): 1488-94. |
| [15] | Patel R, Babady E, Theel ES, Storch GA, Pinsky BA, St George K, et al. Report from the American Society for Microbiology COVID-19 International Summit, 23 March 2020: Value of Diagnostic Testing for SARS-CoV-2/COVID-19. mBio. 2020; 11 (2). |
APA Style
Khaldoun Al-Romaih, Ibtihaj Alsharif, Razan Bakheet, Lina Mahmoud, Najla Alharbi, et al. (2021). Full-in-House Method (FinHM) for SARS-COV-2 Automated Viral RNA Extraction, Followed by in-House ‘Primer-Probe’ Based RT-qPCR Detection; Low Cost Mass Testing. American Journal of Internal Medicine, 9(1), 1-10. https://doi.org/10.11648/j.ajim.20210901.11
ACS Style
Khaldoun Al-Romaih; Ibtihaj Alsharif; Razan Bakheet; Lina Mahmoud; Najla Alharbi, et al. Full-in-House Method (FinHM) for SARS-COV-2 Automated Viral RNA Extraction, Followed by in-House ‘Primer-Probe’ Based RT-qPCR Detection; Low Cost Mass Testing. Am. J. Intern. Med. 2021, 9(1), 1-10. doi: 10.11648/j.ajim.20210901.11
AMA Style
Khaldoun Al-Romaih, Ibtihaj Alsharif, Razan Bakheet, Lina Mahmoud, Najla Alharbi, et al. Full-in-House Method (FinHM) for SARS-COV-2 Automated Viral RNA Extraction, Followed by in-House ‘Primer-Probe’ Based RT-qPCR Detection; Low Cost Mass Testing. Am J Intern Med. 2021;9(1):1-10. doi: 10.11648/j.ajim.20210901.11
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author = {Khaldoun Al-Romaih and Ibtihaj Alsharif and Razan Bakheet and Lina Mahmoud and Najla Alharbi and Sara Bin Judia and Layla Alharbi and Abdulaziz Alzayed and Amjad Jabaan and Jawahar Alotaibi and Alaiya Ayodele and Zakiya Shinwari and Hani Alhadrami and Ahmed Albarrag and Esam Azhar and Haya Al-Saud and Tahani Alrahbini and Maha Al-Mozaini},
title = {Full-in-House Method (FinHM) for SARS-COV-2 Automated Viral RNA Extraction, Followed by in-House ‘Primer-Probe’ Based RT-qPCR Detection; Low Cost Mass Testing},
journal = {American Journal of Internal Medicine},
volume = {9},
number = {1},
pages = {1-10},
doi = {10.11648/j.ajim.20210901.11},
url = {https://doi.org/10.11648/j.ajim.20210901.11},
eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajim.20210901.11},
abstract = {Background: Sever acute respiratory syndrome Coronavirus-2 (SARS-COV-2) spread prompted mass testing. The main method for testing is by any FDA approved kits for RNA extraction followed by One-Step RT-qPCR based on primer-probe assays. Yet, the high demand for these kits created a global bottleneck in the testing capacity. Methods: We developed a Full-In-House Method (FinHM) suitable for automated viral RNA extraction using full in-house solutions utilizing the MagMaxTM beads followed by an In-House RT-qPCR based on the CDC/WHO recommended ‘primer-probe’ assay targeting the following genes; E, RdRp2, and RdRp4. FinHM was validated by an FDA approved kit that targets S, N, and ORF1b genes made by Thermo Fisher Scientific (TF). Results: The sensitivity and specificity of the automated RNA extraction were evaluated on serial dilutions of in-laboratory propagated SARS-COV-2 with a successful detection down to 46 copies in both assays (P>0.05). Moreover, automated FinHM was successful in extraction of SARS-COV-2 RNA in 266 clinical samples, in which the test results replicated the FDA approved test results (>99% similarity, P>0.05). The In-House RT-qPCR assay had low limit of detection (5 RNA templates), with significant negative correlation between the Ct values and RNA titrations as shown by Pearson correlation (-0.8, -0.8 and -0.7 for E, RdRp2 and RdRp4, respectively). Finally, FinHM was also successful in extraction of SARS-COV-2-spiked plasma and patient plasma samples. Conclusion: We report a reliable, reproducible, specific, sensitive and low-cost platform for automated RNA extraction and detection from SARS-COV-2 and other viruses which is suitable for clinical and mass testing.},
year = {2021}
}
TY - JOUR T1 - Full-in-House Method (FinHM) for SARS-COV-2 Automated Viral RNA Extraction, Followed by in-House ‘Primer-Probe’ Based RT-qPCR Detection; Low Cost Mass Testing AU - Khaldoun Al-Romaih AU - Ibtihaj Alsharif AU - Razan Bakheet AU - Lina Mahmoud AU - Najla Alharbi AU - Sara Bin Judia AU - Layla Alharbi AU - Abdulaziz Alzayed AU - Amjad Jabaan AU - Jawahar Alotaibi AU - Alaiya Ayodele AU - Zakiya Shinwari AU - Hani Alhadrami AU - Ahmed Albarrag AU - Esam Azhar AU - Haya Al-Saud AU - Tahani Alrahbini AU - Maha Al-Mozaini Y1 - 2021/01/04 PY - 2021 N1 - https://doi.org/10.11648/j.ajim.20210901.11 DO - 10.11648/j.ajim.20210901.11 T2 - American Journal of Internal Medicine JF - American Journal of Internal Medicine JO - American Journal of Internal Medicine SP - 1 EP - 10 PB - Science Publishing Group SN - 2330-4324 UR - https://doi.org/10.11648/j.ajim.20210901.11 AB - Background: Sever acute respiratory syndrome Coronavirus-2 (SARS-COV-2) spread prompted mass testing. The main method for testing is by any FDA approved kits for RNA extraction followed by One-Step RT-qPCR based on primer-probe assays. Yet, the high demand for these kits created a global bottleneck in the testing capacity. Methods: We developed a Full-In-House Method (FinHM) suitable for automated viral RNA extraction using full in-house solutions utilizing the MagMaxTM beads followed by an In-House RT-qPCR based on the CDC/WHO recommended ‘primer-probe’ assay targeting the following genes; E, RdRp2, and RdRp4. FinHM was validated by an FDA approved kit that targets S, N, and ORF1b genes made by Thermo Fisher Scientific (TF). Results: The sensitivity and specificity of the automated RNA extraction were evaluated on serial dilutions of in-laboratory propagated SARS-COV-2 with a successful detection down to 46 copies in both assays (P>0.05). Moreover, automated FinHM was successful in extraction of SARS-COV-2 RNA in 266 clinical samples, in which the test results replicated the FDA approved test results (>99% similarity, P>0.05). The In-House RT-qPCR assay had low limit of detection (5 RNA templates), with significant negative correlation between the Ct values and RNA titrations as shown by Pearson correlation (-0.8, -0.8 and -0.7 for E, RdRp2 and RdRp4, respectively). Finally, FinHM was also successful in extraction of SARS-COV-2-spiked plasma and patient plasma samples. Conclusion: We report a reliable, reproducible, specific, sensitive and low-cost platform for automated RNA extraction and detection from SARS-COV-2 and other viruses which is suitable for clinical and mass testing. VL - 9 IS - 1 ER -
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