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Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing.
Ann Saudi Med. 2020 Sep-Oct; 40(5):373-381.AS

Abstract

BACKGROUND

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has prompted a need for mass testing to identify patients with viral infection. The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2.

OBJECTIVES

Report on the use of a DNA extraction kit, after modifications, to extract viral RNA that could then be detected using an FDA-approved SARS-COV-2 RT-qPCR assay.

MATERIALS AND METHODS

Initially, automated RNA extraction was performed using a modified DNA kit on samples from control subjects, a bacteriophage, and an RNA virus. We then verified the automated extraction using the modified kit to detect in-lab propagated SARSCOV-2 titrations using an FDA approved commercial kit (S, N, and ORF1b genes) and an in-house primer-probe based assay (E, RdRp2 and RdRp4 genes).

RESULTS

Automated RNA extraction on serial dilutions SARS-COV-2 achieved successful one-step RT-qPCR detection down to 60 copies using the commercial kit assay and less than 30 copies using the in-house primer-probe assay. Moreover, RT-qPCR detection was successful after automated RNA extraction using this modified protocol on 12 patient samples of SARS-COV-2 collected by nasopharyngeal swabs and stored in viral transport media.

CONCLUSIONS

We demonstrated the capacity of a modified DNA extraction kit for automated viral RNA extraction and detection using a platform that is suitable for mass testing.

LIMITATIONS

Small patient sample size.

CONFLICT OF INTEREST

None.

Authors+Show Affiliations

From the Saudi Human Genome Project, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia.From the National Centre of Genomic Technology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.From the National Centre of Genomic Technology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.From the National Centre of Genomic Technology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.From the Biomedical Physics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.From the National Centre of Genomic Technology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.From the National Centre of Genomic Technology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.From the National Centre of Genomic Technology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.From the Saudi Human Genome Project, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia.From the Saudi Human Genome Project, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia.From the Department of of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia.From the Center of Disease Control, Saudi Arabia Ministry of Health, Riyadh, Saudi Arabia.From the Department of of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia.From the Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32954791

Citation

Al-Saud, Haya, et al. "Automated SARS-COV-2 RNA Extraction From Patient Nasopharyngeal Samples Using a Modified DNA Extraction Kit for High Throughput Testing." Annals of Saudi Medicine, vol. 40, no. 5, 2020, pp. 373-381.
Al-Saud H, Al-Romaih K, Bakheet R, et al. Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing. Ann Saudi Med. 2020;40(5):373-381.
Al-Saud, H., Al-Romaih, K., Bakheet, R., Mahmoud, L., Al-Harbi, N., Alshareef, I., Judia, S. B., Aharbi, L., Alzayed, A., Jabaan, A., Alhadrami, H., Albarrag, A., Azhar, E. I., & Al-Mozaini, M. A. (2020). Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing. Annals of Saudi Medicine, 40(5), 373-381. https://doi.org/10.5144/0256-4947.2020.373
Al-Saud H, et al. Automated SARS-COV-2 RNA Extraction From Patient Nasopharyngeal Samples Using a Modified DNA Extraction Kit for High Throughput Testing. Ann Saudi Med. 2020 Sep-Oct;40(5):373-381. PubMed PMID: 32954791.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing. AU - Al-Saud,Haya, AU - Al-Romaih,Khaldoun, AU - Bakheet,Razan, AU - Mahmoud,Lina, AU - Al-Harbi,Najla, AU - Alshareef,Ibtihaj, AU - Judia,Sara Bin, AU - Aharbi,Layla, AU - Alzayed,Abdulaziz, AU - Jabaan,Amjad, AU - Alhadrami,Hani, AU - Albarrag,Ahmed, AU - Azhar,Essam I, AU - Al-Mozaini,Maha Ahmad, Y1 - 2020/10/01/ PY - 2020/9/22/pubmed PY - 2020/10/10/medline PY - 2020/9/21/entrez SP - 373 EP - 381 JF - Annals of Saudi medicine JO - Ann Saudi Med VL - 40 IS - 5 N2 - BACKGROUND: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has prompted a need for mass testing to identify patients with viral infection. The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2. OBJECTIVES: Report on the use of a DNA extraction kit, after modifications, to extract viral RNA that could then be detected using an FDA-approved SARS-COV-2 RT-qPCR assay. MATERIALS AND METHODS: Initially, automated RNA extraction was performed using a modified DNA kit on samples from control subjects, a bacteriophage, and an RNA virus. We then verified the automated extraction using the modified kit to detect in-lab propagated SARSCOV-2 titrations using an FDA approved commercial kit (S, N, and ORF1b genes) and an in-house primer-probe based assay (E, RdRp2 and RdRp4 genes). RESULTS: Automated RNA extraction on serial dilutions SARS-COV-2 achieved successful one-step RT-qPCR detection down to 60 copies using the commercial kit assay and less than 30 copies using the in-house primer-probe assay. Moreover, RT-qPCR detection was successful after automated RNA extraction using this modified protocol on 12 patient samples of SARS-COV-2 collected by nasopharyngeal swabs and stored in viral transport media. CONCLUSIONS: We demonstrated the capacity of a modified DNA extraction kit for automated viral RNA extraction and detection using a platform that is suitable for mass testing. LIMITATIONS: Small patient sample size. CONFLICT OF INTEREST: None. SN - 0975-4466 UR - https://www.unboundmedicine.com/medline/citation/32954791/Automated_SARS_COV_2_RNA_extraction_from_patient_nasopharyngeal_samples_using_a_modified_DNA_extraction_kit_for_high_throughput_testing_ L2 - https://www.annsaudimed.net/doi/10.5144/0256-4947.2020.373?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -