Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing

• Published in: Annals of Saudi medicine Vol. 40; no. 5; pp. 373 - 381
• Main Authors: Al-Saud, Haya, Al-Romaih, Khaldoun, Bakheet, Razan, Mahmoud, Lina, Al-Harbi, Najla, Alshareef, Ibtihaj, Judia, Sara Bin, Aharbi, Layla, Alzayed, Abdulaziz, Jabaan, Amjad, Alhadrami, Hani, Albarrag, Ahmed, Azhar, Essam I., Al-Mozaini, Maha Ahmad
• Format: Journal Article
• Language: English
• Published: Saudi Arabia King Faisal Specialist Hospital and Research Centre 01.09.2020
• Subjects: Animals; Automation; Betacoronavirus - genetics; Chlorocebus aethiops; Clinical Laboratory Techniques; Coronavirus Envelope Proteins; Coronavirus Infections - diagnosis; Coronavirus Nucleocapsid Proteins; Coronavirus RNA-Dependent RNA Polymerase; COVID-19; COVID-19 Testing; Encephalomyocarditis virus - genetics; High-Throughput Nucleotide Sequencing - methods; Humans; Levivirus - genetics; Nasopharynx - virology; Nucleocapsid Proteins - genetics; Original; Pandemics; Phosphoproteins; Pneumonia, Viral - diagnosis; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - analysis; RNA, Viral - isolation & purification; RNA-Dependent RNA Polymerase - genetics; SARS-CoV-2; Sequence Analysis, RNA; Spike Glycoprotein, Coronavirus - genetics; Vero Cells; Viral Envelope Proteins - genetics; Viral Nonstructural Proteins - genetics
• ISSN: 0256-4947; 0975-4466; 0975-4466
• DOI: 10.5144/0256-4947.2020.373

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. 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. 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). 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. 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. Small patient sample size. None.