Open AccessAutomated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing
Author(s) -
Haya Al-Saud,
Khaldoun Al-Romaih,
Razan Bakheet,
Lina Mahmoud,
Najla Al-Harbi,
Ibtihaj Alshareef,
Sara Bin Judia,
Layla Aharbi,
Abdulaziz Alzayed,
Amjad Jabaan,
Hani A. Alhadrami,
Ahmed Albarrag,
Essam I. Azhar,
Maha Al-Mozaini
Publication year - 2020
Publication title -
annals of saudi medicine/annals of saudi medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.373
H-Index - 44
eISSN - 0975-4466
pISSN - 0256-4947
DOI - 10.5144/0256-4947.2020.373
Subject(s) - rna extraction , rna , real time polymerase chain reaction , medicine , primer (cosmetics) , virology , dna , polymerase chain reaction , serial dilution , coronavirus , reverse transcription polymerase chain reaction , reverse transcriptase , extraction (chemistry) , biology , covid-19 , gene , chromatography , messenger rna , chemistry , pathology , biochemistry , genetics , alternative medicine , disease , organic chemistry , infectious disease (medical specialty)
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.