Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing
| Autor: | Hani A. Alhadrami, Amjad Jabaan, Sara Bin Judia, Lina Mahmoud, Najla Al-Harbi, Abdulaziz Alzayed, Maha Al-Mozaini, Haya Al-Saud, Ahmed Albarrag, Khaldoun Al-Romaih, Essam I. Azhar, Layla Aharbi, Ibtihaj Alshareef, Razan Bakheet |
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| Rok vydání: | 2020 |
| Předmět: |
2019-20 coronavirus outbreak
Coronavirus disease 2019 (COVID-19) Modified dna Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Pneumonia Viral lcsh:Medicine Viral Nonstructural Proteins Viral infection Automation Betacoronavirus Coronavirus Envelope Proteins 03 medical and health sciences COVID-19 Testing 0302 clinical medicine Viral Envelope Proteins Nasopharynx Chlorocebus aethiops Animals Coronavirus Nucleocapsid Proteins Humans Medicine 030212 general & internal medicine Encephalomyocarditis virus Pandemics Vero Cells Levivirus 030304 developmental biology 0303 health sciences Coronavirus RNA-Dependent RNA Polymerase Clinical Laboratory Techniques Reverse Transcriptase Polymerase Chain Reaction SARS-CoV-2 Sequence Analysis RNA business.industry lcsh:R Extraction (chemistry) COVID-19 High-Throughput Nucleotide Sequencing General Medicine Nucleocapsid Proteins Phosphoproteins RNA-Dependent RNA Polymerase Virology Spike Glycoprotein Coronavirus RNA Viral Original Article RNA extraction Coronavirus Infections business |
| Zdroj: | Annals of Saudi Medicine, Vol 40, Iss 5, Pp 373-381 (2020) Annals of Saudi Medicine |
| ISSN: | 0975-4466 0256-4947 |
| DOI: | 10.5144/0256-4947.2020.373 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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