tetano
Editor, Senior Moderator
Ann Saudi Med
. 2020 Sep 20;373-381.
doi: 10.5144/0256-4947.2020.373. Online ahead of print.
Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing
Haya Al-Saud[SUP] 1 [/SUP], Khaldoun Al-Romaih[SUP] 2 [/SUP], Razan Bakheet[SUP] 2 [/SUP], Lina Mahmoud[SUP] 2 [/SUP], Najla Al-Harbi[SUP] 3 [/SUP], Ibtihaj Alshareef[SUP] 2 [/SUP], Sara Bin Judia[SUP] 2 [/SUP], Layla Aharbi[SUP] 2 [/SUP], Abdulaziz Alzayed[SUP] 1 [/SUP], Amjad Jabaan[SUP] 1 [/SUP], Hani Alhadrami[SUP] 4 [/SUP], Ahmed Albarrag[SUP] 5 [/SUP], Essam I Azhar[SUP] 4 [/SUP], Maha Ahmad Al-Mozaini[SUP] 6 [/SUP]
Affiliations
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.
. 2020 Sep 20;373-381.
doi: 10.5144/0256-4947.2020.373. Online ahead of print.
Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing
Haya Al-Saud[SUP] 1 [/SUP], Khaldoun Al-Romaih[SUP] 2 [/SUP], Razan Bakheet[SUP] 2 [/SUP], Lina Mahmoud[SUP] 2 [/SUP], Najla Al-Harbi[SUP] 3 [/SUP], Ibtihaj Alshareef[SUP] 2 [/SUP], Sara Bin Judia[SUP] 2 [/SUP], Layla Aharbi[SUP] 2 [/SUP], Abdulaziz Alzayed[SUP] 1 [/SUP], Amjad Jabaan[SUP] 1 [/SUP], Hani Alhadrami[SUP] 4 [/SUP], Ahmed Albarrag[SUP] 5 [/SUP], Essam I Azhar[SUP] 4 [/SUP], Maha Ahmad Al-Mozaini[SUP] 6 [/SUP]
Affiliations
- PMID: 32954791
- DOI: 10.5144/0256-4947.2020.373
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.