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10.5144/0256-4947.2020.373

http://scihub22266oqcxt.onion/10.5144/0256-4947.2020.373
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suck abstract from ncbi


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pmid32954791      Ann+Saudi+Med 2020 ; 40 (5): 373-381
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  • Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing #MMPMID32954791
  • Al-Saud H; Al-Romaih K; Bakheet R; Mahmoud L; Al-Harbi N; Alshareef I; Judia SB; Aharbi L; Alzayed A; Jabaan A; Alhadrami H; Albarrag A; Azhar EI; Al-Mozaini MA
  • Ann Saudi Med 2020[Sep]; 40 (5): 373-381 PMID32954791show ga
  • 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.
  • |Animals[MESH]
  • |Automation[MESH]
  • |Betacoronavirus/*genetics[MESH]
  • |COVID-19[MESH]
  • |COVID-19 Testing[MESH]
  • |Chlorocebus aethiops[MESH]
  • |Clinical Laboratory Techniques[MESH]
  • |Coronavirus Envelope Proteins[MESH]
  • |Coronavirus Infections/*diagnosis[MESH]
  • |Coronavirus Nucleocapsid Proteins[MESH]
  • |Coronavirus RNA-Dependent RNA Polymerase[MESH]
  • |Encephalomyocarditis virus/genetics[MESH]
  • |High-Throughput Nucleotide Sequencing/*methods[MESH]
  • |Humans[MESH]
  • |Levivirus/genetics[MESH]
  • |Nasopharynx/*virology[MESH]
  • |Nucleocapsid Proteins/genetics[MESH]
  • |Pandemics[MESH]
  • |Phosphoproteins[MESH]
  • |Pneumonia, Viral/*diagnosis[MESH]
  • |RNA, Viral/analysis/*isolation & purification[MESH]
  • |RNA-Dependent RNA Polymerase/genetics[MESH]
  • |Reverse Transcriptase Polymerase Chain Reaction/*methods[MESH]
  • |SARS-CoV-2[MESH]
  • |Sequence Analysis, RNA[MESH]
  • |Spike Glycoprotein, Coronavirus/genetics[MESH]
  • |Vero Cells[MESH]
  • |Viral Envelope Proteins/genetics[MESH]


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