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10.3389/fmed.2020.567572

http://scihub22266oqcxt.onion/10.3389/fmed.2020.567572
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suck abstract from ncbi


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pmid33178714      Front+Med+(Lausanne) 2020 ; 7 (ä): 567572
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  • Analytical and Clinical Validation for RT-qPCR Detection of SARS-CoV-2 Without RNA Extraction #MMPMID33178714
  • Miranda JP; Osorio J; Videla M; Angel G; Camponovo R; Henriquez-Henriquez M
  • Front Med (Lausanne) 2020[]; 7 (ä): 567572 PMID33178714show ga
  • Background: The recent COVID-19 pandemic has posed an unprecedented challenge to laboratory diagnosis, based on the amplification of SARS-CoV-2 RNA. With global contagion figures exceeding 4 million persons, the shortage of reagents for RNA extraction represents a bottleneck for testing globally. We present the validation results for an RT-qPCR protocol without prior RNA extraction. Due to its simplicity, this protocol is suitable for widespread application in resource-limited settings. Methods: Optimal direct protocol was selected by comparing RT-qPCR performance under a set of thermal (65, 70, and 95 degrees for 5, 10, and 30 min) and amplification conditions (3 or 3.5 uL loading volume; 2 commercial RT-qPCR kits with a limit of detection below 10 copies/reaction) in nasopharyngeal swabs stored at 4 degrees C in sterile Weise's buffer pH 7.2. The selected protocol was evaluated for classification concordance with a standard protocol (automated RNA extraction) in 130 routine samples and 50 historical samples with Cq values near to the clinical decision limit. Results: Optimal selected conditions for direct protocol were: thermal shock at 70 degrees C for 10 min, loading 3.5 ul in the RT-qPCR. Prospective evaluation in 130 routine samples showed a 100% classification concordance with the standard protocol. The evaluation in historical samples, selected because their Cqs were at the clinical decision limit, showed 94% concordance with our confirmatory standard, which includes manual RNA extraction. Conclusions : Our results validate the use of this direct RT-qPCR protocol as a safe alternative for SARS-CoV-2 diagnosis in the case of a shortage of reagents for RNA extraction, with minimal clinical impact.
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