PropertyValue
?:abstract
  • 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° 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°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°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.
is ?:annotates of
?:creator
?:doi
?:doi
  • 10.3389/fmed.2020.567572
?:journal
  • Front_Med_(Lausanne)
?:license
  • cc-by
?:pdf_json_files
  • document_parses/pdf_json/9570f32ee105c5dbd441f3845a82be29752bdc34.json
?:pmc_json_files
  • document_parses/pmc_json/PMC7593567.xml.json
?:pmcid
?:pmid
?:pmid
  • 33178714.0
?:publication_isRelatedTo_Disease
?:sha_id
?:source
  • Medline; PMC
?:title
  • Analytical and Clinical Validation for RT-qPCR Detection of SARS-CoV-2 Without RNA Extraction
?:type
?:year
  • 2020-10-15

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