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OBJECTIVE: To develop a novel quadruplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, differential diagnosis and detection of co-infections. METHODS: A one-step quadruplex rRT-PCR assay was developed for simultaneous detection and differentiation of SARS-CoV-2 ORF1ab and N genes, influenza A virus (hIAV) and influenza B virus (hIBV). RESULTS: The quadruplex rRT-PCR assay had good sensitivity and specificity. Correlation coefficients and amplification efficiencies of all singleplex and quadruplex rRT-PCR reactions were within acceptable ranges. The 95% lower limits of detection for plasmid standards and positive nucleic acid extracts of the quadruplex rRT-PCR assay were 57.38–95.11 copies/μL and 114.65–154.25 copies/μL, respectively. Excellent results were attained for inter- and intra-assay reproducibility. Among these clinical samples, only four samples showed results inconsistent with the singleplex rRT-PCR assays. CONCLUSIONS: To the authors’ knowledge, this is the first study to report a quadruplex rRT-PCR assay for the detection of two SARS-CoV-2 genes, hIAV and hIBV with perfect clinical performance.
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10.1016/j.ijid.2020.12.027
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document_parses/pdf_json/10b26fa11f4ce552a7c071d983e2c2b3912b6990.json
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document_parses/pmc_json/PMC7836965.xml.json
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Simultaneous detection and differentiation of SARS-CoV-2, influenza A virus and influenza B virus by one-step quadruplex real-time RT-PCR in patients with clinical manifestations
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