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Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: RT-ddPCR showed higher sensitivity in exposed individuals and allowed the detection of sequence variability. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: DISCUSSION Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: MATERIALS AND METHODS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Children: Item: Title: Human samples. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Mouse samples. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: RNA extraction. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: SARS-CoV-2 RT-qPCR detection and viral load quantification. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Item: Title: SARS-CoV-2 RT-ddPCR quantification. 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Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@491cc5c9 Item: Title: SUPPLEMENTAL MATERIAL Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@74ad1f1f Item: Title: ACKNOWLEDGMENTS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6a1aab78 Item: Title: REFERENCES Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@462d5aee Info: Title: Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA Author: Sara Morón-López;Eva Riveira-Muñoz;Victor Urrea;Lucia Gutiérrez-Chamorro;Carlos Ávila-Nieto;Marc Noguera-Julian;Jorge Carrillo;Oriol Mitjà;Lourdes Mateu;Marta Massanella;Ester Ballana;Javier Martinez-Picado Subject: Research Article Keywords: genomic SARS-CoV-2; subgenomic SARS-CoV-2; RT-qPCR; RT-ddPCR; viral persistence; long COVID; post-COVID-19 condition Creator: PTC Arbortext Advanced Print Publisher 11.1.4012/W Producer: Adobe LiveCycle PDF Generator; modified using iText® 5.5.13.2 ©2000-2020 iText Group NV (AGPL-version) CreationDate: Tue Apr 04 18:55:48 CEST 2023 ModDate: Tue Apr 04 22:17:19 CEST 2023 ID: 0x890ba60e968d8204c6d0a3808433600f, 0x83668b7ce2fcfb5b265f6b9421be7eef Filters: FilterPipeline: FlateDecode Images: Image: NisoImageMetadata: FormatName: image/png ImageWidth: 67 ImageHeight: 67 BitsPerSample: 8 BitsPerSampleUnit: integer Filter: FlateDecode Fonts: Type1: Font: BaseFont: EAABMB+AdvOT3b30f6db.B FontSubset: true FirstChar: 45 LastChar: 121 FontDescriptor: FontName: EAABMB+AdvOT3b30f6db.B Flags: Nonsymbolic FontBBox: -39, -250, 875, 897 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EAABMC+AdvOT46dcae81 FontSubset: true FirstChar: 35 LastChar: 252 FontDescriptor: FontName: EAABMC+AdvOT46dcae81 Flags: Nonsymbolic FontBBox: -46, -250, 838, 879 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EAABMD+AdvOT46dcae81+fb FontSubset: true FirstChar: 1 LastChar: 3 FontDescriptor: FontName: EAABMD+AdvOT46dcae81+fb Flags: Symbolic FontBBox: 0, 0, 742, 721 FontFile3: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: EAABPE+AdvOT46dcae81+20 FontSubset: true FirstChar: 146 LastChar: 150 FontDescriptor: FontName: EAABPE+AdvOT46dcae81+20 Flags: Nonsymbolic FontBBox: -9, -205, 1126, 868 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EAACAF+AdvOT3b30f6db.B+fb FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: EAACAF+AdvOT3b30f6db.B+fb Flags: Symbolic FontBBox: 0, 0, 838, 721 FontFile3: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: EAACAG+AdvOT65f8a23b.I FontSubset: true FirstChar: 67 LastChar: 118 FontDescriptor: FontName: EAACAG+AdvOT65f8a23b.I Flags: Nonsymbolic FontBBox: -112, -250, 894, 873 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EAADIC+AdvPSUnvGMP FontSubset: true FirstChar: 49 LastChar: 49 FontDescriptor: FontName: EAADIC+AdvPSUnvGMP Flags: Nonsymbolic FontBBox: -20, -229, 1010, 812 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EAACAH+AdvP80675 FontSubset: true FirstChar: 44 LastChar: 50 FontDescriptor: FontName: EAACAH+AdvP80675 Flags: Nonsymbolic FontBBox: -20, -229, 1010, 812 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EAADIB+AdvP4C4E59 FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: EAADIB+AdvP4C4E59 Flags: Symbolic FontBBox: -31, -250, 1000, 750 FontFile3: true EncodingDictionary: Differences: true Font: BaseFont: EAADEB+AdvP7DA6 FontSubset: true FirstChar: 109 LastChar: 109 FontDescriptor: FontName: EAADEB+AdvP7DA6 Flags: Nonsymbolic FontBBox: -20, -208, 1000, 770 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EAADEC+AdvP4C4E74 FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: EAADEC+AdvP4C4E74 Flags: Symbolic FontBBox: -20, -958, 1114, 770 FontFile3: true EncodingDictionary: Differences: true Font: BaseFont: EAABHB+AdvOTce3d9a73 FontSubset: true FirstChar: 40 LastChar: 243 FontDescriptor: FontName: EAABHB+AdvOTce3d9a73 Flags: Nonsymbolic FontBBox: -42, -250, 858, 889 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EAABMA+AdvOT07517017 FontSubset: true FirstChar: 40 LastChar: 243 FontDescriptor: FontName: EAABMA+AdvOT07517017 Flags: Nonsymbolic FontBBox: -49, -250, 816, 867 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EAABNE+AdvOT07517017+fb FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: EAABNE+AdvOT07517017+fb Flags: Symbolic FontBBox: 0, 0, 683, 721 FontFile3: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: EAACIE+AdvOT8817665d FontSubset: true FirstChar: 35 LastChar: 57 FontDescriptor: FontName: EAACIE+AdvOT8817665d Flags: Nonsymbolic FontBBox: -30, -210, 1000, 779 FontFile3: true Encoding: WinAnsiEncoding XMP: Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA 10.1128/spectrum.04159-22 Sara Morón-López Eva Riveira-Muñoz Victor Urrea Lucia Gutiérrez-Chamorro Carlos Ávila-Nieto Marc Noguera-Julian Jorge Carrillo Oriol Mitjà Lourdes Mateu Marta Massanella Ester Ballana Javier Martinez-Picado Research Article Research Article genomic SARS-CoV-2 subgenomic SARS-CoV-2 RT-qPCR RT-ddPCR viral persistence long COVID post-COVID-19 condition American Society for Microbiology Morón-López et al. Copyright © 2023 Morón-López et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. 20230321 2023 ABSTRACT Most individuals acutely infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit mild symptoms. However, 10 to 20% of those infected develop long-term symptoms, referred to as post-coronavirus disease 2019 (COVID-19) condition (PCC). One hypothesis is that PCC might be exacerbated by viral persistence in tissue sanctuaries. Therefore, the accurate detection and quantification of SARS-CoV-2 are not only necessary for viral load monitoring but also crucial for detecting long-term viral persistence and determining whether viral replication is occurring in tissue reservoirs. In this study, the sensitivity and robustness of reverse transcription (RT)-droplet digital PCR (ddPCR) and RT-quantitative PCR (qPCR) techniques have been compared for the detection and quantification of SARS-CoV-2 genomic and subgenomic RNAs from oropharyngeal swabs taken from confirmed SARS-CoV-2-positive, SARS-CoV-2-exposed, and nonexposed individuals as well as from samples from mice infected with SARS-CoV-2. Our data demonstrated that both techniques presented equivalent results in the mid- and high-viral-load ranges. Additionally, RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, allowing the accurate detection of positive results in individuals exposed to the virus. Overall, these data suggest that RT-ddPCR might be an alternative to RT-qPCR for detecting low viral loads in samples and for assessing viral persistence in samples from individuals with PCC. IMPORTANCE We developed one-step reverse transcription (RT)-droplet digital PCR (ddPCR) protocols to detect SARS-CoV-2 RNA and compared them to the gold-standard RT-quantitative PCR (RT-qPCR) method. RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, while both techniques were equivalent in the mid- and high-viral-load ranges. Overall, these results suggest that RT-ddPCR might be a viable alternative to RT-qPCR when it comes to detecting low viral loads in samples, which is a highly relevant issue for determining viral persistence in as-yet-unknown tissue reservoirs in individuals suffering from post-COVID conditions or long COVID. 20221025 20230224 2165-0497 Pages: Page: Label: 1 Annotations: Annotation: Subtype: Link Rect: 50, 565, 121, 574 Annotation: Subtype: Link Rect: 50, 554, 131, 563 Annotation: Subtype: Link Rect: 50, 543, 100, 552 Annotation: Subtype: Link Rect: 120, 543, 209, 552 Annotation: Subtype: Link Rect: 435, 133, 530, 140 Annotation: Subtype: Link Rect: 400, 124, 456, 131 Annotation: Subtype: Link Rect: 444, 24, 526, 31 Annotation: Subtype: Link Rect: 508, 683, 544, 719 Page: Label: 2 Annotations: Annotation: Subtype: Link Rect: 238, 690, 243, 699 Destination: 112 Annotation: Subtype: Link Rect: 153, 665, 158, 674 Destination: 112 Annotation: Subtype: Link Rect: 162, 665, 167, 674 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