Multiplex Fragment Analysis for Flexible Detection of All SARS-CoV-2 Variants of Concern.
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge, and effective tracking requires rapid return of results. Surveillance of variants is typically performed by whole genome sequencing (WGS), which can be financially prohibitive and requires specializ...
| Publicado en: | Clinical Chemistry Vol. 68; no. 8; pp. 1042 - 1053 |
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| Autores principales: | , , , , , , , , , , , , , , , , , , |
| Formato: | Journal Article |
| Publicado: |
Oxford University Press / USA
Aug2022
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=158258535&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 158258535 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 00099147 10CS jtl: Clinical Chemistry issn: 00099147 maglogo: N pubinfo: dt: Aug2022 vid: 68 iid: 8 pid: 622 pub: Oxford University Press / USA artinfo: ui: 158258535 10.1093/clinchem/hvac081 158258535 ppf: 1042 ppct: 11 formats: fmt: @attributes: type: P tig: atl: Multiplex Fragment Analysis for Flexible Detection of All SARS-CoV-2 Variants of Concern. aug: au: Clark, Andrew E. Zhaohui Wang Ostman, Emily Hui Zheng Huiyu Yao Cantarel, Brandi Kanchwala, Mohammed Chao Xing Li Chen Pei Irwin Yan Xu Oliver, Dwight Lee, Francesca M. Gagan, Jeffrey R. Filkins, Laura Muthukumar, Alagarraju Park, Jason Y. Sarode, Ravi SoRelle, Jeffrey A. affil: Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA sug: ab: BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge, and effective tracking requires rapid return of results. Surveillance of variants is typically performed by whole genome sequencing (WGS), which can be financially prohibitive and requires specialized equipment and bioinformatic expertise. Genotyping approaches are rapid methods formonitoring SARS-CoV-2 variants but require continuous adaptation. Fragment analysis may represent an approach for improved SARS-CoV-2 variant detection. METHODS: A multiplex fragment analysis approach (CoVarScan) was validated using PCR targeting variants by size and fluorescent color. Eight SARS-CoV-2 mutational hot spots in variants of concern (VOCs) were targeted. Three primer pairs (recurrently deleted region [RDR] 1, RDR2, and RDR3-4) flank RDRs in the S-gene. Three allele-specific primers target recurrent spike receptor binding domain mutants. Lastly, 2 primer pairs target recurrent deletions or insertions in ORF1A and ORF8. Fragments were resolved and analyzed by capillary electrophoresis (ABI 3730XL), and mutational signatures were compared to WGS results. RESULTS: We validated CoVarScan using 3544 clinical respiratory specimens. The assay exhibited 96% sensitivity and 99% specificity compared to WGS. The limit of detection for the core targets (RDR1, RDR2, and ORF1A) was 5 copies/reaction. Variants were identified in 95% of samples with cycle threshold (CT), 30 and 75% of samples with a CT 34 to 35. Assay design was frozen April 2021, but all subsequent VOCs have been detected including Delta (n=2820), Mu, (n=6), Lambda (n=6), and Omicron (n=309). Genotyping results are available in as little as 4 h. CONCLUSIONS: Multiplex fragment analysis is adaptable and rapid and has similar accuracy to WGS to classify SARS-CoV-2 variants. pubtype: Academic Journal doctype: Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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