{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2024,9,6]],"date-time":"2024-09-06T16:51:52Z","timestamp":1725641512541},"reference-count":50,"publisher":"Oxford University Press (OUP)","issue":"2","license":[{"start":{"date-parts":[[2020,9,16]],"date-time":"2020-09-16T00:00:00Z","timestamp":1600214400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"funder":[{"name":"Wenzhou Municipal City","award":["ZY202001\u20134"]},{"name":"Zhejiang Provincial Science and Technology Bureau","award":["2020C03128"]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,3,22]]},"abstract":"Abstract<\/jats:title>\n The pandemic of coronavirus disease 2019 (COVID-19) urgently calls for more sensitive molecular diagnosis to improve sensitivity of current viral nuclear acid detection. We have developed an anchor primer (AP)-based assay to improve viral RNA stability by bioinformatics identification of RNase-binding site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and implementing AP dually targeting the N gene of SARS-CoV-2 RNA and RNase 1, 3, 6. The arbitrarily primed polymerase chain reaction (AP-PCR) improvement of viral RNA integrity was supported by (a) the AP increased resistance of the targeted gene (N gene) of SARS-CoV-2 RNA to RNase treatment; (b) the detection of SARS-CoV-2 RNA by AP-PCR with lower cycle threshold values (\u22122.7\u00a0cycles) compared to two commercially available assays; (c) improvement of the viral RNA stability of the ORF gene upon targeting of the N gene and RNase. Furthermore, the improved sensitivity by AP-PCR was demonstrated by detection of SARS-CoV-2 RNA in 70\u201380% of sputum, nasal, pharyngeal swabs and feces and 36% (4\/11) of urine of the confirmed cases (n\u00a0=\u2009252), 7% convalescent cases (n\u00a0=\u200954) and none of 300 negative cases. Lastly, AP-PCR analysis of 306 confirmed and convalescent cases revealed prolonged presence of viral loading for >20\u00a0days after the first positive diagnosis. Thus, the AP dually targeting SARS-CoV-2 RNA and RNase improves molecular detection by preserving SARS-CoV-2 RNA integrity and reveals the prolonged viral loading associated with older age and male gender in COVID-19 patients.<\/jats:p>","DOI":"10.1093\/bib\/bbaa193","type":"journal-article","created":{"date-parts":[[2020,8,11]],"date-time":"2020-08-11T12:11:16Z","timestamp":1597147876000},"page":"1215-1224","source":"Crossref","is-referenced-by-count":6,"title":["Identification of the RNase-binding site of SARS-CoV-2 RNA for anchor primer-PCR detection of viral loading in 306 COVID-19 patients"],"prefix":"10.1093","volume":"22","author":[{"given":"Tao","family":"Xu","sequence":"first","affiliation":[]},{"given":"Jingu","family":"Wang","sequence":"additional","affiliation":[]},{"given":"Bingjie","family":"Hu","sequence":"additional","affiliation":[]},{"given":"Guosi","family":"Zhang","sequence":"additional","affiliation":[]},{"given":"Wu","family":"Zhou","sequence":"additional","affiliation":[]},{"given":"Meiqin","family":"Zheng","sequence":"additional","affiliation":[]},{"given":"Bo","family":"Shen","sequence":"additional","affiliation":[]},{"given":"Baochang","family":"Sun","sequence":"additional","affiliation":[]},{"given":"Yanjun","family":"Zhang","sequence":"additional","affiliation":[]},{"given":"Yin","family":"Chen","sequence":"additional","affiliation":[]},{"given":"Jian","family":"Yu","sequence":"additional","affiliation":[]},{"given":"Min","family":"Liang","sequence":"additional","affiliation":[]},{"given":"Jingye","family":"Pan","sequence":"additional","affiliation":[]},{"given":"Chengshui","family":"Chen","sequence":"additional","affiliation":[]},{"given":"Haixiao","family":"Chen","sequence":"additional","affiliation":[]},{"given":"Minghua","family":"Jiang","sequence":"additional","affiliation":[]},{"given":"Liangde","family":"Xu","sequence":"additional","affiliation":[]},{"given":"Jia","family":"Qu","sequence":"additional","affiliation":[]},{"given":"Jiang-Fan","family":"Chen","sequence":"additional","affiliation":[]}],"member":"286","published-online":{"date-parts":[[2020,9,16]]},"reference":[{"key":"2021032314264614400_ref1","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1056\/NEJMoa2001017","article-title":"A novel coronavirus from patients with pneumonia in China, 2019","volume":"382","author":"Zhu","year":"2020","journal-title":"N Engl J Med"},{"key":"2021032314264614400_ref2","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1016\/S0140-6736(20)30251-8","article-title":"Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding","volume":"395","author":"Lu","year":"2020","journal-title":"Lancet"},{"key":"2021032314264614400_ref3","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1056\/NEJMoa2002032","article-title":"Clinical characteristics of coronavirus disease 2019 in China","volume":"382","author":"Guan","year":"2020","journal-title":"N Engl J Med"},{"key":"2021032314264614400_ref4","doi-asserted-by":"crossref","first-page":"2000045","DOI":"10.2807\/1560-7917.ES.2020.25.3.2000045","article-title":"Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR","volume":"25","author":"Corman","year":"2020","journal-title":"Euro Surveill"},{"key":"2021032314264614400_ref5","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.jpha.2020.02.010","article-title":"Recent advances and perspectives of nucleic acid detection for coronavirus","volume":"10","author":"Shen","year":"2020","journal-title":"J Pharm Anal"},{"key":"2021032314264614400_ref6","doi-asserted-by":"publisher","DOI":"10.1101\/2020.03.12.988246","article-title":"High sensitivity detection of SARS-CoV-2 using multiplex PCR and a multiplex-PCR-based metagenomic method","author":"Li","year":"2020","journal-title":"bioRxiv"},{"key":"2021032314264614400_ref7","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1080\/22221751.2020.1725399","article-title":"RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak","volume":"9","author":"Chen","year":"2020","journal-title":"Emerg Microbes Infect"},{"key":"2021032314264614400_ref8","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1038\/nrmicro.2016.182","article-title":"Clinical and biological insights from viral genome sequencing","volume":"15","author":"Houldcroft","year":"2017","journal-title":"Nat Rev Microbiol"},{"key":"2021032314264614400_ref9","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1016\/j.jmoldx.2020.03.006","article-title":"Development of reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays targeting SARS-CoV-2","volume":"22","author":"Park","year":"2020","journal-title":"J Mol Diagn"},{"key":"2021032314264614400_ref10","doi-asserted-by":"crossref","first-page":"E32","DOI":"10.1148\/radiol.2020200642","article-title":"Correlation of chest CT and RT-PCR testing in coronavirus disease 2019 (COVID-19) in China: A report of 1014 cases","volume":"296","author":"Ai","year":"2020","journal-title":"Radiology"},{"key":"2021032314264614400_ref11","doi-asserted-by":"crossref","first-page":"1280","DOI":"10.2214\/AJR.20.22954","article-title":"Coronavirus disease 2019 (COVID-19): Role of chest CT in diagnosis and management","volume":"214","author":"Li","year":"2020","journal-title":"AJR Am J Roentgenol"},{"key":"2021032314264614400_ref12","doi-asserted-by":"crossref","first-page":"e28","DOI":"10.1016\/j.jinf.2020.03.051","article-title":"Serological immunochromatographic approach in diagnosis with SARS-CoV-2 infected COVID-19 patients","volume":"81","author":"Pan","year":"2020","journal-title":"J Infect"},{"key":"2021032314264614400_ref13","doi-asserted-by":"publisher","DOI":"10.1002\/jmv.25727","article-title":"Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis","author":"Li","year":"2020","journal-title":"J Med Virol"},{"key":"2021032314264614400_ref14","doi-asserted-by":"crossref","first-page":"977","DOI":"10.1093\/clinchem\/hvaa099","article-title":"Limits of detection of six approved RT-PCR kits for the novel SARS-coronavirus-2 (SARS-CoV-2)","volume":"66","author":"Wang","year":"2020","journal-title":"Clin Chem"},{"key":"2021032314264614400_ref15","doi-asserted-by":"crossref","first-page":"903","DOI":"10.1002\/jmv.25786","article-title":"Stability issues of RT-PCR testing of SARS-CoV-2 for hospitalized patients clinically diagnosed with COVID-19","volume":"92","author":"Li","year":"2020","journal-title":"J Med Virol"},{"key":"2021032314264614400_ref16","doi-asserted-by":"crossref","first-page":"e00310-20","DOI":"10.1128\/JCM.00310-20","article-title":"Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp\/Hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens","volume":"58","author":"Chan","year":"2020","journal-title":"J Clin Microbiol"},{"key":"2021032314264614400_ref17","doi-asserted-by":"crossref","first-page":"e00557\u201320","DOI":"10.1128\/JCM.00557-20","article-title":"Comparative performance of SARS-CoV-2 detection assays using seven different primer\/probe sets and one assay kit","volume":"58","author":"Nalla","year":"2020","journal-title":"J Clin Microbiol"},{"key":"2021032314264614400_ref18","doi-asserted-by":"crossref","first-page":"2000152","DOI":"10.2807\/1560-7917.ES.2020.25.9.2000152","article-title":"Evaluation of a quantitative RT-PCR assay for the detection of the emerging coronavirus SARS-CoV-2 using a high throughput system","volume":"25","author":"Pfefferle","year":"2020","journal-title":"Euro Surveill"},{"key":"2021032314264614400_ref19","doi-asserted-by":"crossref","first-page":"304","DOI":"10.7883\/yoken.JJID.2020.061","article-title":"Development of genetic diagnostic methods for novel coronavirus 2019 (nCoV-2019) in Japan","volume":"73","author":"Shirato","year":"2020","journal-title":"Jpn J Infect Dis"},{"key":"2021032314264614400_ref20","doi-asserted-by":"crossref","first-page":"793","DOI":"10.1093\/cid\/ciaa345","article-title":"Quantitative detection and viral load analysis of SARS-CoV-2 in infected patients","volume":"71","author":"Yu","year":"2020","journal-title":"Clin Infect Dis"},{"key":"2021032314264614400_ref21","doi-asserted-by":"crossref","first-page":"600","DOI":"10.1016\/j.virol.2015.02.007","article-title":"Emerging roles for RNA degradation in viral replication and antiviral defense","volume":"479-480","author":"Abernathy","year":"2015","journal-title":"Virology"},{"key":"2021032314264614400_ref22","doi-asserted-by":"crossref","first-page":"E162","DOI":"10.1073\/pnas.1718806115","article-title":"Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA","volume":"115","author":"Ferron","year":"2018","journal-title":"Proc Natl Acad Sci U S A"},{"key":"2021032314264614400_ref23","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1038\/s41591-020-0820-9","article-title":"The proximal origin of SARS-CoV-2","volume":"26","author":"Andersen","year":"2020","journal-title":"Nat Med"},{"key":"2021032314264614400_ref24","doi-asserted-by":"crossref","first-page":"9241","DOI":"10.1073\/pnas.2004999117","article-title":"Phylogenetic network analysis of SARS-CoV-2 genomes","volume":"117","author":"Forster","year":"2020","journal-title":"Proc Natl Acad Sci U S A"},{"key":"2021032314264614400_ref25","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1093\/bioinformatics\/btv629","article-title":"catRAPID signature: identification of ribonucleoproteins and RNA-binding regions","volume":"32","author":"Livi","year":"2016","journal-title":"Bioinformatics"},{"key":"2021032314264614400_ref26","first-page":"1","article-title":"Landscape of SNPs-mediated lncRNA structural variations and their implication in human complex diseases","volume":"2018","author":"Wang","journal-title":"Brief Bioinform"},{"key":"2021032314264614400_ref27","doi-asserted-by":"crossref","first-page":"D277","DOI":"10.1093\/nar\/gkq1108","article-title":"PRIDB: a Protein-RNA interface database","volume":"39","author":"Lewis","year":"2011","journal-title":"Nucleic Acids Res"},{"key":"2021032314264614400_ref28","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1186\/1471-2105-12-489","article-title":"Predicting RNA-Protein interactions using only sequence information","volume":"12","author":"Muppirala","year":"2011","journal-title":"Bmc Bioinformatics"},{"key":"2021032314264614400_ref29","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1007\/978-1-4939-6433-8_10","article-title":"Experiment-assisted secondary structure prediction with RNAstructure","volume":"1490","author":"Xu","year":"2016","journal-title":"Methods Mol Biol"},{"key":"2021032314264614400_ref30","doi-asserted-by":"crossref","first-page":"866","DOI":"10.1093\/cid\/ciaa386","article-title":"New measures for COVID-19 response: a lesson from the Wenzhou experience","volume":"71","author":"Ruan","year":"2020","journal-title":"Clin Infect Dis"},{"key":"2021032314264614400_ref31","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1016\/j.jinf.2020.02.016","article-title":"Clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (COVID-19):A multi-center study in Wenzhou city, Zhejiang, China","volume":"80","author":"Yang","year":"2020","journal-title":"J Infect"},{"key":"2021032314264614400_ref32","doi-asserted-by":"publisher","DOI":"10.26434\/chemrxiv.11860137","article-title":"A single and two stage, closed-tube, molecular test for the 2019 novel coronavirus (COVID-19) at home, clinic, and points of entry","author":"Mohamed","year":"2020","journal-title":"ChemRxiv"},{"key":"2021032314264614400_ref33","doi-asserted-by":"crossref","first-page":"e00310-20","DOI":"10.1128\/JCM.00310-20","article-title":"Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp\/Hel real-time reverse transcription-PCR assay validated in vitro and with clinical specimens","volume":"58","author":"Chan","year":"2020","journal-title":"J Clin Microbiol"},{"key":"2021032314264614400_ref34","doi-asserted-by":"crossref","first-page":"1259","DOI":"10.1080\/22221751.2020.1772678","article-title":"ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens","volume":"9","author":"Suo","year":"2020","journal-title":"Emerg Microbes Infect"},{"key":"2021032314264614400_ref35","article-title":"A protocol for detection of COVID-19 using CRISPR diagnostics","author":"Zhang","year":"2020"},{"key":"2021032314264614400_ref36","doi-asserted-by":"crossref","first-page":"507","DOI":"10.1016\/S0140-6736(20)30211-7","article-title":"Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study","volume":"395","author":"Chen","year":"2020","journal-title":"Lancet"},{"key":"2021032314264614400_ref37","article-title":"Human Kidney is a Target for Novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection","author":"Diao","year":"2020","journal-title":"medRxiv"},{"key":"2021032314264614400_ref38","doi-asserted-by":"publisher","DOI":"10.1126\/science.abc3208","article-title":"How does coronavirus kill? Clinicians trace a ferocious rampagethrough the body, from brain to toes","author":"Wadman","year":"2020","journal-title":"Science"},{"key":"2021032314264614400_ref39","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1038\/s41591-020-0877-5","article-title":"Clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 (COVID-19) in the United States","volume":"26","author":"Team","year":"2020","journal-title":"Nat Med"},{"key":"2021032314264614400_ref40","first-page":"1843","article-title":"Detection of SARS-CoV-2 in different types of clinical specimens","volume":"323","author":"Wang","year":"2020","journal-title":"JAMA"},{"key":"2021032314264614400_ref41","doi-asserted-by":"crossref","first-page":"2","DOI":"10.3390\/v9010002","article-title":"Attacked from all sides: RNA decay in antiviral defense","volume":"9","author":"Molleston","year":"2017","journal-title":"Viruses"},{"key":"2021032314264614400_ref42","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1093\/cid\/ciaa351","article-title":"Factors associated with prolonged viral RNA shedding in patients with COVID-19","volume":"71","author":"Xu","year":"2020","journal-title":"Clin Infect Dis"},{"key":"2021032314264614400_ref43","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1016\/S1473-3099(20)30196-1","article-title":"Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study","volume":"20","author":"To","year":"2020","journal-title":"Lancet Infect Dis"},{"key":"2021032314264614400_ref44","doi-asserted-by":"crossref","first-page":"1767","DOI":"10.1016\/S0140-6736(03)13412-5","article-title":"Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study","volume":"361","author":"Peiris","year":"2003","journal-title":"Lancet"},{"key":"2021032314264614400_ref45","doi-asserted-by":"crossref","first-page":"1303","DOI":"10.1056\/NEJMc1511695","article-title":"Viral load kinetics of MERS coronavirus infection","volume":"375","author":"Oh","year":"2016","journal-title":"N Engl J Med"},{"key":"2021032314264614400_ref46","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1038\/s41586-020-2196-x","article-title":"Virological assessment of hospitalized patients with COVID-2019","volume":"581","author":"Wolfel","year":"2020","journal-title":"Nature"},{"key":"2021032314264614400_ref47","doi-asserted-by":"crossref","first-page":"6490","DOI":"10.18632\/aging.103052","article-title":"Biomarkers of biological age as predictors of COVID-19 disease severity","volume":"12","author":"Lauc","year":"2020","journal-title":"Aging (Albany NY)"},{"key":"2021032314264614400_ref48","doi-asserted-by":"crossref","DOI":"10.1093\/gerona\/glaa089","article-title":"Clinical characteristics and outcomes of older patients with coronavirus disease 2019 (COVID-19) in Wuhan, China (2019): a single-centered, retrospective study","author":"Chen","year":"2020","journal-title":"J Gerontol A Biol Sci Med Sci"},{"key":"2021032314264614400_ref49","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1111\/acel.12326","article-title":"How sex and age affect immune responses, susceptibility to infections, and response to vaccination","volume":"14","author":"Giefing-Kroll","year":"2015","journal-title":"Aging Cell"},{"key":"2021032314264614400_ref50","doi-asserted-by":"crossref","first-page":"1237","DOI":"10.1038\/s41374-018-0084-x","article-title":"Sex dimorphism in ANGII-mediated crosstalk between ACE2 and ACE in diabetic nephropathy","volume":"98","author":"Clotet-Freixas","year":"2018","journal-title":"Lab Invest"}],"container-title":["Briefings in Bioinformatics"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/academic.oup.com\/bib\/article-pdf\/22\/2\/1215\/36654027\/bbaa193.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"http:\/\/academic.oup.com\/bib\/article-pdf\/22\/2\/1215\/36654027\/bbaa193.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,3,23]],"date-time":"2021-03-23T14:30:11Z","timestamp":1616509811000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/bib\/article\/22\/2\/1215\/5906196"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,9,16]]},"references-count":50,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2020,9,16]]},"published-print":{"date-parts":[[2021,3,22]]}},"URL":"http:\/\/dx.doi.org\/10.1093\/bib\/bbaa193","relation":{},"ISSN":["1467-5463","1477-4054"],"issn-type":[{"value":"1467-5463","type":"print"},{"value":"1477-4054","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2021,3]]},"published":{"date-parts":[[2020,9,16]]}}}
