Identification of the RNase-binding site of SARS-CoV-2 RNA for anchor primer-PCR detection of viral loading in 306 COVID-19 patients. Issue 2 (16th September 2020)

• Record Type: Journal Article
• Authors: Xu, Tao; Wang, Jingu; Hu, Bingjie; Zhang, Guosi; Zhou, Wu; Zheng, Meiqin; Shen, Bo; Sun, Baochang; Zhang, Yanjun; Chen, Yin; Yu, Jian; Liang, Min; Pan, Jingye; Chen, Chengshui; Chen, Haixiao; Jiang, Minghua; Xu, Liangde; Qu, Jia; Chen, Jiang-Fan
• Is Part Of: Briefings in bioinformatics. Volume 22:Issue 2(2021)
• Page Start: 1215
• Page End: 1224
• Publication Date: 2020-09-16
• Subjects: SARS-CoV-2 -- COVID-19 -- RNA -- anchor primer -- bioinformatics analysis; Genetics -- Data processing -- Periodicals; Molecular biology -- Data processing -- Periodicals; Genomes -- Data processing -- Periodicals; 572.80285
• DOI: 10.1093/bib/bbaa193
• Languages: English
• ISSNs: 1467-5463

Abstract

Abstract: 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 (−2.7 cycles) 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 70–80% of sputum, nasal, pharyngeal swabs and feces and 36% (4/11) of urine of the confirmed cases ( n = 252), 7% convalescent cases ( n = 54) and none of 300 negative cases. Lastly, AP-PCR analysis of 306 confirmed and convalescent cases revealed prolonged presence of viral loading for >20 days 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.