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.

中文翻译：

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鉴定 SARS-CoV-2 RNA 的 RNase 结合位点，用于锚定引物 PCR 检测 306 名 COVID-19 患者的病毒载量

2019冠状病毒病（COVID-19）的大流行迫切需要更灵敏的分子诊断，以提高当前病毒核酸检测的灵敏度。我们开发了一种基于锚定引物 (AP) 的检测方法，通过生物信息学鉴定严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) RNA 的 RNase 结合位点，并实施 AP 双重靶向 SARS 的 N 基因，以提高病毒 RNA 的稳定性-CoV-2 RNA 和 RNase 1, 3, 6。任意引物聚合酶链式反应 (AP-PCR) 对病毒 RNA 完整性的改善得到 (a) AP 增加 SARS 目标基因（N 基因）的抵抗力的支持 - CoV-2 RNA 转 RNase 治疗；(b) 与两种市售检测方法相比，通过 AP-PCR 检测 SARS-CoV-2 RNA 的循环阈值较低（−2.7 个循环）；(c)靶向N基因和RNase后ORF基因的病毒RNA稳定性得到改善。此外，在确诊病例的 70-80% 痰液、鼻腔、咽拭子和粪便以及 36% (4/11) 尿液中检测到 SARS-CoV-2 RNA，证明了 AP-PCR 灵敏度的提高（n  = 252 例），7% 为康复病例（n  = 54），300 例阴性病例中无一例。最后，对 306 例确诊和康复病例的 AP-PCR 分析显示，在首次阳性诊断后，病毒载量持续存在超过 20 天。因此，双重靶向 SARS-CoV-2 RNA 和 RNase 的 AP 通过保留 SARS-CoV-2 RNA 完整性来改善分子检测，并揭示与 COVID-19 患者年龄较大和男性相关的病毒载量延长。