PDF(Pubmed)
Abstract：
Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a considerable threat to global public health. This study developed and evaluated a rapid, low-cost, expandable, and sequencing-free high-resolution melting (HRM) assay for the direct detection of SARS-CoV-2 variants. A panel of 64 common bacterial and viral pathogens that can cause respiratory tract infections was employed to evaluate our method\'s specificity. Serial dilutions of viral isolates determined the sensitivity of the method. Finally, the assay\'s clinical performance was assessed using 324 clinical samples with potential SARS-CoV-2 infection. Multiplex HRM analysis accurately identified SARS-CoV-2 (as confirmed with parallel reverse transcription-quantitative PCR [qRT-PCR] tests), differentiating between mutations at each marker site within approximately 2 h. For each target, the limit of detection (LOD) was lower than 10 copies/reaction (the LOD of N, G142D, R158G, Y505H, V213G, G446S, S413R, F486V, and S704L was 7.38, 9.72, 9.96, 9.96, 9.50, 7.80, 9.33, 8.25, and 8.25 copies/reaction, respectively). No cross-reactivity occurred with organisms of the specificity testing panel. In terms of variant detection, our results had a 97.9% (47/48) rate of agreement with standard Sanger sequencing. The multiplex HRM assay therefore offers a rapid and simple procedure for detecting SARS-CoV-2 variants. IMPORTANCE In the face of the current severe situation of increasing SARS-CoV-2 variants, we developed an upgraded multiplex HRM method for the predominant SARS-CoV-2 variants based on our original research. This method not only could identify the variants but also could be utilized in subsequent detection of novel variants since the assay has great performance in terms of flexibility. In summary, the upgraded multiplex HRM assay is a rapid, reliable, and economical detection method, which could better screen prevalent virus strains, monitor the epidemic situation, and help to develop measures for the prevention and control of SARS-CoV-2.
摘要：
冠状病毒疾病2019，由严重急性呼吸道综合症冠状病毒2（SARS-CoV-2）引起，对全球公共卫生构成相当大的威胁。这项研究开发并评估了一种快速、低成本,可扩展,和用于直接检测SARS-CoV-2变体的无测序高分辨率熔解(HRM)测定。一组可引起呼吸道感染的64种常见细菌和病毒病原体被用来评估我们的方法的特异性。病毒分离物的连续稀释确定了该方法的灵敏度。最后,使用324份潜在SARS-CoV-2感染的临床样本评估了该试验的临床表现。多重HRM分析准确鉴定了SARS-CoV-2（如平行逆转录定量PCR[qRT-PCR]测试所证实），在大约2小时内区分每个标记位点的突变。对于每个靶标，检测限(LOD)低于10拷贝/反应(N的LOD，G142D,R158G,Y505H,V213G,G446S,S413R,F486V,S704L为7.38、9.72、9.96、9.96、9.50、7.80、9.33、8.25和8.25拷贝/反应，分别)。与特异性测试小组的生物体没有发生交叉反应性。在变异检测方面，我们的结果与标准Sanger测序的符合率为97.9%(47/48).因此,多重HRM测定提供了用于检测SARS-CoV-2变体的快速且简单的程序。重要性面对当前SARS-CoV-2变种增多的严峻形势,我们开发了一种升级的多重HRM方法的主要SARS-CoV-2变种基于我们的原始研究。该方法不仅可以鉴定变体,而且还可以用于新变体的后续检测,因为该测定在灵活性方面具有很好的性能。总之,升级后的多重HRM测定是一种快速的，可靠,和经济的检测方法，可以更好地筛选流行的病毒株，监测疫情，并帮助制定SARS-CoV-2的预防和控制措施。
