PDF(Pubmed)
Abstract：
The emergence of SARS-CoV-2 mutations poses significant challenges to diagnostic tests, as these mutations can reduce the sensitivity of commonly used RT-PCR assays. Therefore, there is a need to design diagnostic assays with multiple targets to enhance sensitivity. In this study, we identified a novel diagnostic target, the nsp10 gene, using nanopore sequencing. Firstly, we determined the analytical sensitivity and specificity of our COVID-19-nsp10 assay. The COVID-19-nsp10 assay had a limit of detection of 74 copies/mL (95% confidence interval: 48-299 copies/mL) and did not show cross-reactivity with other respiratory viruses. Next, we determined the diagnostic performance of the COVID-19-nsp10 assay using 261 respiratory specimens, including 147 SARS-CoV-2-positive specimens belonging to the ancestral strain and Alpha, Beta, Gamma, Delta, Mu, Eta, Kappa, Theta and Omicron lineages. Using a LightMix E-gene RT-PCR assay as the reference method, the diagnostic sensitivity and specificity of the COVID-19-nsp10 assay were found to be 100%. The median Cp values for the LightMix E-gene RT-PCR and our COVID-19-nsp10 RT-PCR were 22.48 (range: 12.95-36.60) and 25.94 (range 16.37-36.87), respectively. The Cp values of the COVID-19-nsp10 RT-PCR assay correlated well with those of the LightMix E-gene RT-PCR assay (Spearman\'s ρ = 0.968; p < 0.0001). In conclusion, nsp10 is a suitable target for a SARS-CoV-2 RT-PCR assay.
摘要：
SARS-CoV-2突变的出现对诊断测试提出了重大挑战,因为这些突变会降低常用RT-PCR检测的灵敏度。因此,需要设计具有多个靶标的诊断测定法以增强灵敏度。在这项研究中,我们确定了一个新的诊断目标，nsp10基因,使用纳米孔测序。首先,我们确定了COVID-19-nsp10检测的分析灵敏度和特异性.COVID-19-nsp10检测的检出限为74拷贝/mL(95%置信区间：48-299拷贝/mL)，与其他呼吸道病毒没有交叉反应。接下来,我们使用261个呼吸道标本确定了COVID-19-nsp10测定法的诊断性能，包括147个SARS-CoV-2阳性标本，属于祖先菌株和α，Beta,Gamma,Delta,穆,埃塔,Kappa,Theta和Omicron谱系。使用LightMixE基因RT-PCR检测作为参考方法，COVID-19-nsp10检测的诊断敏感性和特异性为100%.LightMixE基因RT-PCR和我们的COVID-19-nsp10RT-PCR的Cp中位数分别为22.48（范围：12.95-36.60）和25.94（范围16.37-36.87），分别。COVID-19-nsp10RT-PCR测定的Cp值与LightMixE基因RT-PCR测定的Cp值密切相关(Spearman'sρ=0.968；p<0.0001)。总之,nsp10是SARS-CoV-2RT-PCR测定的合适靶标。
