Analytical and Clinical Performance of Droplet Digital PCR in the Detection and Quantification of SARS-CoV-2.
Analytical and Clinical Performance of Droplet Digital PCR in the Detection and Quantification of SARS-CoV-2.
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DOI：
10.1007/s40291-021-00547-1
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发表时间：
2021-09
影响因子：
4
通讯作者：
Kim M
中科院分区：
文献类型：
--
作者：
Kim KB;Choi H;Lee GD;Lee J;Lee S;Kim Y;Cho SY;Lee DG;Kim M
自2019年底首次爆发冠状病毒病（COVID-19）以来，逆转录实时聚合酶链反应（RT-qPCR）已成为检测严重急性呼吸综合征相关冠状病毒2（SARS-CoV-2）的金标准测试。然而，需要一种更敏感和准确的诊断工具。因此，液滴数字聚合酶链反应（ddPCR）被建议作为一种替代方法。在这里，我们评估了ddPCR检测SARS-CoV-2的性能，并将其与RT-qPCR的性能进行了比较。使用阳性和阴性SARS-CoV-2参比物质确定分析性能，包括空白限和检测限。从四个机构共收集了366份RNA提取物（173份RT-qPCR阳性，193份RT-qPCR阴性），并使用Bio-Rad SARS-CoV-2 ddPCR试剂盒进行检测，该试剂盒使用N1和N2引物检测SARS-CoV-2基因组。空白限设为0，N1和N2的检测限分别为1.99拷贝/μL和5.18拷贝/μL。使用系列稀释样品评价线性，结果良好（R2：0.999，线性范围：N1为5.88-6825.25拷贝/μL，R2：0.999，N2为5.53-5855.47拷贝/μL）。ddPCR和RT-qPCR的结果显示基本一致（Cohen’s kappa：0.639，p < 0.01）。ddPCR阳性但RT-qPCR阴性的63份样本显示拷贝数较低，其中55%的样本有COVID-19相关症状。液滴数字聚合酶链反应检测SARS-Cov-2具有良好的灵敏度，并与常规RT-qPCR的结果一致。此外，ddPCR提供的定量数据可用于监测COVID-19患者病毒载量的变化。在线版本包含补充材料，可通过10.1007/s40291-021-00547-1获得。
Since the initial coronavirus disease outbreak in late 2019 (COVID-19), reverse-transcription real-time polymerase chain reaction (RT-qPCR) has become the gold standard test to detect severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). However, a more sensitive and accurate diagnostic tool was required. Therefore, droplet digital polymerase chain reaction (ddPCR) was suggested as an alternative method. Here, we evaluated the performance of ddPCR to detect SARS-CoV-2 and compared it to the performance of RT-qPCR. The analytical performances, including limit of blank and limit of detection, were established using positive and negative SARS-CoV-2 reference materials. A total of 366 RNA extracts (173 positive and 193 negative by RT-qPCR) were collected from four institutions and tested with a Bio-Rad SARS-CoV-2 ddPCR kit that detects the SARS-CoV-2 genome using primers for N1 and N2. Limit of blank was set at 0, and the limits of detection of N1 and N2 were 1.99 copies/μL and 5.18 copies/μL, respectively. Linearity was evaluated using serial dilution samples, which demonstrated good results (R2: 0.999, linear range: 5.88–6825.25 copies/μL for N1 and R2: 0.999, 5.53–5855.47 copies/μL for N2). The results of ddPCR and RT-qPCR revealed substantial agreement (Cohen’s kappa: 0.639, p < 0.01). The 63 samples with positive ddPCR but negative RT-qPCR showed low copy numbers, and 55% of them had COVID-19-related symptoms. Droplet digital polymerase chain reaction demonstrated excellent sensitivity for SARS-Cov-2 detection and consistently agreed with the results from conventional RT-qPCR. Furthermore, ddPCR provided quantitative data that can be used to monitor changes in the viral load of patients with COVID-19. The online version contains supplementary material available at 10.1007/s40291-021-00547-1.
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影响因子：
48
作者：
Hindson, Christopher M.;Chevillet, John R.;Briggs, Hilary A.;Gallichotte, Emily N.;Ruf, Ingrid K.;Hindson, Benjamin J.;Vessella, Robert L.;Tewari, Muneesh
通讯作者：
Tewari, Muneesh
DOI：
10.1016/j.bbrc.2020.10.069
发表时间：
2021-01-29
影响因子：
3.1
作者：
Lai CKC;Lam W
通讯作者：
Lam W
ddPCR：一种在低病毒载量样本中检测 SARS-CoV-2 的更准确工具。
影响因子：
13.2
作者：
Suo, Tao;Liu, Xinjin;Chen, Yu
通讯作者：
Chen, Yu
影响因子：
9.3
作者：
Huggett, Jim F.;Whale, Alexandra S.;Vandesompele, Jo
通讯作者：
Vandesompele, Jo
影响因子：
9.4
作者：
Nalla, Arun K.;Casto, Amanda M.;Greninger, Alexander L.
通讯作者：
Greninger, Alexander L.