Evaluation of a fully automated high-throughput SARS-CoV-2 multiplex qPCR assay with built-in screening functionality for del-HV69/70- and N501Y variants such as B.1.1.7.
Evaluation of a fully automated high-throughput SARS-CoV-2 multiplex qPCR assay with built-in screening functionality for del-HV69/70- and N501Y variants such as B.1.1.7.
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DOI：
10.1016/j.jcv.2021.104894
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发表时间：
2021-08
期刊：
影响因子：
--
通讯作者：
Lütgehetmann M
中科院分区：
文献类型：
--
作者：
Nörz D;Grunwald M;Olearo F;Fischer N;Aepfelbacher M;Pfefferle S;Lütgehetmann M
新的SARS-CoV-2变种具有更高的传播性，如首次在英国发现的B.1.1.7或首次在南非发现的B.1.351，引起了全世界的极大关注。为了遏制这些谱系的传播，拥有快速、灵敏和高通量的检测方法至关重要。对一组RT-qPCR测定进行了修改，用于诊断SARS-CoV-2多重测定，包括在cobas 6800平台上检测del-HV 69/70和N501 Y突变。通过连续稀释评估野生型SARS-CoV-2和B.1.1.7谱系的分析灵敏度。对于临床性能，共对176份临床样本进行了检测，并将结果与商业手动分型PCR检测试剂盒和作为金标准的下一代测序进行了比较。该方法对临床标本中SARS-CoV-2 RNA的检测具有较高的灵敏度，检出限为6.16 cp/ml（CI：4.00-8.31）。检测HV 69/70缺失（85.92，CI：61-194.41）和N501 Y SNP（105.99 cp/ml，CI：81.59 - 183.66）的LoD略高。共检测了176份临床样本，包括50份含有B.1.1.7谱系的SARS-CoV-2的样本，1份含有B.1.351和85份非B.1.1.7/B.1.351谱系的样本，其中3份还含有HV 69/70缺失。所有这些都通过多重测定正确鉴定。我们在此描述了一种高灵敏度的全自动多重PCR检测方法，用于同时检测del-HV 69/70和N501 Y突变，可以区分B.1.1.7和其他谱系。该测定允许在测序之前高通量筛选临床样品中当前相关的变体。
New SARS-CoV-2 variants with increased transmissibility, like B.1.1.7, first detected in England or B.1.351, first detected in South Africa, have caused considerable concern worldwide. In order to contain the spread of these lineages, it is of utmost importance to have rapid, sensitive and high-throughput detection methods at hand. A set of RT-qPCR assays was modified for a diagnostic SARS-CoV-2 multiplex assay including detection of the del-HV69/70 and N501Y mutations on the cobas6800 platform. Analytical sensitivity was assessed for both wild-type SARS-CoV-2 and B.1.1.7 lineage by serial dilution. For clinical performance, a total of 176 clinical samples were subjected to the test and results compared to a commercial manual typing-PCR assay and next generation sequencing as gold standard. The multiplex assay was highly sensitive for detection of SARS-CoV-2 RNA in clinical samples, with an LoD of 6.16 cp/ml (CI: 4.00–8.31). LoDs were slightly higher for detection of the HV69/70 deletion (85.92, CI: 61–194.41) and the N501Y SNP (105.99 cp/ml, CI: 81.59 – 183.66). A total of 176 clinical samples were tested with the assay, including 50 samples containing SARS-CoV-2 of the B.1.1.7 lineage, one containing B.1.351 and 85 non-B.1.1.7/B.1.351 lineage, of which three also harbored a HV69/70 deletion. All were correctly identified by the multiplex assay. We describe here a highly sensitive, fully automated multiplex PCR assay for the simultaneous detection of the del-HV69/70 and N501Y mutations that can distinguish between B.1.1.7 and other lineages. The assay allows for high-throughput screening for currently relevant variants in clinical samples prior to sequencing.
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DOI：
10.1016/s2213-2600(21)00005-9
发表时间：
2021-03
期刊：
The Lancet. Respiratory medicine
影响因子：
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