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首页> 外文期刊>Journal of Clinical Microbiology >Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern
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Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern

机译：单次扩增子多重实时逆转录转录-PCR与平铺探针检测SARS-COV-2 <斜体切换=“是”>尖峰与关注变体相关的突变

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ABSTRACT To provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (SNP) assay, to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348-bp region of spike , and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log _(10) genome equivalents (GE)/ml for the three initial targets (～1 to 2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with threshold cycle ( C _(T) ) values?of &30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 C _(T) values?of ≥30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in the receptor binding domain, and it can be quickly modified to detect new mutations that emerge.
机译：摘要提供一种可进入和廉价的方法对严重急性呼吸综合征冠状病毒2（SARS-COV-2）突变的突出，我们开发了一种多重实时逆转录-PCR（RRT-PCR）测定，尖刺单核苷酸多态性（SNP）测定，检测尖峰受体结合结构域中的特定突变。设计单个引物对以扩增348-BP区域的穗区，并且最初设计探针以检测K417，E484K和N501Y。使用表征变体样品池和残留的鼻咽样品评估测定。 SARS-COV-2基因组测序在样品的子集中证实了变体调用。随后，设计了第四探针以检测L452R。 95％检测的下限为2.46至2.48 log _（10）基因组当量（Ge）/ ml对于三个初始靶标（〜1至2ge /反应）。在具有可检测的SARS-COV-2 RNA的253次残留的鼻咽拭子中，刺激SNP测定在238（94.1％）样品中为阳性。所有220个样品，具有阈值循环（C _（t））值？＆amp; LT; 30用于SARS-COV-2 N2靶标，而18/33用N2 C _（T）值的样品≥30被发现了。通过在50/50样品（100％）中测序来证实刺SNP结果。添加452R探针并未影响原始目标的性能。尖峰SNP测定精确地识别受体结合结构域中的SARS-COV-2突变，并且可以快速修改以检测出现的新突变。

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