Highly sensitive and specific detection of the SARS-CoV-2 Delta variant by double-mismatch allele-specific real time reverse transcription PCR.
Highly sensitive and specific detection of the SARS-CoV-2 Delta variant by double-mismatch allele-specific real time reverse transcription PCR.
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DOI：
10.1016/j.jcv.2021.105049
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发表时间：
2022-01
期刊：
影响因子：
--
通讯作者：
McClure MO
中科院分区：
文献类型：
--
作者：
Garson JA;Badru S;Parker E;Tedder RS;McClure MO
SARS-CoV-2 的高度传播性 Delta 变种 (B.1.617.2) 首次在印度发现，目前正在世界许多地区取代现有的变种。为了帮助指导公共卫生政策，有效监测其传播非常重要。基因组测序是鉴定 Delta 的黄金标准，但耗时、昂贵且在许多地区无法实现。开发和评估一种快速、简单且廉价的 Delta 鉴定测序替代方案。开发了双错配等位基因特异性 RT-PCR (DMAS-RT-PCR)。该技术利用等位基因特异性引物，针对同一扩增子内的两个刺突基因突变 L452R 和 T478K。位于从 3' 端算起的第四个核苷酸处的额外错配增强了每个引物的区分能力。通过测试经过充分表征的细胞培养物衍生的病毒分离株和临床样本来评估特异性，其中大部分样本此前已进行过完全测序。在所有情况下，通过 DMAS-RT-PCR 进行的病毒基因分型结果与测序结果完全一致，并且该检测方法能够可靠地区分 Delta 变体和其他变体（Alpha 和 Beta）以及“野生型”SARS-CoV-2。甲型流感和 RSV 在检测中没有反应。 DMAS-RT-PCR 的灵敏度与诊断 SARS-CoV-2 RT-qPCR 筛查测定的灵敏度相匹配。一些因病毒不足而无法测序的样本通过 DMAS-RT-PCR 成功进行了基因分型。我们描述的方法很容易在任何可以进行 PCR 检测的实验室中建立，并且应该极大地促进监测 Delta 变种在全球的传播。
The highly transmissible Delta variant of SARS-CoV-2 (B.1.617.2), first identified in India, is currently replacing pre-existing variants in many parts of the world. To help guide public health policies it is important to monitor efficiently its spread. Genome sequencing is the gold standard for identification of Delta, but is time-consuming, expensive, and unavailable in many regions. To develop and evaluate a rapid, simple and inexpensive alternative to sequencing for Delta identification. A double-mismatch allele-specific RT-PCR (DMAS-RT-PCR) was developed. The technique exploits allele-specific primers, targeting two spike gene mutations, L452R and T478K, within the same amplicon. The discriminatory power of each primer was enhanced by an additional mismatch located at the fourth nucleotide from the 3′ end. Specificity was assessed by testing well characterised cell culture-derived viral isolates and clinical samples, most of which had previously been fully sequenced. In all cases the results of viral genotyping by DMAS-RT-PCR were entirely concordant with the results of sequencing, and the assay was shown to discriminate reliably between the Delta variant and other variants (Alpha and Beta), and ‘wild-type’ SARS-CoV-2. Influenza A and RSV were non-reactive in the assay. The sensitivity of DMAS-RT-PCR matched that of the diagnostic SARS-CoV-2 RT-qPCR screening assay. Several samples that could not be sequenced due to insufficient virus were successfully genotyped by DMAS-RT-PCR. The method we describe would be simple to establish in any laboratory that can conduct PCR assays and should greatly facilitate monitoring of the spread of the Delta variant globally.
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DOI：
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发表时间：
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期刊：
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