One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection
One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection
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DOI：
10.3855/jidc.12726
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发表时间：
2020-07-01
影响因子：
1.9
通讯作者：
Delgado-Enciso, Ivan
中科院分区：
文献类型：
--
作者：
Meza-Robles, Carmen;Barajas-Saucedo, Carlos E.;Delgado-Enciso, Ivan
简介：由于冠状病毒大流行，识别感染者已成为限制其传播的关键。病毒核酸实时RT-PCR检测已成为目前的标准诊断方法，但高需求可能导致短缺。因此，我们提出了一种检测策略，使用一步巢式RT-PCR。方法：ORF 1ab基因中的核苷酸区域，具有最大的差异之间的人冠状病毒和蝙蝠冠状病毒的选择。在该序列之后设计引物。所有诊断引物都是种特异性的，因为序列的3 '端不同于其他物种的序列。引物组还产生合成阳性对照。扩增产物在2.5%琼脂糖凝胶中可见，以及在SYBR Green为基础的实时RT-PCR.Results：扩增阳性对照和特定区域在两种techniques.Conclusions：这种新技术是灵活的，易于实施。它不需要实时热循环仪，可以在琼脂糖凝胶中进行解释，并适用于定量病毒基因组。它的优点是，如果冠状病毒在关键扩增核苷酸之一发生突变，至少有一对仍然可以扩增，这要归功于四个诊断引物。
Introduction: Due to the coronavirus pandemic, identifying the infected individuals has become key to limiting its spread. Virus nucleic acid real-time RT-PCR testing has become the current standard diagnostic method but high demand could lead to shortages. Therefore, we propose a detection strategy using a one-step nested RT-PCR.Methodology: The nucleotide region in the ORF1ab gene that has the greatest differences between the human coronavirus and the bat coronavirus was selected. Primers were designed after that sequence. All diagnostic primers are species-specific since the 3 ' end of the sequence differs from that of other species. A primer set also creates a synthetic positive control. Amplified products were seen in a 2.5% agarose gel, as well as in an SYBR Green-Based Real-Time RT-PCR.Results: Amplification was achieved for the positive control and specific regions in both techniques.Conclusions: This new technique is flexible and easy to implement. It does not require a real-time thermocycler and can be interpreted in agarose gels, as well as adapted to quantify the viral genome. It has the advantage that if the coronavirus mutates in one of the key amplification nucleotides, at least one pair can still amplify, thanks to the four diagnostic primers.