Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR
Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR
复制标题
DOI：
10.1038/s12276-020-0452-7
复制
发表时间：
2020-06-16
影响因子：
12.8
通讯作者：
Lee, C. Justin
中科院分区：
文献类型：
--
作者：
Park, Myungsun;Won, Joungha;Lee, C. Justin
SARS-CoV-2传染性极强，已在全球迅速传播。由于各种有症状和无症状的病例以及无症状传播的可能性，迫切需要一种快速灵敏的检测方案来诊断无症状者。许多公司和国家卫生机构已经提供了各种SARS-CoV-2诊断试剂盒。然而，缺乏关于这些诊断试剂盒的公开信息。为了应对日益增长的需求和缺乏信息，我们开发并提供了一种低成本，易于访问，基于实时PCR的协议，用于早期检测病毒在以前的研究。在检测方案的开发过程中，我们发现未优化的引物组可能会无意中显示假阳性结果，这增加了商业上可用的诊断试剂盒也可能包含产生假阳性结果的引物组的可能性。在这里，我们为特异性引物组的设计和优化提供了三步指南。这三个步骤包括（1）选择目标基因组（SARS-CoV-2）中靶基因（RdRP、N、E和S）的引物组，（2）引物和扩增子序列的计算机验证，和（3）PCR条件的优化（即，引物浓度和退火温度）用于引物和靶基因之间的特异性杂交，以及消除假引物二聚体。此外，我们已经扩大了以前开发的实时PCR为基础的协议，更传统的PCR为基础的协议，并应用多重PCR为基础的协议，允许同时测试引物集为RdRP，N，E，andSall在一个反应。我们新优化的方案应该有助于在没有任何高规格设备的情况下，对无症状人群进行大规模、高保真的筛查，进一步预防传播，并实现对快速传播病毒的早期干预和治疗。诊断微生物学：增强对COVID-19检测的信心病毒检测测试的设计策略可能会提高对SARS-CoV-2冠状病毒的检测。实验室目前进行SARS-CoV-2诊断分析的基础上，一种技术称为聚合酶链反应（PCR），它可以有针对性地扩增病毒核酸。PCR检测是敏感的，但可能受到假阳性或假阴性的困扰。C.韩国大田基础科学研究所的Justin Lee及其同事设计了一种优化分析性能的方案。PCR检测的成功取决于“引物”的选择，即决定扩增哪个基因组序列的短DNA链。研究人员确定了一种设计有效引物组的策略，并选择了最佳利用这些引物的反应条件。虽然这里的重点是SARS-CoV-2，但这种方法应该适用于其他病毒。
SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP,N,E, andS) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets forRdRP,N,E, andSall in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.Diagnostic microbiology: Boosting confidence in COVID-19 detection A design strategy for virus detection tests could lead to improved detection of SARS-CoV-2 coronavirus. Laboratories currently perform SARS-CoV-2 diagnostic assays based on a technique called the polymerase chain reaction (PCR), which enables targeted amplification of viral nucleic acids. PCR assays are sensitive but can be plagued by false positives or false negatives. C. Justin Lee of the Institute for Basic Science, Daejon, Republic of Korea, and coworkers have devised a protocol for optimizing assay performance. The success of a PCR assay is determined by the selection of 'primers', short DNA strands that determine which genomic sequence gets amplified. The researchers identify a strategy for designing effective primer sets, and selecting reaction conditions that make best use of those primers. Although the focus here is on SARS-CoV-2, this approach should be applicable to other viruses.