Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR

Abstract

© 2020, The Author(s). 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