The most critical part of a molecular detection assay is proper amplification of sequences specific to the target virus. The first step will be to design primer sequences that amplify regions present only in the chosen virus, and also allow efficient replication of the sequences. This can be a difficult and time-consuming process; however, in many cases consensus sequences are rapidly published by leading infectious disease laboratories such as the Centers for Disease Control and Prevention (CDC). For example, primer sequences were made available for the 2019-COVID (2019 nCoV) virus by the CDC within two months of the first case identified in China.
After establishing primer sequences, a lab should consider PCR or qPCR reagents that allow rapid, consistent and efficient amplification of the target amplicon. Given that most viruses are RNA-based, we will focus on selection of a real-time RT-qPCR reagent set. Commercially available products can come in both 1-step and 2-step versions, allowing users the flexibility to create their protocol as needed. However, most laboratories will prefer the use of a 1-step RT-qPCR product. Utilization of dUTP incorporated into the amplification products is recommended as the resulting amplicons are susceptible to degradation by uracil-DNA glycosylase (UNG); allowing users to incorporate UNG into subsequent reactions for control of possible carryover contamination. Assay designers may also wish to incorporate an RNase inhibitor to minimize loss of target.
Identifying the presence of the target requires a quick, reliable method of detection. Real-time qPCR instruments are readily available from multiple suppliers, but considerations should be given to speed and color detection capabilities. Simple machines are sufficient if you will be detecting a single target per well, but as assays become more complex (with multiplexed targets or internal controls incorporated) more complex or expensive instrumentation is required. Balance of signal per target and proper dye selection for filter sets available on a given instrument must be considered.