Considerations for PCR-Based Viral Assay Development

The development of any viral detection assay begins with extraction of viral nucleic acids.  Choosing a protocol can be challenging, with the need to balance the chemistry’s ability to break apart the virus particle without damaging the nucleic acids, while at the same time maximizing recovery of genetic material. Sample matrices can vary widely from plasma or serum to swabs or other media that may contain PCR inhibitors. Fortunately, there are a wide variety of commercial extraction kits available to laboratories with established protocols. Users should consider whether they want to isolate only DNA or RNA, or total nucleic acid. Pre-processing requirements may also vary depending on the starting material. In addition laboratories will need to decide between manual and automated extraction methods based on sample processing needs, including throughput, sample number and time required. For automated platforms, finding instruments that fit in existing airflow hoods is also important to minimize risk of exposure.

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.

With any detection assay, appropriate controls are required to ensure the test is working as expected. For negative controls, it is recommended that the well contain a known-negative nucleic acid target and not just water, as exclusion of any nucleic acid in the control would miss false-positive results from non-specific primer binding or amplification. Positive controls can be incorporated internally within the assay tube (with alternative sequences or reporter dyes) or performed in a separate well run simultaneously with the unknown sample. Incorporation of an internal positive control is the surest method for confirming that all aspects of the assay are working correctly, but can result in decreased sensitivity due to multiplexing (1).

With the development of any assay, the user must verify that the test is performing as expected.  Measuring accuracy (compared to a reference technique), precision (reproducible results), sensitivity (false negatives, specificity (false positives), and range of detection. Guidance for testing these parameters is available from resources such as the Association for Molecular Pathology .

There are many parameters to consider when developing an assay for virus detection. After establishing the base technology (extraction method, amplification reagents and detection methods), detection of future targets is more straightforward. Utilization of readily available commercial products with proven characteristics will further enhance the process. However, with proper planning and research a laboratory can rapidly create a reproducible method.