SARS-CoV-2, the causative agent of COVID-19 (coronavirus disease 2019), is a member of the SARS-related species of coronaviruses. This strain is the most recent addition to the list of seven known coronaviruses that are pathogenic to humans. Included here are SARS-CoV and MERS-CoV, which cause the SARS and MERS diseases, respectively. Coronaviruses are enveloped RNA viruses that cause upper respiratory tract diseases in humans. A more detailed explanation of the SARS-CoV-2 life cycle has been previously discussed. 

Two general methods are used in the diagnostic detection of COVID-19: the sequence-specific molecular nucleic acid test and the antigen-specific immunoassay. These techniques are the current gold standards for diagnosing acute infection and for monitoring immune response. There are now over 70 COVID-19 diagnostic tests that have been authorized for emergency use by the U.S. FDA. The vast majority are molecular assays, while approximately one-fifth are antibody immunoassays. In this editorial, we will describe the general workflow for these methods with a focus on the various tools involved in the process.

Molecular Assays

The SARS-CoV-2 molecular assay operates on the detection of specific genetic sequences within the viral genome, typically through the use of gene-specific primers. Molecular diagnostic tests that have been released generally utilize real-time reverse-transcription PCR (also known as real-time RT-PCR or qRT-PCR). However, other variations have also been developed.

Sample collection and RNA extraction

Because coronaviruses contain RNA genomes, RNA serves as the starting material for the assay. Samples are collected from upper respiratory fluids that may contain viral particles. This may include swab samples or aspirates from the nasal cavity, nasopharynx, and throat, as well as saliva. Total viral RNA must then be extracted from the specimen samples. To ensure rapid and reproducible collection of high-quality RNA, some diagnostic tests have validated the use of RNA purification kits. These kits are ready to use and contain all necessary components, such as cell lysis reagents, RNase inhibitors, collection tubes, and binding beads.

Real-time RT-PCR

Real-time RT-PCR is a multi-step technique for quantifying sequences within RNA samples. Using reverse transcriptase, extracted RNA is converted to cDNA, serving as the template for the following PCR-based amplification. A DNA polymerase guided by sequence-specific primers will amplify the genetic regions of interest while fluorescent probes instantaneously bind to the newly synthesized fragments. Each amplification cycle results in a measurable intensity of fluorescence that, when compared to a standard curve, can be used to quantify the target sequence. For specific detection of SARS-CoV-2 RNA, highly specific genes to the strain would be most ideal. Among the approved diagnostic tests, common targets include genetic regions within the SARS-CoV-2 nucleocapsid (N), envelope (E), spike (S), and ORF1ab. 

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The defining instrument required for this application is the real-time PCR system, which functions as a PCR thermal cycler as well as a fluorescence detector. There are a variety of these options in the market with varying features, such as plate format, fluorescence channels, and validated use in diagnostics. Similarly, a number of coronavirus qRT-PCR assays are increasingly becoming available for both diagnostic and research use. These complete kits contain the key components required for the molecular assay, including primer and probe sets, enzymes, buffers, and nucleotides.

Other molecular assays

With more assay manufacturers providing new options for molecular assays, we also begin to see tests that use methods outside of qRT-PCR. One example is digital PCR (dPCR), whose principle is to carry out PCR reactions within thousands of individually partitioned droplets. Each reaction is digitally detected, enabling absolute quantification of DNA. This method has been reported to accurately quantify samples that suffer from low amounts of nucleic acid or from variable levels of protein contamination. Other molecular diagnostic methods that have been recently granted emergency authorization include endpoint RT-PCR, rapid isothermal amplification, and RT-LAMP. 

Assay-enhancing tools

While most instruments can accommodate 96-well formats, some are designed to also work with 384-well microplates, which drastically increases the number of samples that can be analyzed. The number of fluorescent channels equipped in the instrument can also be considered, as this allows the multiplexed detection of multiple targets at once. For even greater throughput, steps within the assay workflow can be automated by robotic workstations, such as for nucleic acid extraction and PCR-related sample prep. For any type of molecular work, the use of PCR hoods is encouraged, as these enclosures minimize the flow of air within the experimental area. Note that when dealing with pathogenic agents, appropriate safety precautions should be observed. Other real-time PCR–related accessories or consumables can also be explored here. 

Immunoassays

Immunoassays work on the principle of specific antibody-to-antigen binding, in which antibodies are used to detect and measure unique signatures of pathogen infection. Viruses can be directly recognized through targeting of specific viral proteins or antigens. Alternatively, serum immunoglobulins (such as IgG, IgA, and IgM) that an individual has produced as part of an immune response can also be measured. These types of immunoassays are known as antibody tests. A number of immunoassays have received approval for diagnostic use for COVID-19, utilizing formats like ELISA and lateral flow. 

ELISA

The enzyme-linked immunosorbent assay (ELISA) uses enzyme-substrate reactions to produce measurable signals proportional to the concentration of the target analyte. A defining feature of the ELISA is the immobilization of the target antigen upon a solid surface, usually in microplate wells. This can occur by coating the surface with specific antibodies. If the targets are immunoglobulins, the surface is coated with recombinant proteins instead. The general procedure follows in a set of sequential steps: sample application and analyte binding, addition of enzymes conjugated to specific antibodies (usually alkaline phosphatase or horseradish peroxidase), incubation with the chemical substrate, and detection on a microplate reader. ELISA kits are sensitive, quantitative tests that can be used to measure a variety of analyte types within complex samples. 

Lateral flow immunoassays

In this chromatography type of assay, capture antibodies are immobilized onto the surface of a support membrane (in antibody tests, recombinant proteins are immobilized). When the sample is applied, its various components will travel across the membrane. The target analytes, however, will become trapped in the designated region, producing a color observable in plain sight or with a detection instrument. Compared to the ELISA, lateral flow assays are generally qualitative, rather than quantitative. However, the relative simplicity of the procedure has led to the widespread development of various point-of-care and direct-to-consumer devices.

Immunoassay tools

Many tools are available that make it possible to run immunoassay experiments or even develop new, customized tests. At the heart of the assay are specific SARS-CoV-2 antibodies for targeting various antigens. Recombinant SARS-CoV-2 proteins can be used to develop controls or targets or to serve as antigens for immunoglobulins. Plate readers, essential in the analysis of microplate-based assays, come in many models with an assortment of features. For more related reagents and accessories, check out this comprehensive ELISA product listing.

Contrasting roles in diagnostics

There are several fundamental differences between molecular tests and immunoassays. Molecular assays are sensitive to viral nucleic acids, while immunoassays detect proteins and antigens. The experimental workflows also differ. Molecular tests generally require more steps and instruments, thus taking more time to complete. PCR amplification, which involves repetitive temperature cycling, is a rate-limiting step that takes roughly 1–2 hours to complete. Including sample prep, RNA extraction, and data analysis, a full manual procedure can take several hours. In contrast, immunoassay procedures are relatively simpler and require shorter incubations. ELISAs can take a few hours while some rapid disposable tests can produce results within the hour. Notably, both ELISAs and real-time RT-PCR assays in multi-well formats are scalable and can undergo automated processes, greatly increasing throughput. 

As for diagnostic roles, only molecular tests are currently intended to diagnose acute infection, as these recognize viral components directly from respiratory samples. On the other hand, antibody tests inform whether the body has responded to an infection but not when the infection has taken place. While these tests cannot diagnose infected individuals, they can be used to conduct serosurveys—studies to determine which people have developed antibodies against SARS-CoV-2. Serosurveys are important in that they can be used to determine sources of convalescent plasma as well as to track the spread of the disease within a population. The growing development centered around these two contrasting test methods presents a hopeful avenue in mitigating the COVID-19 pandemic, which has now claimed over 320,000 lives worldwide. 

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