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SARS-CoV-2 PCR screening test by nasal swab in Strasbourg on August 21, 2020.

In the previous chapter, the most common testing methodologies for COVID-19 and other coronaviruses were discussed in detail. The prevailing method (often called the "gold standard") among them all is real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays for testing. Broadly speaking, PCR is useful in pharmaceutical, biotechnology, and genetic engineering endeavors, as well as clinical diagnostics. As such, labs in those industries that already have PCR infrastructure in place have a theoretical step-up over labs that do not.

PCR technology has advanced to the point where it is more efficient and user-friendly than prior, yet "the high cost of the instruments, servicing contracts, and reagents pose major challenges for the market, especially to the price-sensitive academics." Writing about the thirty-fifth anniversary of PCR in 2018, science writer Alan Dove not only highlighted these cost issues but also the size and energy requirements for running the equipment. "As a result, one of the defining techniques of modern molecular biology has remained stubbornly inaccessible to educators and unusable in many remote locations." Various efforts have been made over the years to bring costs down by modifying how heating and temperature control are performed, but many of those system aren't typically optimal during a pandemic when turnaround time is critical.

Amidst the pandemic, additional challenges also exist to those wanting to conduct PCR testing for COVID-19 and other viruses. As was discussed at the end of the previous chapter, supplies of reagents and consumables are not particularly robust mid-pandemic, with various shortages being reported off and on since the start of the pandemic. Some of these shortages have gradually worked themselves out over time, but they highlight the need for other varying methods that don't necessarily depend on the same reagents and consumables that are in short supply.

For those labs wishing to adopt PCR testing of viruses—particularly COVID-19—into their workflow while providing reasonable turnaround times, all is not lost. However, careful planning is required. For example, you'll want to keep in mind that some PCR machines require vendor-specific reagents. If you're going to acquire a particular instrument, you'll want to do due diligence by verifying not only the supported reagents but also those reagents' overall availability (real and projected). You'll also want to consider factors such as anticipated workload (tests per day), what your workflow will look like, and how to balance overall investment with the need for reasonable turnaround times.

An increasing body of research is being produced suggesting ways to improve turnaround times with PCR testing for COVID-19, with many research efforts focusing on cutting out RNA extraction steps entirely. Alcoba-Florez et al. propose direct heating of the sample-containing nasopharyngeal swab at 70 °C for 10 minutes in place of RNA extraction. Adams et al. have proposed an "adaptive PCR" method using a non-standard reagent mix that skips RNA extraction and can act "as a contingency for resource‐limited settings around the globe." Wee et al. skip RNA extraction and nucleic acid purification by using a single-tube homogeneous reaction method run on a lightweight, portable thermocycler. Other innovations include tweaking reagents and enzymes to work with one step, skipping the reverse transcription step, and using saliva-based molecular testing that skips RNA extraction.

Saliva as a specimen

The saliva molecular tests in particular are intriguing. Talk of the potential utility of using saliva as a specimen for COVID-19 was occurring as early as April 2020, and the first saliva-based COVID-19 test, produced by Spectrum Solutions in cooperation with RUCDR Infinite Biologics Laboratory and Vault Health, was given an FDA EUA in April 2020. On August 15, 2020, Yale School of Public Health was given an EUA for it SalivaDirect molecular test. Although still PCR-based (and a CLIA high-complexity test), SalivaDirect is being touted as a means to improve specimen collection safety, consume fewer reagents, prove compatible with high-throughput workflow, and cut overall turnaround time. Not only is saliva easier to collect and safer for healthcare staff, the test is essentially "open sourced," not requiring proprietary equipment from Yale, making the test more flexible by being validated to reliably function with a wider array of reagents and instruments. When compared to using a nasopharyngeal swab specimen using the ThermoFisher Scientific TaqPath COVID-19 combo kit, results were comparable 94.1% of the time. While sensitivity and specificity may be slightly less comparable to other PCR options, the overall advantages during reagent shortages and a definitive need for broader testing likely outweigh the slightly lesser sensitivity and specificity. In November 2020, public health agencies in Arizona and Minnesota reportedly began running trials of free saliva-based molecular testing.

As the pandemic has progressed into 2021, saliva testing has become even more attractive, in particular for at-home over-the-counter testing. In August 2021, Spectrum Solutions received an EUA for its Spectrum Solutions SDNA-1000 saliva collection system, specifically designed "to avoid user collection errors" and eliminate "the requirement for any bio-sample temperature-controlled storage or transport," arguably upping the game for new saliva-based test kits going forward. Additionally, as variants of COVID-19 continue to crop up, additional saliva-based at-home tests are coming into development. For example, researchers at the Wyss Institute, the Massachusetts Institute of Technology, and Boston-area hospitals have been working on a laboratory-developed test called Minimally Instrumented SHERLOCK (miSHERLOCK) based on CRISPR (clustered regularly interspaced short palindromic repeats) technology. The researchers claim that the test, able to be used with typical off-the-shelf components, "works as well as the gold standard PCR tests and could cost as little as $3 per test."