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3. Adding COVID-19 and other virus testing to your laboratory

Does using one method make the most sense, or will your lab turn to multiple methods for virus testing?

3.1 What methodologies will you use?

3.1.1 PCR

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 was 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 ifrastructure in place have a theoretical step-up over a lab that doesn't.

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 requirments 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 shortages being reported since March 2020. These shortages may eventually work themselves out, 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 investent with the need for reasonable turnaround times.

As of August 2020, 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.

3.1.2 Pooled testing

Another method some labs are taking to speed up turnaround time is using pooled testing. The general concept involves placing two or more test specimens together and testing the pool as one specimen. The most obvious adventage to this is that the process saves on reagents and other supplies, particularly when supply chains are disrupted. This methodology is best used "in situations where disease prevalence is low, since each negative pool test eliminates the need to individually test those specimens and maximizes the number of individuals who can be tested over a given amount of time." However, it's best left to situations where expectations are that less than 10 percent of the population being tested is affected by what's being tested for.

The downside of pooled testing comes with the issues of dillution, contamination, and populations with 10 or more percent infected. A target-positive specimen that comingles with other target-free specimens is itself diluted and in some cases may cause issues with the limit of detection for the assay. Additionally, if the pool tests positive, target-free specimens may become contaminated by a target-positive specimen. This may cause issues with any individual specimen assays that get ran. And the workflows involving pooling must be precise, as a technician working with multiple specimens at the same time increases the chance of lab errors.Finally, at least in the U.S., an Emergency Use Authorization for a validated pooled testing method is required. (Validation of pooled methods may differ in other countries.) The U.S. Centers for Disease Control and Prevention (CDC) has published interim guidance on pooled testing strategies for SARS-CoV-2.

3.1.3 Rapid antigen testing

An antigen is a substance—often a protein but may also be an environmental like a virus—that provokes the immune system to produce an antibody against it. As such, another approach to testing for the presence of a virus in a specimen is to test for the antigen rather than the antibody. On one hand, antigen testing is more rapid and convenient because the extraction and amplification steps of PCR are not use, but on the other, antigen testing is less sensitive for the same reason: you test only what's there (rather than amplifying the amount for greater sensitivity).

A theory increasingly gaining traction, however, is that "[a] higher frequency of testing makes up for poor sensitivity.”

• Also see Nature article below

3.1.4 LAMP and CRISPR

Alternatives such as LAMP and CRISPR:

• https://www.fiercebiotech.com/medtech/oxford-researchers-develop-portable-covid-19-test-costing-less-than-25 (LAMP)
• (LAMP, sequencing, CRISPR, antigen)
• https://thejewishvoice.com/2020/08/10-ways-israeli-scientists-are-improving-corona-testing-part-1/ (Rapid saliva, breath, AI, spectrometry, ultrahigh frequency radiation)

3.1.5 Point-of-care testing

POLs:

• https://hitconsultant.net/2020/08/03/talis-nih-contract-for-point-of-care-covid-19-testing/
• https://www.nih.gov/news-events/news-releases/nih-delivering-new-covid-19-testing-technologies-meet-us-demand

3.2 What equipment and supplies will you need?

Instruments

Eppendorf Mastercycler Pro S, a thermal cycler for PCR and other applications

Thermal cyclers (a.k.a. PCR machines) - standard PCR systems, RT PCR - advantages of digital PCR systems such as precision, sensitivity, accuracy, reproducibility, direct quantification and multiplexing, and speed of the analysis systems, and digital PCR systems "The market is witnessing an emerging trend of digital and droplet digital PCR technology, which is sensitive and accurate than the traditional method."

https://www.thermofisher.com/search/browse/category/us/en/602552/PCR+Machines+%28Thermal+Cyclers%29+%26+Accessories https://blog.biomeme.com/how-do-you-test-for-covid-19

Reagents

template DNA, PCR primers and probes, dNTPs, PCR buffers, enzymes, and master mixes

Consumables

PCR tubes, plates, and other accessories https://www.sigmaaldrich.com/labware/labware-products.html?TablePage=9577275

Vendors

Major players operating in the global PCR market are Bio-Rad Laboratories, Inc., QIAGEN N.V., F. Hoffmann-La Roche AG, Thermo Fisher Scientific, Inc. Becton, Dickinson and Company, Abbott, Siemens Healthcare GmbH (Siemens AG), bioMérieux SA, Danaher Corporation, and Agilent Technologies. Merck KGaA, Promega