The COVID-19 pandemic has exploded globally in a matter of months, accumulating over one million reported cases by the beginning of April. The new respiratory disease, caused by the zoonotic coronavirus, SARS-CoV-2, is a close relative of an earlier SARS virus first described in 2002. 

The scientific community has quickly risen to the occasion and reports of new developments have arrived at an unprecedented scale—both in academic findings and in the biomedical industry. Of note is the response to the need for widespread diagnostic testing. Currently, there is a growing list of over 400 SARS-CoV-2 diagnostic tests worldwide either in development or approved for clinical use. 

As tools that analyze human samples with direct effects on health, in vitro diagnostic tests around the world undergo varying levels of regulation. In the United States, such tests must be independently validated by the Food and Drug Administration (FDA) before and after entry into the market. This process can range from several months to a few years. 

Because extraordinary times call for special measures, the US in February approved the emergency use of medical countermeasures, enabling the emergency use authorization (EUA) for SARS-CoV-2 diagnostic tests. With the new EUA designation, new diagnostic tests can reach the market a lot faster. 

Within the first of such tests to be approved was the Centers for Disease Control and Prevention's (CDC) diagnostic panel. The FDA has since granted emergency authorizations for a growing list of tests. To expedite the approval process, a submission template has been created, outlining requirements needed for EUA approval. Validation must be reported in these key areas:

  • Limit of Detection (LoD) — the LoD must be determined for the device utilizing the entire test system from sample preparation to detection. 
  • Inclusivity — inclusivity studies must demonstrate 100% detection of all SARS-CoV-2 strains.

  • Cross-reactivity — studies must demonstrate that the test does not react with related pathogens, other prevalent disease agents, or other coincidental pathogenic flora. 

  • Clinical evaluation — while natural clinical specimens are preferred, representative reactive specimens can be used. Acceptance criteria is defined at 95% agreement at 1x-2x LoD, and 100% agreement at all other concentrations and for negative specimens.

  • Instrument requirements — the manufacturer must specify the instruments used with the test, including RT-PCR systems, software, and any automation equipment. 

The chart below lists the in vitro diagnostic tests that have been granted emergency use authorization by the US FDA. Scroll right to see notes on the assay type, target, methodology, and required equipment. 

Approved In Vitro Diagnostic Tests

Most EUA diagnostic tests utilize real-time reverse transcription PCR (also known as rRT-PCR or qRT-PCR), which uses sequence-specific primers to detect the presence of SARS-CoV-2 viral RNA. The general workflow proceeds from RNA extraction from swab specimen samples, reverse transcription, PCR amplification and detection using a real-time PCR instrument. 

Assay targets include regions from the following SARS-CoV-2 genes: nucleoprotein (N), envelope (E), and the polyprotein ORF1ab, which includes the replicase RdRP. The human RNase P gene is a common control for effective RNA extraction. 

Materials typically included in the tests are the primer and probe sets, an enzyme mix containing reverse transcriptase, DNA polymerase and dNTPs, and controls. Any materials not included are further specified, such as RNA extraction reagents, general consumables, and equipment. 

As more tests receive EUA approval, new variations emerge in terms of scale, methodology, and speed. The earliest tests have relied on manual methods, using kit-based RNA extraction followed by detection on a real-time PCR system. Increasingly, new tests are approved that are validated to use automated extraction instruments, as well as fully integrated systems for complete process automation. A handful of tests also feature disposable intermediaries, such as cassettes for directly testing specimens, omitting nucleic acid extraction. 

We may expect to see more diagnostic tests being granted EUA in the near future and hopefully, these can make coronavirus testing more widespread and accessible. Clinical laboratories and testing institutions still deciding on a particular test may weigh certain aspects, such as throughput or access to instruments, that best suit their operational capabilities. 

Additional Resources