“Testing, testing, testing!” is a widely touted slogan for controlling the COVID-19 pandemic. The World Health Organization and other federal/state agencies around the world have all publicized this familiar catchphrase. Indeed, testing human populations rapidly is the cornerstone for controlling a pandemic like COVID-19.
Extensive testing provides a rational basis for implementing public health strategies that can prevent further spread of the disease. It allows authorities to make informed decisions about mitigating policies such as social distancing, stay-at-home and, in extreme cases, curfew. In the wake of an air-borne disease like COVID-19, these crucial public health initiatives are vital for reducing the pressure on health care systems and saving human lives.
Genome-based analysis is the key to designing a test kit specific to SARS-CoV-2. This is the case whether we are designing DNA-based kits (PCR, isothermal amplification, CRISPR) or generating antigens for serological detection. A global non-profit organization has collated a list of all the SARS-CoV-2 tests that are commercially available. Here we will discuss the two main techniques that are currently in use: DNA-based testing and antibody-based testing.
Current testing for COVID-19 performed on viral genetic material from nose and throat swabs uses a common molecular biology technique called Reverse Transcription Polymerase Chain Reaction (RT-PCR). Another test employs the more recent isothermal nucleic acid amplification technology developed by Abbot Labs1. Both tests are highly sensitive. They work by amplifying a region of the genome that is specific to the SARS-CoV-2 virus. This amplification is primed by a pair of oligonucleotides (also called primers) that are complementary to the viral sequences.
The current RT-PCR method includes extraction of viral RNA from nose and throat swabs and reverse transcribing the RNA to DNA, followed by a PCR reaction. The PCR method uses a temperature-cycling strategy that involves denaturing the template and annealing short primers to complementary sequences on the template. Extension of the primer-template complex, facilitated by the polymerase enzyme, leads to exponential amplification of the target amplicons. The results are obtained within a few hours. Detailed protocols on the PCR method are shared by WHO and CDC. On the contrary, the isothermal method from Abbott labs is not limited by constraint of thermal cycling and the positive results for COVID-19 are obtained in 5 minutes, while negative results are obtained in 13 minutes1.
The unique regions in SARS-CoV-2 identified by comparative genome analyses serve as distinctive markers for designing primers or probes used in DNA-based kits. SARS-CoV-2 is a single-stranded RNA virus with a genome of nearly 30,000 bases. The viral genome encodes four structural proteins, namely, the envelope (E), the membrane (M), the nucleocapsid (N) and the spike (S) proteins.
Comparative genome analyses have identified different regions unique to SARS-CoV-2. The uniqueness of the sequence is validated by searching for similar sequences using the BLAST (Basic Local Alignment Search Tool) algorithm in the GenBank database, a global repository of all sequenced genomes. The unique regions in genes such as ORF1ab, N, RdRp and S are currently used as primers in SARS-CoV-2 RT-PCR diagnosis2, while Abbott’s ID NOW™ COVID-19 rapid test targets the RdRp gene.
New Methods on the Horizon
CRISPR, a popular gene-editing technology, is considered a scalable option for population testing. This method uses CRISPR machinery’s ability to recognize specific genetic sequences and cut them. CRISPR also cuts a reporter molecule added to the reaction which can quickly reveal the presence of viral genetic material. The CRISPR-based SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) is described as a paper dipstick method that can produce results in one hour. SHERLOCK was co-developed by Feng Zhang from the Broad Institute of MIT and Harvard3. Another method developed by Jennifer Doudna at the University of California, Berkeley, usurps the isothermal pre-amplification with DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) for coronavirus. DETECTR can provide results in 30 minutes4.
Sequence-based methods provide critical information about the presence or absence of the virus in a patient, information that is especially valuable when developing public health and safety policies. In the U.S. we currently only test people who are exhibiting symptoms for COVID-19. However, with a disease such as COVID-19 there is a serious threat that asymptomatic people could be spreading the virus in the community.
How do we know if a person has already had the SARS-CoV-2 infection and has developed immunity?
Antibody testing identifies asymptomatic individuals who already had infection and are probably immune. Antibody testing is also the best strategy for screening the entire population and alleviating the fear of community spread. Individuals with positive antibody results could also be a potential source of plasma that can be injected into COVID-19 patients, an approach that is currently being tested in the U.S.
Antibody tests require some knowledge of the proteins that are crucial for the virus, e.g., the viral coat protein. Ideally, those viral proteins that trigger the immune system are the best candidates, because they initiate the production of antibodies that flag or neutralize the virus. It is then necessary to produce those portions or sections of the viral protein in the laboratory and transfect them into cell lines for inclusion in an immunoassay such as ELISA that detects the presence of antibodies.
Such immunoassays can potentially form the basis for home testing kits to detect immunity to diseases like COVID-19. However, developing these kits takes time. The most challenging bottleneck in this process is expressing the protein or protein segment in the right conformation.
The SARS-CoV-2 spike protein presents one potential avenue for the development of diagnostics since it has few unique regions. Several teams are testing the receptor binding domain of the S protein, while a few are investigating the whole spike protein2. Other potential candidates include the nucleocapsid protein and the S protein. The FDA is assessing different antibody testing methods and has recently approved the first test, which will help to determine how many people in the population have immunity5.
- Coronavirus tests: researchers chase new diagnostics to fight the pandemic. Subbaraman N. Nature. 2020 Mar 23. doi: 10.1038/d41586-020-00827-6.