The PCR Test:

Long gone are the days of apocalyptic panic many felt in the initial stages of the pandemic. Can you imagine something more alarming than the COVID-19 pandemic? How about those big gnashing jaws of the T-Rex in Jurassic Park, the original?

Well, it turns out they have something in common; Michael Crichton, the imagination behind the park of prehistoric dinosaurs, had PCR technology on his mind when he wrote the novel. It’s how Crichton envisioned dinosaurs could be brought back from the dead. Indeed, in real life, mammoth DNA has been resurrected and placed at the fingertips of eager scientists as a result of PCR technology, so perhaps Crichton was only a stone’s throw from the truth.

It so happens that in 1993, the year Jurassic Park was released to audiences worldwide, a brilliant, eccentric, and slightly drunk man ascended the stage in Stockholm, Sweden, to accept the Nobel Prize in Chemistry for his role in the invention of the polymerase chain reaction (PCR) technique. The man’s name was Kary Mullis.

Scientists and television viewers alike would soon grasp the genius of his technology. If you’ve ever watched a crime drama or criminal prosecution and heard the phrase “DNA found at the crime scene,” then you can begin to appreciate the magnitude and reach of this revolutionary discovery, at least in the realm of criminal justice.

While DNA detection had existed before its invention, the advent of PCR made it quick and cost-effective. Not long after PCR’s invention, the technique took center stage during the famous examination of the glove at OJ Simpson’s trial. Thanks to the pandemic, PCR has become a globally recognized household name for its role in identifying the novel coronavirus (SARS-CoV-2). But how exactly does it do this?  

Years after being awarded the Nobel Prize for PCR, Mullis would credit the psychedelic drug lysergic acid diethylamide (LSD) with the vision of his discovery, giving him the ability to realize and reframe the problem he faced: the human genome was enormous. A single cell in your body could produce six feet of unraveled DNA. The DNA in all of your cells, unwound and strung out end to end, could travel to the moon and back 150,000 times.

Looking for a single sequence or gene was like looking for a needle in a haystack in every silo in the state of Wisconsin! If not worse. To find the correct sequence he would have to find a way to isolate the “needle” and replicate it so many times that it would be impossible to miss. To do this would require him to target and isolate specific segments of DNA. But how to do this?  

Together with a team of researchers (and another team working in parallel), Mullis used artificial strands of DNA, known as primers, that would, essentially, target and “bookend” specific sequences of DNA. But before they could target the DNA site, they would have to “melt” or physically separate the DNA double helix at high temperatures in a process called nucleic acid denaturation.

Once separated, the two strands of DNA, coaxed by cooling temperatures, partner with the primer in the test tube or cassette. Together, each double-stranded pair of DNA and primer creates a template by which an enzyme known as DNA polymerase can assemble a new DNA strand. This step, however, would prove problematic. 

The researchers quickly found that DNA polymerase, the very enzyme needed to duplicate the DNA strands, was destroyed by the heat used to replicate it. The answer to this problem lurked in the bubbling waters of geysers, such as Old Faithful, in Yellowstone National Park.

Deep beneath the earth, magma chambers from the core heat and pressurize water into eruptions of steam mere yards from the feet of curious tourists on the surface. Floating in the tumultuous, steaming waters of these geysers were billions of microscopic heat-loving bacteria known as Thermus aquaticas who evolved, over millions of years, precisely the solution the team was looking for: a DNA polymerase enzyme resistant to heat.      

Equipped with this knowledge and the new DNA polymerase enzyme, the researchers were able to complete the process. For every new DNA strand that was produced, the same process could occur, exponentially amplifying the number of DNA strands (like John Hammond’s clones in Jurassic Park) until, tiny as they may be, the strand of DNA became detectable. Within a span of an hour or less, the technique could multiply a single sequence a millionfold. Rain or shine, Cepheid or Accula, commercial laboratory or production trailer, these machines make miracles, transforming amino acids into what Bill Clinton called “the language in which God created life.” 

You can think of this process as photocopying a film script. The DNA is like the original script, hot off the press from the writers’ room. In order to make sure the entire production understands the intricacies of each scene, you will need hundreds of scripts. So perhaps you put a production assistant to work, printing and watermarking the many copies, making sure the right scenes get into the right hands. In this analogy, the production assistant is like the DNA primer, choosing which section of the script to copy. The photocopier is analogous to the DNA polymerase, which generates all the copies of the DNA. And the new DNA strands are like the new copies of the script ready to be read and acted upon.    

Today, most people are aware of PCR through the context of the novel coronavirus. The technique has come a long way since its origins thanks to the additions and modifications of a multitude of remarkable people. Almost anywhere on the planet, a person can produce a sample of saliva or nasal swab and within a week receive a result confirming whether they are infectious or not. Thousands of laboratories around the world, and millions of machines, convert the coronavirus’s genetic material, known as RNA, into “readable” DNA, using an enzyme similar to DNA polymerase called reverse transcriptase (If you are interested in how reverse transcriptase may help coronavirus integrate into the human genome read our article, The Curious Case of the Persistent Positive). Scientists employ primers designed specifically to target and “complement” the viral DNA, and avoid amplifying human DNA. Then the amplification process can begin, exponentially duplicating the DNA copies until they are observable or considered ostensibly nonexistent. 

This cycle of heating, cooling and replication occurs 20-30 times and results in a cycle threshold (CT) value. This value is then read by a healthcare provider and, together with symptoms, will tell them not only whether you are infectious, but, often, just how infectious you may be. If the CT value is low, it means it took very little time for the analyzer to detect the viral DNA, suggesting that there is a copious amount of viral DNA in your sample. This indicates a peak level of infection. If your CT value is high, and viral DNA has been detected, it means you have a low amount of viral load. This can mean you are either in the early stages of an infection or at the tail end. The only way to know for certain is to speak with your physician. 

Of all of PCR’s achievements since its creation, keeping the COVID-19 pandemic under control ranks among its most remarkable, taking us from apocalyptic panic to the calm many of us feel today. It has played a crucial role in flattening the curve of cases and limiting needless deaths. That there is so much more to the story is a testament to this technology’s paramount importance to science and society, and its much-deserved status as a household name. 

The Rapid Antigen Tests:

By now, the rapid test is a mainstay of the American experience: swirl your nasal swab in a vial, squeeze several drops of the mucus-mixed medium onto an oversized domino-shaped assay, wait fifteen or twenty minutes, and, bam, you, incredibly, have a fairly-reliable read on your internal “state of affairs”.

This process, which seems miraculous, can cite as inspiration a long history, spanning thousands of years, of using fluids as diagnostic tools, particularly as predictors of otherwise unknowable events.

The ancient Chinese, for instance, forced suspected criminals to consume handfuls of rice and, while observing whether the suspect could swallow or not, cited ‘lack of saliva’ as a sign of guilt. In ancient Egypt, the prototype of the pregnancy test had women peeing on wheat and barley, and, by the judge of which plant grew, predicted a boy, girl, or no baby at all. In Europe, urine analysis gave rise to a cult of so-called “piss-prophets” who determined ailments and fates by “reading” patterns of bubbles produced by a splash of urine in the water.   

While seemingly still unable to shake their reputation as unreliable, fluid-based diagnostic tests, such as the antigen test, have traveled lightyears in terms of accuracy since the ancient Chinese and Egyptians, and Europe’s so-called “piss-prophets”. To get from drop to diagnosis, your mixture of mucus must make a journey up what’s called an immunochromatographic test strip. This movement of molecules is a migration worthy of David Attenborough’s narration for a virus that is about a million times smaller than the period at the end of this sentence. Understanding how this technology works takes a basic appreciation of the relationship between viruses and the immune system. 

Unlike molecular tests, such as PCR, antigen tests detect a completely different component of the virus’s anatomy. Instead of targeting the virus’s genetic code, the antigen tests target tiny proteins protruding on the outer surface of the virus. These tiny proteins, collectively called antigens serve several different purposes, the most important of which is to break into the host cell and inject the virus’s genetic material. In the case of the coronavirus, the S protein essentially allows the virus to “jimmy the lock” on the doors into your cell to gain entrance while the N protein holds the RNA genome that is injected inside.  

Antigen tests target these proteins in an ingenious way. When a virus is detected in your body, your immune system produces proteins called antibodies. These antibodies essentially attach themselves to virus antigens to prevent the virus from breaking into your cells. The antibodies then flag these viruses for destruction by your immune system. Antigen tests actually use antibodies, either derived from an immune host or artificially produced in a laboratory to detect antigens. These tiny immobilized antibodies are laid out in lines along the antigen test pad. A chemical is added so that when an antibody comes in contact with the antigen on the test strip, a chemical reaction occurs and creates a colorful line that is typically read by a clinician.  

Between PCR and Antigen Tests, Which is best to diagnose COVID-19?

How do clinicians use PCR and antigen test results to diagnose COVID-19 infection? While the reputation of poor sensitivity has plagued antigen tests since the start of the pandemic, this is not precisely true. Both PCR and antigen testing have their limitations and their strengths.

Rapid tests truly shine when a patient demonstrates symptoms. Symptoms are a clear sign that someone has enough viral load to spread the disease, and antigen tests can determine whether the antigens derive from SARS-CoV-2 or not.

Where PCR tests shine and the rapid antigen test fails is in detecting either earlier or later stages of infection, when viral loads are lower. Since PCR tests use technology to exponentially increase the number of copies of viral RNA, it can act as a predictor of whether the virus may replicate, becoming contagious within the next 48-72 hours.

That said, the PCR test may have trouble determining whether the low viral load is from a developing infection or a receding one. This is where a clinician steps in to analyze CT values of the test and a variety of other factors.