PCR Tests

There is no test for COVID-19, the disease of the pandemic.  Instead, tests look for the presence of SARS-CoV-2, the virus known to cause COVID-19, or the body’s response to SARS-CoV-2.  This last group, known as “antibody” tests, have found very little utility during the pandemic. There are different kinds of tests in the first group, the tests for the virus, and the reliability of these tests vary widely based on methodology.  Today, we will discuss the gold-standard method: polymerase chain reaction, known as “PCR”.

PCR is a several step process.  It begins by exposing the sample to the components necessary for replication of nucleotide segments.  Nucleotide segments are pieces of DNA or RNA which carry the genetic material of cells.  By exposing the sample in this environment to a series of 30 to 40 heating and cooling cycles, all of the DNA or RNA sequences contained in the sample are amplified—and not just 30 to 40 times, but one to 2³⁰ to 2⁴⁰ times.  That’s one billion to one hundred billion! This produces a highly sensitive test able to detect minute quantities of genetic material.

But, you might ask, if all genetic material is being amplified, won’t other genetic material in the sample not related to the virus be amplified also?  Yes, it will, but that’s where the second part of the test becomes important.

Probes are incubated with the amplified sample that bind to a specific nucleic acid sequence that is known to be unique to the target—in this case the SARS-CoV-2 virus.  If that sequence exists, then the probe binds, and a signal is sent to the test system.  If the sequence doesn’t exist, then there is no signal.  It’s like looking for a specific password to a website among all the passwords that could possibly be entered.

But, you might ask again, couldn’t that nucleic acid sequence exist in the sample just by chance?  That would be like you guessing the password to a billionaire’s bank account; unlikely, but not entirely out of the question.  

That is why PCR tests for SARS-CoV-2 include a second set of probes, specific a different nucleic acid sequence that is also unique to the virus.  Only if both signals are detected is the test positive.  Some PCR tests even use a third set of probes.  The result is a highly sensitive (meaning detection of tiny amounts of viral RNA) and a highly specific (meaning false positive rates close to zero) test system.  

But like any human endeavor, PCR is not perfect.  False negative results can occur if the sample is improperly collected, resulting in no viral RNA to amplify.  Sloppy analytic practices can lead to false positive results by a process referred to as “carry-over”—literally, the genetic material from one sample is carried-over to another sample.  

There is another source of apparent false positives: a patient who is not sick and not infectious but has a positive test.  I call this pseudo-false positive, because it’s not a false positive result at all.  Instead, it’s a failure to interpret the result correctly.  Remember that laboratories do not test for the COVID-19 disease; they test for the SARS CoV-2 virus itself, and in the case of PCR tests, for viral RNA.  Viral RNA is known to persist in detectable quantities for weeks, even months, after the patient has recovered from the disease.  How is this possible?  Because to be infectious, the virus must have the genetic code wrapped in a capsule that binds to human cells.  Without the capsule, the RNA is inert.  When the body clears an infection, it denatures the capsule.  Remnants of the viral RNA persist for a long time afterwards, just like a bombed-out city after a war.

It gets even more complicated.  There are tests which have unfortunately received Emergency Use Authorization (EUA) from the FDA which are not as sensitive or specific as PCR tests. These are many of the super rapid tests (<15 minutes), some of which have sensitivity rates as low as 67%.  These tests are known as “antigen” tests, and we will address these tests soon.  Next, we will discuss the meaning of positive and negative tests.