I was asked to write some comments for a fact check article about some of the myths going around about PCR-based testing and whether PCR tests can detect “the COVID virus”. In particular, an agglomeration of them in the form of a Facebook video by a guy in his car. I wrote a few hundred too many words – so here are most of them.
Don’t listen to a car dude
First up – the comments which generated the need for this article were from someone who appeared to have zero understanding about PCR or PCR test design or PCR use. Some of the comments were wrongly attributed to the creator of the PCR test, Kary Mullis, who, as we know, died before this pandemic began.
The speaker – who I will not specifically oxygenate here – also seemed to have no scientific experience, or any expertise in science (he was unfamiliar with routine words used by anyone in the biological sciences) and I’d guess that he hasn’t ever worked in a laboratory, designed a pair of PCR primers, constructed, optimised or validated a PCR test, regularly consumed or written scientific literature or generated or interpreted any scientific data. In sort – this guy is not an expert so please take what he said with a grain of NaCl (you know what that is, right?). These are important factors when someone is lecturing at you and using their platform to pull down or threaten experts on the topic. This sets the stage. It provides context.
The purpose of these sorts of comments seems to be disruption and the erosion of trust in expertise and science. There is possibly something here about the speaker feeling that they know something special or something different from the real experts. I’m sure there’s a much longer treatise on who this person just wanting his five minutes of fame, that his mum told him he was special and that he felt unheard or misunderstood. But during a major public health event where lives are on the line, these selfish reasons aren’t enough.
The claim that PCR tests can be contaminated
Contamination of PCR is a real issue and I’ve covered this here just recently. Labs that routinely use PCR and RT-PCR are extremely careful to consider and account for and prevent that from being an issue. These expert labs also include controls in every run which tell them if a contamination problem has occurred.
RT = reverse transcription. This is an enzymatic step that precedes the PCR (polymerase chain reaction) cycling, still in the same tube, producing a DNA copy of an RNA target, such as a small portion of the RNA which makes up the genes and genome of SARS-CoV-2 and most other respiratory viruses that infect humans. The PCR is an enzymatic process that produces a new chain of nucleotides that are a mirror image of the original target region’s nucleotide sequence (genetic code) through a process of heating and cooling controlled by a programmable thermal cycler. It’s a cyclical process which copies (amplifies) that small piece of DNA millions of times until it reaches a level we can record using fluorescence detectors. Without the amplification, there is too little viral genetic material to detect.
The claim that PCR tests only detect a small part of the virus
Each RT-PCR test is designed to be specific to the intended virus (there are examples where that may not be the intention). So labs have a number of these tests; reference labs may have dozens!
The types of RT-PCR used to test for virus only seek out a small part of the complete genome sequence. This is to make the tests work as quickly (an hour or less) and efficiently as possible.
In particular, the “real-time” PCR method has been used widely since the 2009 fluA/H1N1 pandemic. It works best with shorter target regions. Real-time RT-PCR (RT-rPCR) includes a fluorescence-producing (the fluorogenic bit) DNA probe. The probe adds another layer of target-specificity to the test. It also generates a signal when the viral RNA is present (but not, when it isn’t). The probe doesn’t take part in the PCR amplification – it just binds to the DNA as it builds up…in real-time! When enough DNA is present, the probe will produce a detectable fluorescent signal. The signal grows a bit more during each cycle until the PCR finishes.
That small target sequence is usually – but not always – chosen to be highly specific to the target; both primers and probe bind only to the intended viral target.
PCR gleans its extreme specificity from the primers. At each and every position of a new DNA primer, we have one of four nucleotides to choose from, dATP, dCTP, dGTP and dTTP.
If we designed a sequence-specific primer of 20nt nucleotides in length (a ’20mer’), the chance that the same exact sequence will occur randomly in nature would be (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4) x (1/4). That means there’s a 1 in 1012 chance of a 100% identical sequence occurring randomly in nature. Pretty small chance. That last bit is relevant to how incredibly specific RT-PCR tests can be.
Our test is designed to detect a particular virus. We can look at the genetic sequences of all the other known viruses and gauge, with quite a bit of confidence, that this test will not interact with their genetic material. This helps us avoid one form of false result (rarely are things absolute in biology).
So rather than next to impossible, it’s actually highly probable that expert primer and RT-PCR test designers do detect what we design our primers and probe (see below) to detect.
The claim that PCR tests don’t detect “the COVID virus”
RT-PCR tests in use today are extremely effective at very sensitively and specifically detecting SARS-CoV-2, the virus that causes COVID-19. Some are a bit better than others, but they very much can detect SARS-CoV-2. Some early RT-rPCR tests were designed intentionally to allow for sequence variation just in case the virus did a lot of changing early on (it didn’t).
Additionally, SARS-CoV-2 tests do not detect other coronaviruses (see that caveat above and it really only holds for other closely related SARS-like and bat CoVs), or rhinoviruses or adenoviruses, parainfluenza viruses, influenza viruses, respiratory syncytial viruses and so on, that may be found in the same patient sample.
But we test developers also check this in the real world. We test other related and unrelated viruses (like those👆) and different types of human samples (snot, saliva, sputum, swabs, liquids etc) during a process called validation. This way we know what the primers – and thus the RT-PCR test – can do and can’t do. This is all part of the expertise developed over decades of our use of PCR methods.
Of course, a badly designed RT-PCR can have problems, as can anything in science, or life. False positives and negatives can happen because of test design issues (not just because of sampling and timing issues), but professional laboratories watch for this, minimise the risk of this and can identify and rectify this. And they spend time on test design, optimisations and that validation thing.
Expertly designed and used SARS-CoV-2 tests have been well-proven to detect this virus. The leading tests have also been tested on SARS-CoV-2 RNA purified from virus isolated and grown in cells in the lab – so we know it is this virus that we’re detecting. We can also design and order viral sequences in the lab and have them made commercially, so again, we know the tests detect the viral sequences we expect them to and not other viruses.
The claim that PCR tests don’t detect the whole genome
But that is by design, not an error or an oversight. We have only been able to quickly and relatively easily (compared to even a decade ago) detect entire viral genomes – a key part of the science of genomics – in very recent times.
It is still costly, less sensitive and slower to determine the entire sequence of a virus compared to simply detecting its presence using an RT-rPCR test though.
For cluster, outbreak, epidemic and pandemic control and for patient management – detection the presence of viral RNA (which real-time RT-PCRs do) is all that is needed. For more extensive tracking of virus change over time and to better understand where an outbreak may have come from – genomics is great, but only a small fraction of viruses usually get this treatment. That will change with time.
A positive result doesn’t mean virus is present – but we can be pretty sure it was
A positive RT-PCR result doesn’t prove that replicating (also called infectious or viable) virus is present. It proves that its genetic material – the RNA – is present.
But we have learned much about this virus in 7 months and SARS-CoV-2, like other viruses we detect with RT-rPCR, is present before and during the peak symptomatic period. These are also the times we get sampled and tested because we feel sick. We know from experience that when detectable viral RNA is present in a patient’s sample, there is often an infectious virus present as well. RNA is not so stable that it hangs around without a constant source producing more of it.
Even though it’s really sensitive, RT-rPCR has a limit of detection – as we have been seeing in the story of “false negatives” among patients being tested for SARS-CoV-2 well after they became clinically well.
So we use the RT-rPCR result as a surrogate indicator of the presence of the infectious virus.
Our other option would be to try and “grow” virus in the lab, but this has problems. It is slower, more expensive (lots of labour costs), affected by poor sample handling/storage/cold chain, needs a high level of expertise (actually a dying art) and is much less sensitive than real-time RT-PCR.
For some non-experts, including medical Doctors in some cases, this caused a big problem in the early years of PCR as it moved from a research tool to an aid for patient diagnosis.
They couldn’t understand what a PCR positive result meant because they were used to virus culture results….which were less often positive because the method was less sensitive.
Now you are more expert than you were
So now you may know a bit more about this subject than you did before. Hopefully, I’ve explained that PCR tests can detect “the COVID virus”. I feel confident in saying that you will know a lot more than a certain guy in a certain car in a certain video. Always be willing to learn a new thing. Thanks for reading.