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We’ve received lots of letters questioning the use of PCR (polymerase chain reaction) tests. They claimed that PCR tests are manipulated to inflate COVID-19 numbers. Or that the PCR cycles have been sped up and are therefore unreliable. So how reliable is PCR testing?
We said:
This is a good question, especially because we often hear about false or weak positive results from people who’ve never had COVID-19. We’ve written about how PCR testing works previously.
PCR was first invented in 1985 and is a well-established, common, standard laboratory practice for molecular biology, genetics and medical diagnostics. PCR is highly accurate and sensitive and is considered the gold standard DNA/RNA identification and SARS-CoV-2 diagnosis.
The technique is so accurate at building the right DNA strand that it is used to build DNA for use in CRISPR and other cloning techniques.
Occasionally, false negatives or positives will arise. Regardless, the rates are extremely low and usually happen because of a low quality or old sample – the problem is the sample collection, rather than the test itself. One study of PCR SARS-CoV-2 tests found that just five patients in 96,000 came back with a false negative result.
Thankfully, it is usually easy to flag which results might be a false negative because low quality samples are visible to experts, and they are able to retest the sample. This means that the actual number of false negatives upon COVID diagnosis is even lower.
The test is also very sensitive and only needs tiny volumes of sample, such as what is on a swab, where other techniques need a higher volume (eg blood).
It is also a relatively quick test. It only takes a couple of hours to run, and multiple samples can be run together.
The speed, ease, sensitivity, and accuracy of PCR is very fine-tuned, and is therefore an unshakeable standard in the world of molecular biology. It has been used for decades and will continue to be used for decades to come.
We’ll expand on that to answer your questions about whether the cycles can be sped up.
When a PCR process is completed, it is visualised through a technique called gel electrophoresis, which looks like this:
Image Hirzahoseini et al., 2009
At the top, there are numbers that denote different samples. On the left and right there’s a kind of molecular ruler – called a ladder – that shows examples of standard molecules with a known size.
In the middle, the white lines show different samples after PCR; we can compare the location of that piece of DNA to the ladder and estimate the size of the DNA fragment.
We already know what size we’re looking for, so this is a good, quick way of determining a positive or a negative. If it’s the wrong size, it’s probably not covid.
Of course, there is some error of margin, but it’s very, very low.
Is PCR still reliable if we speed up the process?
PCR has three main stages: breaking the DNA open so it can be read; attaching the molecules that read and amplify the DNA; and growing the DNA chain for observation.
Each of these has a specific temperature and time it needs to run, and will affect how reliable the final PCR result is.
The last stage is the most flexible because it depends directly on the length of the DNA fragment you are trying to read. If you made this step shorter, the DNA won’t grow all the way to its correct length.
Think of it like a cake: a big cake needs more time in the oven. If it isn’t in the oven for the right amount of time, it won’t cook properly.
This means the final PCR can sometimes show the wrong result if the timing is wrong and the fragment is thus smaller than expected.
If the final stage usually takes 40 seconds and you lower it to 30 seconds, the final gel electrophoreses will not look very clear, and you’ll end up wasting time to repeat the test.
Instead, you might get a gel that has extra bands, like this:
To get more samples through the system, more machines are used, but they will all maintain the same cycles.
However, the process isn’t deliberately sped up, and we do know how long is needed, so if there is an unclear result, it is usually because the original sample was poor quality, not because of a change in PCR timing.
The PCR technique has been perfected over the past five decades and further optimised for COVID-19 over the since its emergence in 2020. That means there are standard temperature and time protocols specifically for SARS-CoV-2 tests.
Finally, when a band is really faint, like in this image, the sample is retested.
This means the faint sample will be tested multiple times, and if it has the same result, it will be sent off for sequencing to confirm. Sequencing of a sample is time intensive, and so is only done for positive, or sometimes indecipherable, results.
Altogether, this means that PCR testing is very reliable and undergoes multiple confirmations, so numbers are unlikely to be inflated.
If you have your own questions that you like to submit to the Cosmos team, contact us!
This article was originally published on Cosmos Magazine and was written by Deborah Devis. Deborah Devis is a science journalist at Cosmos. She has a Bachelor of Liberal Arts and Science (Honours) in biology and philosophy from the University of Sydney, and a PhD in plant molecular genetics from the University of Adelaide.
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