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September 21, 2021

How reliable are PCR tests?

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Barden Ridge, Australia – 2021-07-10 Medical stuff making covid test swab at COVID-19 drive through testing clinic. NSW, Australia. Photo Shutterstock.

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:

 

A standard gel elctrophoresis.
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:

A gel electrophoresis with unclear samples that have multiple bands. Image Chegg

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.

A gel electrophoresis showing a bright ladder (marker), but a faint band. This could be a false or weak positive. Image BioRad

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.

Published by The Echo in conjunction with Cosmos Magazine.


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7 COMMENTS

  1. Cosmos Magazine receives funding from South Australian Government, Commonwealth Government and Santos Oil and gas. An unholy alliance to be sure and a serious impediment for me to trust their reporting on politically loaded , contentious issues where there are numerous dissenting (censored) opinions, as is the case with PCR Testing (including from it inventor, Kary Mullis

    • Hi Saul, it’s Gail, editor of Cosmos here. Thanks for asking that question – in today’s world, asking questions is more important than ever! Just wanted to let you know, Cosmos is published by the charity The Royal Institution of Australia. While RiAUS received startup funding back in 2012 from the Commonwealth, the State of South Australia, and Santos Ltd, those orgs have nothing to do with what the people at Cosmos think, say, or do in 2021. The only things we’re influenced by are the facts and research coming out of the amazing world of science and support from readers, which reminds us we’re doing something important (shameless plug: https://riaus.org.au/give).

      Kary Mullis was an intriguing character and his biography makes for rollicking reading. On this topic, though, he’s often taken out of context. It comes from a quote in 1996 article in which he says: “PCR is intended to identify substances qualitatively, but by its very nature is unsuited for estimating numbers. Although there is a common misimpression that the viral load tests actually count the number of viruses in the blood, these tests cannot detect free, infectious viruses at all; they can only detect proteins that are believed, in some cases wrongly, to be unique to HIV. The tests can detect genetic sequences of viruses, but not viruses themselves.”

      Vast leaps in sequencing in the 25 years since mean that there is no doubt as to the sequence of SARS-CoV-2. It was true then and remains true now that while the PCR test can detect the presence of a virus, but not whether a person is infectious or not.

  2. Thanks for your in depth response Gail, which is further food for thought, although I would like to point out that my comment was not a question but rather an expression of concern about the provenance and credibility of ‘undoubtable facts’ promulgated in an over- heated climate (no pun intended) But certainly yes, questions are good.

  3. As a heads up, most labs these days use what’s called real-time PCR. Rather than amplifying through all the cycles and then using gel electrophoresis to see the bands, the amplification and analysis steps are combined.

    When amplifying RNA, we use primers which are specific to portions of the virus’ genome. These primers are attached to a dye which activates when the primer binds with another piece of RNA. The dyes glow under a specific wavelength of light, and each gene we test for has its own unique dye. After each cycle of PCR, the system measures the fluorescence of the sample by shining it with those wavelengths.

    The advantage of this system is that we can more easily determine the strength of a signal: the earlier the fluorescence starts to rise, the more virus particles were in the sample. It also let’s us begin processing positive cases before the run finishes which is a huge time-saver. We can also look at the “curves” the fluorescence makes to spot any weak positives that may not make the cutoff so that we can monitor the testee and see if their levels rise in the future.

    Source: work in a molecular lab testing covid samples

    • So nice to have ‘new real-time test’ and how fateful it was of the ‘old amplifying test’ to manufacture a pandemic and turned the history of the world into a totalitarian reset.

      Recommended read from Luke Massey writing in today’s The Spectator tells us that the estimate “Medicare bill for Covid testing in Queensland has amounted to over $250,000,000.” 
      So for all the Me people with financial interests in the lab testing game – we thank you.

      Imagine what that test and vaccine money could have been.

  4. person taht invented it said it is NOT realiable way to test, also why do you need to have soemthign stuck far up noise or other hole, yet can spread it by breathing….. somehtign is up and even children knwo soemthign is wrong.

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