Think of polymerase chain reaction (PCR) as the toner in a xerox machine used by geneticists. This process makes copies of your DNA or RNA (which it converts into cDNA). Lots of copies. Scientists cannot study or test DNA until they have a lot of genetic material and this simple process of copying (amplifying) a small amount of your genetic material (your DNA) to allow it to be tested has been around for nearly forty years. It works spectacularly well and is incredibly reliable.
This article will explain, at length, in both plain English and in more scientific language, how the test works, the different types of tests and it will discuss at length, reliability.
A quick history
Often heralded as one of the most important scientific advances in molecular biology, PCR revolutionized the study of DNA to such an extent that its creator, Kary B. Mullis, was awarded the Nobel Prize for Chemistry in 1993. The link leads to a short, but fantastic biography of his life and offers deep insights into the man, I highly recommend reading it.
Once amplified, the DNA produced by PCR can be used in many different laboratory procedures. For example, most mapping techniques in the Human Genome Project (HGP) relied on PCR.
PCR is also valuable in a number of laboratory and clinical techniques, including DNA fingerprinting, detection of bacteria or viruses (particularly AIDS), and diagnosis of genetic disorders.
The process that underpins PCR was first discovered by Mullis in 1985. Surprised? It’s been around a long time and over the course of the last 36 years, science has had the opportunity to refine and improve on it, over numerous iterations, to the point where we can now quickly and accurately produce millions of copies from a small DNA sample to enable various tests.
Unsurprisingly, most people were blissfully unaware of the test until the pandemic arrived in 2020. It’s taken a year and no formal training to turn the entire planet into PCR experts. Let’s get to grips first with the terminology used to discuss PCR.
Your Personal PCR Dictionary
This list will provide all the terms you need to enable you to follow the more technical aspects below. Do refer back to it if you get lost further into the article and don’t be put off by the technical bits. The process itself is really simple. Some of the terms below do not apply directly to PCR but have been included to provide a clearer understanding of the differences between PCT and Serum (blood) based tests.
DNA
Deoxyribonucleic acid, more commonly known as DNA, is a complex molecule that contains all of the information necessary to build and maintain an organism. All living things have DNA within their cells. In fact, nearly every cell in a multicellular organism (like us) possesses the full set of DNA required for that organism. DNA contains our hereditary material and our genes — it’s what makes us unique.
cDNA
cDNA, called complementary DNA,is a copy of DNA that can be derived from either prokaryotes or eukaryotes. It is used in genetic engineering to produce clones of other genes. cDNA is synthesized from mRNA using an enzyme called reverse transcriptase. The RT-PCR test uses RNA which it converts into cDNA for testing.
RNA, mRNA, and tRNA
The main function of RNA is to carry information of amino acid sequence from the genes to where proteins are assembled on ribosomes in the cytoplasm. This is done by messenger RNA (mRNA). A single strand of DNA is the blueprint for the mRNA which is transcribed from that DNA strand. More specifically, messenger RNA (mRNA) carries the protein blueprint from a cell’s DNA to its ribosomes, which are the “machines” that drive protein synthesis. Transfer RNA (tRNA) then carries the appropriate amino acids into the ribosome for inclusion in the new protein.
Template
Where you see this term in relation to PCR tests, it refers to the materials you provide via a swab test, in other words, your genetic material and if you’re infected, the virus’s genetic material. This is used as a template (the original from which copies are produced) for the PCR tests.
PCR
Also referred to as “molecular photocopying,” the polymerase chain reaction (PCR) is a fast and inexpensive technique used to “amplify”(copy) small segments of DNA. Because significant amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification¹.
RT-PCR
Reverse transcription PCR, or RT-PCR, allows for the use of RNA as a template. An additional step allows the detection and amplification of RNA. The RNA is reverse transcribed into complementary DNA (cDNA), using reverse transcriptase (RT). The quality and purity of the RNA template is essential for the success of RT-PCR
qPCR and RT-qPCR
A real-time PCR, also known as quantitative Polymerase Chain Reaction, is a laboratory technique of molecular biology based on the polymerase chain reaction. It monitors the amplification of a targeted DNA molecule during the PCR, not at its end, as in conventional PCR. RT-PCR is for amplification, while qPCR is for quantification.
In RT–qPCR, RNA transcripts are quantified by reverse transcribing them into cDNA first, as described above and then qPCR is subsequently carried out. qPCR plays an important role in the detection, quantification, and typing of viral pathogens. This is because detection of important clinical and veterinary viruses using culture methods is time-consuming or impossible, while ELISA tests are not universally available and suffer from comparatively low sensitivity and specificity.
Real Time RT-PCR or Real Time PCR (again, for clarity)
RT–PCR, as we now know, is a variation of PCR. The two techniques use the same process except that RT–PCR has an added step of reverse transcription of RNA to DNA, or RT, to allow for amplification. PCR is used for pathogens, such as viruses and bacteria, that already contain DNA for amplification, whereas RT–PCR is used for those containing RNA that needs to be transcribed to DNA for amplification.
Both techniques can be performed in ‘real time’, which means results are visible via fluorescent markers almost immediately, Conventional PCR and RT-PCR results are only visible at the end of the reaction.
Taq DNA polymerase
When most organisms go above 130 degrees, the proteins in their cells change structure so that they become non-functional, similarly to how the protein in egg white changes when you cook it. Because proteins do almost everything that gets done in our cells, high temperatures are usually fatal.
Because of the higher temperatures required for denaturation DNA Polymerase is not suitable for Polymerase Chain Reaction (PCR) tests. Taq DNA Polymerase, on the other hand, plays an essential role in PCR. Taq DNA Polymerase is a highly thermostable recombinant DNA polymerase. It is named after Thermus aquaticus, the heat-tolerant bacterium from which it isolates itself. It is able to tolerate the heat required for the denaturation process in PCR.
Thermocycler
The machine that runs the actual PCR test. It works by heating and then cooling the DNA sample to allow the DNA sample to be broken apart (denatured) and then reformed(synthesis). Think of it as a biological actual xerox machine and your DNA is the white sheet of paper being copied.
Denature
In biology, this process describes modifying the molecular structure of a protein. Denaturation involves the breaking of many of the weak linkages, or bonds (e.g., hydrogen bonds), within a protein molecule that are responsible for the highly ordered structure of the protein in its natural (native) state. In the PCR test, heat is used to denature DNA, in other words, to separate the two combined strands of DNA
Serological Tests
Serologic tests are blood tests that look for antibodies in your blood. They can involve a number of laboratory techniques. Different types of serologic tests are used to diagnose various disease conditions. Serologic tests have one thing in common. They all focus on proteins made by your immune system. These tests offer an alternative to PCR tests.
Assay or Bioassay
Wondered about this term? A bioassay or biological assay is a biological testing procedure for estimating the concentration of a pharmaceutically active substance in a formulated product or bulk material. In contrast to common physical or chemical methods, a bioassay results in detailed information on the biological activity of a substance.
Antibodies
We’ll explain anyway, although we doubt anyone, anywhere on the planet, doesn’t know by now what antibodies are. Antibody, also called immunoglobulin, is a protective protein produced by the immune system in response to the presence of foreign substances, called antigens. Antibodies recognize and latch onto antigens in order to remove them from the body.
They don’t always protect though. Antibodies that recognize the body’s own proteins, instead of proteins from infectious microbes, can cause harm. In autoimmune diseases, such as lupus, multiple sclerosis and rheumatoid arthritis, people produce antibodies that stick to their body’s own proteins and attack healthy cells.
Serum or Plasma Sample
Serum is the liquid portion of the blood obtained after a serum sample tube has been allowed to clot and is centrifuged. You need 12 mL of whole blood for each 5 mL of serum or plasma. Blood s collected in an appropriate collection tube and placed in a centrifuge for at least 15 minutes at 2200–2500 RPM.
Seropositive
The state of either having or not having detectable antibodies against a specific antigen, as measured by a blood test (serologic test). For example, HIV seropositive means that a person has detectable antibodies to HIV; seronegative means that a person does not have detectable HIV antibodies. Interestingly, new HIV antivirals are so effective that an HIV-positive person will have no detectable antibodies if tested. This means that although they remain HIV-positive, they can no longer transmit the disease while they are on the antiviral medication.
ELISA
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones. Other names, such as enzyme immunoassay (EIA), are also used to describe the same technology. Some examples include diagnosis of HIV infection, pregnancy tests, and measurement of cytokines or soluble receptors in cell supernatant or serum.
How does PCR work?
To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. Next, an enzyme called “Taq polymerase” synthesizes (builds ) two new strands of DNA, using the original strands as templates. This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA.
Then each of these strands can be used to create two new copies, and so on, and so on. The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment.
The entire cycling process of PCR is automated and can be completed in just a few hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis.
There are many different PCR tests made by different manufacturers and each test is unique in its own way, requiring different cycles and temperatures, depending on the manufacturer’s instruction, to function effectively. It is critical that laboratories follow these instructions to ensure the tests return accurate results.
Understanding how PCR detects the coronavirus
PCR is highly sensitive and requires minimal template (your sample) for detection and amplification of specific sequences. At the moment the majority of the current Covid-19 tests are using PCR². They detect the genetic information of the virus, the RNA. That’s only possible if the virus is there and someone is actively infected.
PCR tests are used to directly detect the presence of an antigen, rather than the presence of the body’s immune response, or antibodies. By detecting viral RNA, which will be present in the body before antibodies form or symptoms of the disease are present, the tests can tell whether or not someone has the virus very early on. The PCR test doesn’t depend on finding an immune response in the form of an antibody.
Blood tests would typically check for the antibody, in other words, the body’s response to being infected. PCR tests are therefore able to pick up infected individuals far earlier than traditional serum-based tests. It takes a few days for your body to start producing antibodies in response to the antigen.
The presence of the antigen is, however, immediately detectable with PCR, particularly as swabs are taken from the virus’s ingress point into the body, the nasopharyngeal area.
The contribution PCR makes to immediate detection, isolation, and contact tracing in the midst of a pandemic can not be overstressed. Correctly used, PCR in conjunction with policies to identify and isolate early outbreaks, leads to early containment. Asian countries are the perfect example of the efficacy of this strategy in combatting a viral outbreak.
America sadly portrays the effects of ignoring early warning signs and not taking aggressive and offensive action at the outset of the pandemic.
How is Real Time RT–PCR used?
Real time RT–PCR is a nuclear-derived method for detecting the presence of specific genetic material in any pathogen, including a virus. Originally, the method used radioactive isotope markers to detect targeted genetic materials, but subsequent refining has led to the replacement of isotopic labeling with special markers, most frequently fluorescent dyes. This technique allows scientists to see the results almost immediately while the process is still ongoing, whereas conventional RT–PCR only provides results at the end of the process.
Real time RT–PCR is one of the most widely used laboratory methods for detecting the COVID-19 virus. While many countries have used real time RT–PCR for diagnosing other diseases, such as the Ebola virus and Zika virus, many have needed support in adapting this method for the COVID-19 virus, as well as in increasing their national testing capacities.
Here is an example of the CDC-recommended kit and if you’re wondering about how these are used, you can find the instructions for usage here
The exact process involved in Real-Time RT-PCR³
A sample is collected from the parts of the body where the COVID-19 virus gathers, such as a person’s nose or throat. The sample is treated with several chemical solutions that remove substances such as proteins and fats and that extract only the RNA present in the sample. This extracted RNA is a mix of your genetic material and, if present, the virus’s RNA.
The RNA is reverse transcribed to cDNA using a specific enzyme. Scientists then add additional short fragments of DNA that are complementary to specific parts of the transcribed viral DNA. If the virus is present in a sample, these fragments attach themselves to target sections of the viral DNA.
Some of the added genetic fragments are used for building DNA strands during amplification, while the others are used for building the DNA and adding marker labels to the strands, which are then used to detect the virus.
The mixture is then placed in an RT–PCR machine. The machine cycles through temperatures that heat and cool the mixture to trigger specific chemical reactions that create new, identical copies of the target sections of viral DNA. The cycle is repeated over and over to continue copying the target sections of viral DNA.
Each cycle doubles the previous number: two copies become four, four copies become eight, and so on. A standard real time RT–PCR set-up usually goes through 35 cycles, which means that, by the end of the process, around 35 billion new copies of the sections of viral DNA are created from each strand of the virus present in the sample.
As new copies of the viral DNA sections are built, the marker labels attach to the DNA strands and then release a fluorescent dye, which is measured by the machine’s computer and presented in real-time on the screen. The computer tracks the amount of fluorescence in the sample after each cycle. When a certain level of fluorescence is surpassed, this confirms that the virus is present. Scientists also monitor how many cycles it takes to reach this level in order to estimate the severity of the infection: the fewer the cycles, the more severe the viral infection is.
This key difference of enabling an assessment of the severity of the infection is one of the key benefits of using real time RT-PCR testing over conventional PCR.
Is it foolproof?
There is a flaw, however, that exists in these tests, not because of PCR, but rather as a result of human error.
It is possible for a nasopharyngeal swab sample to be taken from an area that does not contain the antigen (virus), despite the person being infected with the SARS-CoV2 virus. In this instance, the test will return a negative result. The test is not wrong, it is dependent on the quality and integrity of the sample provided.
PCR tests can also be very labor-intensive, with several stages at which errors may occur between sampling and analysis. As a result, false negatives can occur up to 30% of the time with different PCR tests, meaning they’re more useful for confirming the presence of infection than giving a patient the all-clear.
Again, much of this can be ascribed to human error rather than flaws in the test itself.
So just how reliable are PCR tests?
Aah, the million-dollar question, and we don’t have a fixed number answer for you. It isn’t that simple. The answer to this should be provided by data obtained prior to the pandemic and that is not subject to conjecture relating to any covid agendas. This data exists in reams, despite Google’s insistence on placing 30 pages of pandemic-based data and some very questionable articles in front of you. It requires a deep dive and few are willing to go there.
If PCR is shown to work with a reasonably high degree of success, prior to the pandemic, you’ll agree there is no reason to assume PCR suddenly stopped working simply because of the coronavirus? That’s a logic bump that not even Covid doubters can cross.
We’ve tried to paint as honest an image of the technology as possible, and below this, we discuss known factors that do affect current covid testing. The next section is complicated and you can skip over it if you prefer as it is not integral to understanding published results. To understand how the reliability, sensitivity, and accuracy of PCR assays are gauged we need to explore a few concepts to help clarify why a simple 90/100 answer can not be provided.
Analytical Sensitivity (Limit of Detection, LOD and Limit of Quantification, LOQ)
Probit or Logit approaches are preferred for determining the LOD for PCR methods. Briefly, both mathematical functions are regressions used to analyze binomial response variables (positive or negative) and are able to transform the sigmoid dose-response curve, typical of a binomial variable, to a straight line that can then be analyzed by regression either through least squares or maximum likelihood methods.
The final end-point of the analysis is a concentration (coupled with relative intervals of confidence), associated to a probability (e.g., 95%) to detect the nucleic acid. Moreover, Probit regression is exploitable only for normally distributed data, while Logit function can also be used for data not normally distributed; however, in this context, both functions have the same meaning.
An issue that must be addressed for the determination of the Limit of Quantification(LOQ), as well as LOD, is the efficiency of recovery of target molecules during the nucleic acid extraction phases. Generally, nucleic acids are extracted from different complex matrices, like food, feces, or other samples using different procedures. The efficiency of DNA recovery is usually around 30% and lower and neglecting this parameter leads to underestimation of the true number of target microorganisms in the original sample, which is then reflected by the lower LOD and LOQ values.
Therefore, the determination of DNA isolation efficiency should be part of the LOD and LOQ. DNA isolation efficiency is a quotient between the number of microorganisms recovered after the entire procedure (nucleic acid extraction + qPCR) and the number of microorganisms used for spiking the negative matrices.
There are various other mechanisms used to determine the specificity of PCR and these are bound to the type of assay and the endpoint. As an example, here are specifics from tests performed on the Intellicube from Douglas Scientific, dating back to 2014.
The IntelliQube is a fully integrated liquid handling and real-time quantitative PCR (qPCR) instrument optimized for use with miniaturized reactions in 768-well Array Tape®. We evaluated the technical performance of the IntelliQube with human genomic DNA, calibrated against the National Institute of Standards and Technology (NIST) standard reference material (SRM), and measured with the ValidPrime human genome specific assay. In this study, we estimated the limit of detection (LoD), limit of quantification (LoQ) and PCR efficiency. The LoD was three molecules, which corresponds to the theoretical sensitivity limited by sampling ambiguity. LoQ, corresponding to a relative standard deviation of 35%, was 32 molecules, and PCR efficiency was 99.7% (95% confidence range: 97.7–101.6%). The sensitivity of the instrument was tested using a digital PCR setup. When loading an average of two molecules per well, we recovered 1.96 (95% confidence range: 1.80–2.15) molecules. A small loss was observed when loading an average of one molecule per well. Taken together, these data are evidence of excellent performance with a technical error negligible relative to the sampling error, virtually perfect PCR under optimum conditions, and a sensitivity to detect one to two template molecules per well.
Real time PCR for identifying Visceral Leishmaniasis (VL)
This simple example illustrates the efficacy of PCR, particularly real time PCR, PCR technology has been significantly refined, and real-time PCR now has advantages over traditional PCR, with shorter run times (because electrophoresis is no longer required) and reduced risk of contamination (because amplification can be detected while the tube is still closed).
PCR for the detection of parasite DNA in peripheral blood is a non-invasive alternative for diagnosis, especially when the disease is suspected in patients with negative parasitological results. in a paper entitled, ‘Sensitivity of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood’ the authors noted the following.
- real-time PCR using the RV1/RV2 primers exhibited higher discriminative power, a feature that corroborates its advantages over the other techniques investigated
- Real-time PCR was the only technique able to provide positive diagnoses for two of the patients, corroborating the advantages of applying this technique with RV1/RV2 primers for VL diagnosis
- Real-time PCR was sufficiently sensitive to detect as little as 0.001 parasite per reaction using TaqMan probes in human blood samples. A slope of -3.3 corresponds to an efficiency of 100.0%, indicating that the number of amplified molecules doubles with each cycle of PCR. In this study a slope of -3.06 was obtained, demonstrating high efficiency.
- A real-time PCR system with SYBR green was used for the diagnosis of cutaneous leishmaniasis using skin biopsy samples from 100 patients. This technique was found to be more sensitive than microscopy and culture methods.
Medical literature is filled with similar papers pre-pandemic, confirming the efficacy of PCR. Obviously, no medical test is foolproof and depending on the antigen we are testing for, various factors can affect the results we achieve using PCR. To suggest however that the test is hopelessly flawed and not suited to identifying the SARS-CoV2 virus is complete lunacy and not based in reality.
It is clear however that the reliability of the PCR test itself is dependant on various factors.
- Human error in collecting and handling samples. Tardy delivery and lax protocols regarding samples, failure to follow specific PCR instructions on different tests, and other issues can and do affect results.
- Some doubt exists as to the best areas to swab for SARS-CoV2. A study has reported sputum as the most accurate sample for laboratory diagnosis of COVID-19, followed by nasal swabs, while throat swabs were not recommended for the diagnosis.⁴
- False-negative results may occur by mutations in the primer and probe-target regions in the SARS-CoV-2 genome. Variability causing mismatches between the primers and probes and the target sequences can lead to a decrease in assay performance and potential false-negative results. In this regard, multiple target gene amplification could be used to avoid invalid results.⁵
- Different viral load kinetics of SARS-CoV2 in different patients suggest that sampling timing and period of the disease development play an important role in real-time RT-PCR results.
The Centers for Disease Control and Prevention (CDC) has designed a SARS-CoV-2 Real-Time RT-PCR Diagnostic Panel⁶ to minimize the chance of false-positive results. It is the same test referenced earlier in the article. The efficacy of this test is, again, dependent on qualified laboratory staff and following the recommended protocols.
It’s important to note that most literature points predominantly to the risks of false-negatives, rather than false-positives. While false-positives do occur they are outweighed by tests that actually miss the virus. This is completely at odds with the fanciful narrative the conspiracy theorists would have you believe, namely that all the positive cases identified are as a result of the tests being manipulated.
The invention of PCR has greatly boosted research in various areas of biology and this technology has significantly contributed to the current level of human knowledge in many spheres of research.
Why Medika Life wrote this article
PCR testing has become the poster child of Covid conspiracy theorists. It’s their defacto fallback when launching an attack on the very real dangers the coronavirus poses. If they attended rallies, probably next on their twisted to-do lists, PCR slogans would adorn their billboards and T-shirts.
Now while we realize that the anti-vaxxers, conspiracy, and anti-covid clans are for the most part indistinguishable and driven by the same misguided psychology, they nonetheless pose a risk to public health and must be addressed.
Their tactics are simple. Like a circling pack of hyenas, they will identify the weakest member in their targeted group and focus their attack on that individual. In the world of Covid, they seem, for some unfathomable reason to have settled on the PCR test.
I say unfathomable because the PCR test is in fact not the weakest link in the pandemic chain. Not by a long shot. It does however suffer from a critical flaw they’ve exploited. It is complicated. It is also prone to human error, but not in the way they would have you believe. It is more likely to return a false negative than a false positive result
To understand exactly how PCR tests work, you need, if you are a layman, to do a reasonably deep dive into the scientific world to form an understanding of the mechanisms and reliability of the process. Most people don’t and it’s for this reason the test has been earmarked.
It’s easy to discredit PCR with an article composed of a few pages of pseudo-scientific mumbo jumbo and the average reader would be none the wiser. The scientist would see through the lies and the intelligent reader would seek alternate reliable sources to validate the article.
As the pandemic has progressed, the term ‘reliable’ has become vague. Real science, evidence-based, tried and tested data, has become something of a rare beast. Hard to find, amidst the sea of misinformation and conspiracy-driven drivel that floods the digital world.
If we publish an article discrediting conspiracy theorists, the author’s defenders go for the jugular, using their tool of choice. The PCR test. It’s their absolute favorite and they will quote all sorts of nonsense or take existing science and misinterpret it to the point it becomes unrecognizable.
So, rather than waste hours of precious time answering these misguided souls, we chose to write this article to explain, in plain English, why PCR tests are in fact, extremely reliable.
References
- Polymerase Chain Reaction (PCR) Fact Sheet, NIH, National Human Genome Research Institute
- What are the differences between PCR, RT-PCR, qPCR, and RT-qPCR? Enzo Life Sciences, 2017
- How is the COVID-19 Virus Detected using Real Time RT-PCR? International Atomic Energy Agency, March 2020
- Yang Y, Yang M, Shen C, et al. Laboratory diagnosis and monitoring the viral shedding of 2019-nCoV infections. medRxiv preprint. DOI:10.1101/2020.02.11.20021493. [CrossRef] [Google Scholar]
- Kim JY, Ko JH, Kim Y, et al. Viral Load Kinetics of SARS-CoV-2 infection in first two patients in Korea. J Korean Med Sci. 2020. February 24;35(7):e86. . [PMC free article] [PubMed] [Google Scholar]
- [Cited 2021 Mar 25;]. https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-detection-instructions.html
