A deep dive into PCR testing
Though polymerase chain reactions (PCR) tests have been one of the most common laboratory tests in biochemical research for decades, they have only recently become a topic of discussion for the public as they have been at the center of the COVID-19 testing efforts. PCR tests have been seen as the gold standard for proving that a person does not have COVID-19, being required for everything from surgical procedures to international air travel in order to prevent the spread of disease. But what is a PCR test and why are they so effective at proving that someone doesn’t have a disease like COVID-19?
To answer these questions, it is important to understand exactly what a PCR test is. The PCR test is a method of searching for the presence of small amounts of DNA based on the principle of amplification through something called a polymerase chain reaction, according to Dr. Ken Hilt, a lecturer in the UC Davis Department of Molecular and Cellular Biology.
“The polymerase chain reaction makes copies of the piece of DNA that you wish,” Hilt said via email. “It requires primers, short strands of single-stranded DNA that recognize your gene of interest, deoxynucleotides, a heat resistant polymerase (enzyme) and a machine (thermocycler) to raise and lower the reaction temperature several times during one cycle. The machine is programmed to go through many cycles in the course of three hours. During that time, you have made many copies of the gene of interest.”
This polymerase chain reaction allows for the amplification of a single piece of DNA over and over again. This amplification process means that even if only a small amount of the DNA is present in the initial sample, it can be replicated enough times until the DNA is abundant and easily detectable.
When the COVID-19 pandemic began in March 2020, PCR tests were quickly considered the best way to detect the presence of the virus. However, the process of using PCR tests to detect viruses was slightly different than the traditional approach. Viruses use ribonucleic acid (RNA) as their molecular code while PCR tests are designed to look at DNA, so in order to detect the presence of the virus by PCR test, its RNA was first converted to DNA.
Dr. Nam Tran, an associate clinical professor in the Department of Pathology and Laboratory Medicine at UC Davis, explained how PCR tests were adapted to detect SARS-CoV-2.
“PCR works by targeting specific genes within DNA which are then copied over and over,” Tran said. “This allows one to amplify a single piece of DNA many times over so we can detect it. For SARS-CoV-2 testing, the virus is an RNA virus, thus we use reverse transcription PCR, or RT-PCR to first convert the RNA to DNA. Thereafter, we amplify the complementary DNA which is detected.”
While this amplification/replication process of the DNA can theoretically happen nearly infinitely, according to Tran, there is also a tradeoff between how sensitive the test is and how specific the results of the test can be.
“[A]s you increase sensitivity, it comes at a tradeoff of specificity. How sensitive something is helps you determine the number of false negatives,” Train said. “While the specificity helps you determine the number of false positives, there is always a tradeoff between sensitivity and specificity. This is not unique to COVID-19. This is taught to medical students, and health care is well versed with this concept.”
As Tran mentioned, sensitivity refers to the minimum amount of DNA required to get a positive result. In this case, higher sensitivity means more cycles of amplification have occurred, and specificity refers to the ability to correctly identify who is not infected with disease. This is an important concept in clinical medicine because sensitivity and specificity are inversely related—as sensitivity increases, specificity decreases.
Put another way, the more sensitive the test is, the more likely it is to get a false positive (less specificity). This happens because increasing the sensitivity (achieved by increasing the number of times you amplify the DNA) means an individual might take something that is too small to cause infection and amplify it to the point where it would appear to be clinically significant. As a result, the balance of sensitivity and specificity must be carefully determined when using PCR tests as a clinical diagnostic tool.
While PCR tests are seen as the gold standard in detection of DNA, they are not a perfect tool. False positives and false negatives do happen, but false positives are more common in a PCR test because of the idea of sensitivity versus specificity; if the test is too sensitive, then it is possible for a small contaminant to enter at some point in the process which can be amplified to the point of detection.
Dr. Marcelo Prado, the supervisor of the Molecular Pathology Laboratory at UC Davis, and its scientists Carl Sax, Rick Westerman and Mary Grace Laya, explained the complications of a PCR test in a joint statement.
“PCR testing is highly sensitive and target-specific, and false-positive results rarely occur,” Prado said via email. “However, they can occur due to specimen mix-up, processing issues or lab contamination (as opposed to test limitations). All it takes is an improperly sealed PCR plate to aerosolize those amplicons and then all your subsequent runs might be contaminated. That is why molecular testing is performed according to strict standard operating procedures.”
Prado continued, “False negative results can occur from improper specimen collection and can vary depending on the stage of infection.”
False test results are possible, but they are extremely unlikely. The work done in clinical laboratories is precise and careful to prevent these false results from occurring. While false positive results are more common than false negatives, they are still extremely rare and generally only occur when the sensitivity is far too high. For this reason, it is common to report the number of cycles of amplification that the sample went through when reporting a positive PCR test. The clinician is then responsible for interpreting this information and deciding whether the specificity and sensitivity is enough to issue a diagnosis or if further tests are required. If a clinician believes that the sensitivity was too high and that there is a possibility the result is a false positive, they can often order other types of tests that have lower sensitivity, such as an antigen test for COVID-19 which has a higher chance of a false negatives but a lower chance of a false positive. The clinician can then integrate the data from multiple tests to ultimately determine the patient’s diagnosis.
PCR testing is an accurate way to detect the presence of DNA or RNA in a sample—and provided that the sample is collected and stored properly, it can be an extremely effective diagnostic tool for detecting diseases such as COVID-19. As for the pandemic, PCR tests remain an important tool for ensuring that people are not infected by COVID-19.
Written by: Justin Weiner – science@theaggie.org
