What is PCR Testing? | FastLab

In the past, diagnoses were limited to what the attending physician could perceive with his or her own senses. As might be expected, this led to widely varying levels of patient care and unconventional tests and treatments, many of which unfortunately involved animals. Fortunately, the modern era has brought tremendous advances in the field of molecular biology and, in turn, medical diagnosis. Today, there are definitive tests for many diseases and conditions, from infertility to cancer. Recently, it has become apparent that there is an urgent need for a rapid method to determine if an individual is positive for COVID-19. The current gold standard methods for diagnosing COVID-19 are antigen testing and polymerase chain reaction (PCR).

Antigen Tests

COVID-19 antigen testThe most common antigen test is probably familiar to just about everyone, as it is similar to a pregnancy test. COVID-19 antigen tests work in the same way and rely on the specific binding of an antigen (a protein on the surface of the virus) in the sample to antibodies (other proteins, usually of animal origin) immobilized at the sites where the indicator lines appear.1 To facilitate detection, secondary antibodies also bind to the antigen and fluoresce or change color in response.1 The degree of color change or fluorescence can also be used to quantify the amount of antigen present in the sample. In the COVID-19 antigen test, the sample is collected from the patient's upper respiratory tract, usually through an oropharyngeal or nasopharyngeal swab, as shown on the right.

PCR tests make millions of copies of a particular sequence or sequences of genetic material present in the sample so that a much lower initial viral load can be detected. These copied sequences can then be used to diagnose a patient, either by their presence (positive) or absence (negative). In the case of COVID-19, the genetic material is ribonucleic acid (RNA), which is relatively sensitive, unlike deoxyribonucleic acid (DNA), which is far more stable.3 To perform the test, which can only be done with DNA, a conversion from RNA to DNA must occur.3 This is done with the help of an enzyme called reverse transcriptase, which turns DNA into a copy of RNA. Once the complementary DNA, or cDNA, is obtained, PCR can be performed. The main components are the Taq DNA polymerase, the template, the primers, and loose nucleotides in the form of deoxynucleoside triphosphates (dNTPs).3 The polymerase can best be thought of as a machine whose job is to assemble new DNA segments. Like a machine, the polymerase needs instructions, such as where to start and what sequence the new strand should have. The template is the piece of cDNA that is created from the sample and provides the sequence of the new strand. Everyone is familiar with the well-known double helix profile of DNA, which gives a good indication of its structure. DNA consists of two complementary strands with linked nucleotides arranged in pairs to form A/T and C/G. Therefore, if one strand is present, the polymerase can make the opposite strand. Primers provide the starting points and are short stretches of DNA with the opposite sequence of base pairs on the template strand at the beginning of the stretch of interest. Finally, the dNTPs can be thought of as fuel. They are single nucleotides that are assembled by the polymerase to form the final product. The actual process of PCR involves heating and cooling the mixture to separate the complementary strands one by one, attaching the primers, and finally allowing the polymerase to replicate the segment of interest.

3 By repeating this process, the original strand is exponentially amplified.

Originally, these cycles were performed manually by moving samples from one water bath to another. Fortunately, this process has been automated.2 On average, PCR tests go through 40 of these loops, producing over one trillion copies of the initial strand.3 To put this into perspective, one trillion seconds is equivalent to 30,000 years, or going back to the Neolithic era. This enormous amount of amplification allows the detection of the virus at extremely low concentrations, resulting in both smaller sample size and high detection accuracy. This detection is accomplished by using fluorescent markers bound to a probe in a procedure known as quantitative PCR (qPCR). This probe is another short segment of complementary DNA that adheres to the template and is destroyed during copying. When destroyed, the marker fluoresces. When the fluorescence reaches the point where it is detectable, the cycle threshold (Ct) is reached and can be used to calculate the concentration of the original template in the sample.