What is PCR Testing

1. What is PCR Testing? In the past, diagnoses were limited to what could be observed by an attending physician with their own senses. As one might expect, this resulted in wildly varying levels of patient care and unconventional tests and treatments, many of which unfortunately involved livestock. Fortunately, the modern era has seen vast advancements in the area of molecular biology, and by extension, medical diagnosis. Today we have definitive tests for many conditions and diseases ranging from infertility to cancer. Recently, the world has seen a dire need for a rapid means of determining if a subject is positive for COVID-19. The current gold standard methods for diagnosing COVID-19 are antigen testing and polymerase chain reaction (PCR). Antigen Testing The most common antigen test is likely quite familiar to just about everyone as it resembles a pregnancy test. COVID-19 antigen tests function in the same way and rely on the specific binding of an antigen (a protein displayed on the surface of the virus) in the sample to antibodies (yet more proteins, usually animal-sourced) immobilized in the spots where the indicator lines appear.1 To facilitate detection, secondary antibodies also bind to the antigen and either fluoresce or change color in response.1 The level of colour change or fluorescence can also be used to quantify the level of antigen present in the sample. In the case of the COVID-19 antigen test, the sample comes from the patient’s upper respiratory tract, usually through an oropharyngeal or nasopharyngeal swab, as pictured to the right. PCR Testing PCR testing makes millions of copies of a specific sequence or sequences in the genetic material present in the sample allowing for detection of a much lower initial viral load. 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 comprised of Ribonucleic Acid (RNA) which is relatively fragile, as opposed to Deoxyribonucleic Acid (DNA), which is far more stable.3 In order to perform the test, which can only be performed on DNA, a conversion from the former to the latter must be performed.3 This is done through the use of an enzyme called reverse transcriptase, which makes a copy of the RNA out of DNA. Once the complementary DNA, or cDNA, has been obtained, the PCR can proceed. The main components consist of the Taq DNA polymerase, the template, the primers, and loose nucleotides in the form of deoxynucleoside triphosphates (dNTPs).3 The polymerase can best be visualized as a piece of machinery whose job is to assemble new segments of DNA. Like a piece of machinery, the polymerase requires instructions such as where to start and the sequence of the new strand. The template is the piece of cDNA created from the sample and provides the sequence of the new strand. Everyone knows the familiar double helix profile of DNA, which gives a good indication of its structure. DNA is composed of two complementary strands of linked nucleotides in

2. matched pairs of A/T and C/G. Thus if one strand is present, the polymerase can produce the opposite strand. Primers give the start points and are short segments of DNA with the opposing sequence of base pairs on the template strand at the beginning of the segment of interest. Finally, dNTPs can be considered the fuel. They are single nucleotides which the polymerase links together into the final product. The actual process of the PCR involves heating and cooling the mixture in order to sequentially separate the complementary strands, adhere the primers, and finally allow the polymerase to replicate the segment of interest.3 By repeating this process, the original strand is exponentially amplified.3