ABSTRACT
As we adapt to containment measures for COVID-19, inaccurate detection of the SARS-CoV-2 virus need not, and should not, be the new normal. Molecular testing is currently the only traditional way to accurately detect the virus and provide an accurate measurement of active cases in the target population regardless of whether the individual is symptomatic or not. Polymerase chain reaction (PCR) is one of the most widely used diagnostic tests for detecting pathogens, including viruses, that cause diseases such as Ebola, African swine fever, and foot-and-mouth disease. Building upon the principles of PCR, wherein a minute amount of genetic materials can be copied a million times to a detectable level, the real-time reverse transcription PCR (RT-PCR) enables the copying and detection to be carried simultaneously. Since the COVID-19 virus only contains RNA, real time or conventional RT–PCR and digital PCR (dPCR) is used to detect it.
The use of the PCR in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility, and the reduced risk of carry-over contamination. The aim of this chapter is to discuss the factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages, and limitations of highly recommended PCR variants such as RT-PCR and dPCR. This chapter reviews the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of RT-PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to detection of COVID-19 microbiology as well as studies of gene expression and its genetic disease.
