While quantitative polymerase chain reaction, commonly known as real-time PCR or simply as qPCR, is often employed for studies of gene expression, the years since its innovation by Russell Higuchi and colleagues in the early 1990s have seen its use cover a wide range of goals. For many people, qPCR is nearly synonymous with reverse transcription qPCR (RT-qPCR) targeting messenger RNA, usually to explore the changes in gene expression that accompany the variables of an experiment. In fact, there are most likely many scientists who will use qPCR extensively in their careers with only this specific goal in mind. However, its reach is much larger. At the risk of stating the obvious…qPCR can help to answer any question that hinges upon the number of copies of a specific sequence of DNA (or RNA) in a sample.
For much of the world, qPCR (and PCR as a general technique) came into focus during the progression of the COVID-19 pandemic. This focus highlighted qPCR’s highly effective use as a tool for diagnosis of infectious diseases. The technique’s high degree of sensitivity made it the gold standard for COVID-19 testing, even in the face of faster tools such as antigen tests. (It should be noted, though, that qPCR’s “real-time” nature makes it fairly quick at turning around test results; the need for specialized equipment is what has really held it back from becoming even more widespread.)
This molecular diagnostic approach has been one of the most successful applications of qPCR over the years. Previous to the arrival of PCR, specific diagnosis of bacterial or viral pathogens was often dependent upon the successful culture and subsequent identification of the offending microbes. For many bacteria – and especially for many viruses and protozoa – this was, and is, difficult or even impossible. Even as an achievable goal, culturing takes time that isn’t always available when dealing with the progression of an illness. The use of PCR – and especially qPCR, with its speed and sensitivity – has enabled faster and often more accurate diagnoses for years now.
qPCR and Pathogens
The diagnostic capability of qPCR can be further expanded to give more information about the pathogens it targets. The nature of the assay is, of course, to be quantitative, so while assessing whether or not a pathogen is present in an infection, one can also quantify it. This capability has great implications for research, as pathogen abundance is often the most direct measure possible in assessing the magnitude of an infection. Even with this single endpoint in mind, research options are essentially infinite due to the limitless array of models and scenarios can be designed to lead to such a meaningful value.
This approach is also used with great success in evaluating microbial communities. Quantitative PCR can help in understanding the relative abundance of various taxa within an environment. It can even be used to measure the amount of a specific type of gene within a sample, so that a functional understanding of a metagenome can be achieved in the absence of taxonomic details. In addition, these techniques can be combined with the use of a DNA-binding dye to detect only the contributions of live cells in a sample, an approach often referred to as viability PCR.
Like the PCR technique upon which it is built, qPCR is truly limited in its uses only by the imaginations of researchers and clinicians who employ it. Scientists at Visikol regularly complete gene expression studies for clients, usually analyzing variation in expression induced by the application of a therapeutic agent. However, Visikol has also designed studies using it for analysis of bacterial growth within a human tissue model and for determining relative abundance of bacterial species within a biofilm. Most importantly, Visikol is always on the lookout for interesting studies that will expand the frontiers of research in drug development. If you have an idea for using qPCR in your work, simply reach out to Visikol’s experienced team to discuss it further.