Diagnostics use two primary methods to detect infectious diseases: detect the organism or detect antibodies against the organism, serology. Most frequently, they detect infectious agents in biological specimens by culture, cytology, fecal examination, histopathology, immunological techniques and nucleic amplification techniques. Depending on the infectious agent in question, Polymerase Chain Reaction (PCR) assays (DNA or RNA) can be quicker than other available assays. In addition, PCR assay results can often be returned within 24 hours of sample submission, which is generally quicker than culture. Additionally, many organisms in question are difficult to culture, can’t be cultured, or take an exceedingly long time to culture (i.e. viruses). As a result, PCR assays are of particular benefit.
Polymerase Chain Reaction (PCR)
qPCR vs.PCR Testing
In the first step of PCR, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered, and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to sequence around the DNA region, targeted for amplification under specific thermal cycling conditions.
PCR has a number of advantages. It is simple to understand, to use and it produces results rapidly. The technique is highly sensitive with the potential to produce millions to billions of copies of a specific product for sequencing, cloning, and analysis. qPCR shares the same advantages as the PCR, with an added advantage of quantification of the synthesized product. Therefore, it has its uses to analyze alterations of gene expression levels in tumors, microbes, or other disease states.
PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria, parasites or viruses. PCR also permits identification of organisms that cannot be cultured or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.
A limitation of PCR is that even the smallest amount of contaminating DNA can be amplified, resulting in misleading or ambiguous results. qPCR eliminates this by quantizing the PCR product at mid-exponential phase before these contaminating issues can arise and affect the results.
Difference between PCR, qPCR, and RT-qPCR
PCR (original invention) requires 25-30 cycles and the end result must be run on a “gel” or capillary electrophoresis unit requiring extra time and expense. Also, PCR frequently has noise in the end reaction and requires extra interpretation and time usually greater then 24 hr. before results are achieved.
qPCR can detect product during the exponential phases of amplification and collects data at the mid point of the exponential reaction greatly speeding up the results and without the noise which can be seen in traditional PCR.
qPCR is more sensitive then traditional PCR because it detects product in the early stages of amplification.
RT-qPCR is Reverse Transcriptase-qPCR, this technique is used for RNA containing viruses. The RNA must first be converted to DNA via RT and then qPCR is run. This requires some extra reagents and steps and adds slightly to the cost.