Real-Time Polymerase Chain Reaction

Abstract

The real-time PCR, also known as quantitative PCR assay, detects the presence of the target pathogen (virus) in real time during PCR amplification without the need for post-PCR gel electrophoresis. The real-time detection of the accumulating amplicons is made possible by labelling of primers, probes or amplicons with fluorogenic molecules. Quantification of the amplicon in real-time PCR is achieved by the detection of the fluorescence signal produced proportionally during the amplification. Dyes such as SYBR-Green I or ethidium bromide that bind non-specifically to any double-stranded DNA or fluorescent probes that specifically bind to the targeted region are used for real-time detection of the amplicon during amplification. The real-time PCR instrument consists of optical sensor to record the transmission emitted by the fluorogenic agent during amplification. The results of real-time PCR are displayed in the form of a curve that has four different phases which are used to determine the cycle threshold (Ct) and amplification efficiency. In order to quantify or determine copy number of the target DNA molecule produced, a standard curve is initially plotted by using Ct values obtained for different known copy numbers of the targeted amplicon region. Once the graph is done, copy number of the same target amplicon in a test sample can be determined using the standard curve.