Quantitative polymerase chain reaction (Q-PCR) is a method by which the amount of the PCR product can be determined, in real-time, and is very useful for investigating gene expression. Often abbreviated to Q-PCR, this method is sometimes also referred to as real-time PCR or depending on the application, quantitative reverse-transcriptase PCR (both of which are abbreviated to RT-PCR, which can be rather confusing). We will refer to all quantitative PCR methods as Q-PCR in this chapter, as this will distinguish between the two types of RT-PCR as necessary. Q-PCR does not rely on any downstream analysis such as electrophoresis

or densitometry and is extremely versatile, enabling multiple PCR targets to be assessed simultaneously but can sometimes be a little trickier to set up than “standard” PCR; however, if you are sufficiently familiar with “standard” PCR then you are in a good position to successfully undertake Q-PCR.

Real-time PCR (RT-PCR) is also called quantitative PCR or qPCR. The key feature in RT-PCR is that amplification of DNA is detected in real time as PCR is in progress by the use of fluorescent reporter. The fluorescent reporter signal strength is directly proportional to the number of amplified DNA molecules.

In 2001–2010, RT-PCR has been used as a powerful tool for genotyping (Alker, 2004), quantifying viral load, and gene copy number assays. RT-PCR has been the gold standard of gene expression level assay.

There are two detection methods of RT-PCR, the first is based on sequence-specific probe such as TaqMan probe, molecular beacon; the second is based on generic non-sequence-specific double-stranded DNA-binding dye such as SYBR green. RT-PCR is a very sensitive and powerful DNA analysis tool. RT-PCR can be divided into four stages: linear ground phase, early exponential phase, linear exponential phase (log phase) and plateau phase. In the first phase, PCR is just starting, fluorescent signal has not risen above background. The second phase is where fluorescent signal just rise significantly above background, the cycle at which occurs is called cycle threshold (Ct). In linear exponential phase, PCR is in its optimal amplification stage with doubling PCR products in every cycle.

The last phase is when substrates are exhausted and Taq DNA polymerase is in its end of life, fluorescent signal will no long increase. Figure 1 shows the PCR stages.

A variety of RT-PCR assays were developed during the epidemic for SARS-CoV (1,58), including a commercial ready-to-use RT-PCR kit (Artus Biotech, Hamberg, Germany). Early assays based on convention

al designs that required postamplification product processing (e.g., gel electrophoresis), were time-consuming and prone to false-positive results from amplicon contamination. Conversely, real-time RT-PCR assays based on detecting and quantifying a fluorescent signal generated during amplification do not require postamplification processing and therefore eliminate one potential avenue for template contamination.

3 views0 comments