Molecular DiagnosticsReal Time PCR Technique (Quantative PCR or Real Time PCR)




Over the last years, PCR has significantly improved the diagnosis of infectious diseases. However, it still remains time consuming and susceptible to false positive and negative results.In addition, as conventional PCR is a non-quantitative technique, the exact number of
pathogens copies in samples cannot be specified Nevertheless, this parameter is crucial for the control of diseases and until now it was achieved with complicated procedures. This fact emerged the need for the use of a more rapid and sensitive molecular technique, which could quantify pathogens in various animal tissues.

As a result, real-time RCR, the most sensitive technique in molecular biology was developed. PCR in real boosted the evolution of the existing methodologies due to the quantification of the pathogen. It is widely applied in medicine and it is considered as the most sensitive technique amplification of DNA and RNA fragments. It is based on the measurement of a fluorescent substance attached in the double stranded DNA

If we were to compare those methods for their reliability, we should emphasize in the above:

1. The sensitivity of real-time PCR concerning the detection of pathogens has been compared in many studies with those of conventional PCR. In most of these, real-time PCR’s sensitivity was about 10 times higher than conventional PCR
resulting in a decrease in false negative samples as real-time PCR detects the pathogen even in very low concentration in the sample.

2. The instrument is connected to a PC for monitoring the amplification of DNA even from the first steps of the method.

3. In addition, conventional PCR procedure is followed by the coloring (with ethidium bromide) and electrophoresis of the final product for the confirmation of its size. (image). These stages, which are carried out by the lab staff, are critical, because of the risk for contamination of the reagents and products resulting in false positive results and mistakes during the interpretation and recording of the results. On the contrary, in real-time PCR the reading and recording of the results is carried out by the computers’ software and the products of the amplification are not opened in the laboratory invoronment.

4. It has also been estimated that the binding ability of ethidium bromide with the product is less effective than
real-time PCR’s dyes.

From the above, it is obvious that real-time PCR is capable of:

• Detecting subclinical infections

• Estimating the progress of a disease in acute conditions

• Monitoring the efficacy of treatment

To conclude with, real-time PCR surpasses the conventional one and tends to replace it.

In the following figure, the results of both methodologies (real-time and conventional PCR) are given as an example of the way that they are interpreted (read) in each method.

The difference in the use of technology and results’ precision is obvious between those techniques.

Graph. Fluorescence curves resulting from logarithmic dilutions of a well-known pathogen. The x-axis shows the number of cycles, during which the fluorescence intensity exceeds the set limit determined by the real-time PCR device and the y-axis shows the fluorescence during the multiplication of the pathogen DNA. The multiplication cycle in which the linear fluorescence curve begins to increase is used to quantify the agent.


Picture. The result of conventional PCR as obtained from the electrophoresis of the product, based on which the results are read. In the first cell there is a DNA ‘scale’ according to which the size of the amplification product is compared (2nd and 3rd cell).

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Molecular Diagnostics
Real Time PCR Technique (Quantative PCR or Real Time PCR)