Real Time PCR

In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR), is based on the polymerase chain reaction, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence.

The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-stranded DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.

Applications of Real Time Polymerase Chain Reaction

There are numerous applications for real-time polymerase chain reaction in the laboratory. It is commonly used for both diagnostic and research applications.

Diagnostically real-time PCR is applied to rapidly detect the presence of genes involved in:

1-           Infectious diseases (e.g. HCV, HBV, T.B., CMV, HIV & HPV).

2-           Cancer (e.g. Philadelphia chromosome)

3-           Genetic abnormalities (e.g. Different mutations of the Familial Mediterranean Fever (FMF), thalassaemia and the genetic detection of the anticoagulant therapy dose).

4-           Organ transplantation (e.g. HLA type І & ІІ)

5-           Research studies (highly sensitive quantitative measurements of gene transcription)

Real Time PCR in Detection of HCV

Real Time PCR is the most rapid, sensitive and accurate method to detect Hepatitis C Virus RNA, it can detect a virus load (with linearity) in a range from (5) IU/ml to (30,000,000) IU/ml. Detection assays of Real Time PCR have equal sensitivity for the detection of all HCV genotypes.

In contrast, TMA can do a qualitative detection of HCV only (not HBV) while the sensitivity of both Real Time PCR & TMA are the same. So Real Time PCR has the advantage on TMA and all the old types of PCR.