It is based on PCR technology, which is used to amplify and simultaneously detect or quantify a targeted DNA molecule on a real-time basis. Reverse transcriptase real-time PCR formats can detect and quantify RNA molecules of the test organism in the sample on a real-time basis.

It uses a different thermocycler than the conventional PCR. It is very expensive, 5-10 times more than the cost of conventional PCR (Fig. 1).

Fig1. Real time PCR. Source: Department of Microbiology, JIPMER, Puducherry (with permission).

Advantages:

 Real-time PCR has many advantages over a conventional PCR, such as:

- Quantitative: rt-PCR can quantitate the DNA or RNA present in the specimen; hence can be used for monitoring the disease progression in response to treatment, e.g. viral load monitoring in HIV or hepatitis B viral infection

- Takes less time: In rt-PCR, the amplification can be visualized simultaneously during the process of amplification unlike the conventional PCR where there is an extra-step of gel electrophoresis to detect the amplicons

- Contamination rate is extremely less

- Sensitivity and specificity of rt-PCR assays are much more than the conventional PCR.

Detection of amplification products of real-time PCR:

 The detection of amplified nucleic acid in a real-time PCR reaction is carried out by using a variety of fluorogenic molecules which may be either nonspecific or specific.

- Nonspecific methods: They use SYBR green dye that stains any nucleic acid nonspecifically

- Specific methods: They use fluorescent labeled oligonucleotide probe which binds (i.e. hybridizes) only to a particular region of amplified nucleic acid. Three types of hybridization probes are commonly used:

* TaqMan or hydrolysis prob

* Molecular beacon

* Fluorescence resonance energy transfer (FRET) probe.

 Post-amplification melting curve analysis is used for quantitation of the nucleic acid load.