The Real Time PCR combines the new automatic systems for the nucleic acids purification, it offers an ideal platform for the development of a wide variety of molecular tests for the identification and quantification of infectious agents of clinical interest.
This technology is based on the detection of the emission of fluorescence during each amplification cycle, which allows the continuous monitoring of the PCR reaction. An exciter laser beam is transmitted to the sample by an optical fiber.
In the Real Time PCR, the amplification and detection processes occur simultaneously in the same closed vial, without the need for any further action. By fluorescence detection the amount of DNA formed can be measured during the amplification.
There can be two types of fluorescence detection systems, used in real-time PCR:
1. Intercalating agents.
2. Specific probes labeled with fluorochromes.
1. Intercalating agents</>
They are fluorochromes that significantly increase the emission of fluorescence when they bind to double-stranded DNA. The increase of DNA in each cycle is reflected in a proportional increase of the emitted fluorescence. This detection system has the advantage that the optimization of the reaction conditions is very easy. Their drawback is their low specificity, because they bind differently to nonspecifically generated products. To improve this, optimum reaction conditions and primer selection must be employed to decrease the risk of dimer formation.
2. Specific probes labeled with fluorochromes.</>
They are probes labeled with two types of fluorochromes, one donor and one acceptor. The process is based on the transfer of fluorescent energy by resonance between the two molecules.
The most used are the hydrolysis or TaqMan probes, the beacons molecular probes and FRET probes; those of hydrolysis are oligonucleotides labeled with a donor fluorochrome with the 5'end that emits fluorescence upon being excited and an acceptor at the 3' end that absorbs the fluorescence released by the donor. The molecules must be close, if the probe is intact, the fluorescence emitted by the donor is absorbed by the acceptor; during the amplification of target DNA, the hybrid probe with its complementary strand.
FRET probes are composed of two probes that bind to adjacent sequences of the target DNA. One of the probes carries a donor at the 3' end and the other an acceptor at the 5' end. When the probes are hybridized, both fluorochromes are close. Upon being excited, the donor transfers its energy to the acceptor, which, at the same time, emits the fluorescence that detects the reader of the equipment.
The use of probes guarantees the specificity of detection and allows the identification of polymorphisms or specific mutations.
Real-time PCR offers an ideal platform for the development of a wide variety of molecular tests, due to its undoubted advantages, such as ease of use, thanks to its speed; it allows a much larger flow of samples and tests. Another advantage is that, when using closed systems, the risk of contamination significantly decreases. In addition, it allows quantifying the initial concentration of nucleic acid present in the samples more accurately and simply.