Currently, molecular diagnostics developed a mechanism of polymerase chain reaction (PCR), which was the impetus for the development of a large number of methods of diagnosis, based on the determination of the genetic material.For carrying out real-time PCR using gene fragment (in particular the nucleic acid) of the causative agent, which is not found in other individuals.
principle of PCR is the multiplication of a certain pathogen DNA fragment millions of times by DNA polymerase in a special unit, referred to as the thermal cycler, which provides repetition of the temperature cycling regime according to a predetermined program.
Diagnostics PCR makes it possible to repeat a series of amplifications, maintaining the enzyme activity.When the amplification takes place smoothly, in one cycle of the number of copies of a particular stretch of DNA of the pathogen is doubled, after thirty cycles - a hundred and ten.That is why PCR is used to determine the diagnosis of small enough pathogens, the identification of which is impossible without it.
It should be noted that the RNA can not be a template for real-time PCR.Therefore, in order to detect viral RNA before the amplification necessary to obtain a DNA correlated with its corresponding fragment.The polymerase chain reaction allows to measure DNA and RNA of the pathogen.The information thus obtained is used for monitoring the effectiveness of the therapy, and clinical evaluation of the forecast.
In addition, real-time PCR does not require additional action with respect to the decryption of the target DNA or amplicons, which serve as a barrier to the correct diagnosis and lead to false results.This approach allows us not to use the stage of electrophoresis, which leads to a decrease in the risk of contamination of the polymerase chain reaction.Also, due to the decrease in the number of ongoing manipulation of the pathogen, reduces the time of the survey, it simplifies the process, reduces the likelihood of errors.PCR also allows you to specify the initial number of copies of the DNA of the pathogen in a clinical trial to help identify periods of exacerbations and take timely measures to cure the patient.
Today there are many methods of recording real-time amplification.The most commonly used different techniques used correlated with amplicon fluorogenic probes, the radiation intensity of which varies depending on the accumulation of amplicon and recorded in real time using a special instrument, called a thermocycler with an optical module.This provides a high level of sensitivity of the analysis.When this PCR product can be detected, along with a large quantity of by-products while, when using electrophoresis, in some cases it is not noticed.
When real-time PCR using positive and negative control samples.For example, positive and negative samples in the research analyzed as separate trials, reference sample at the same time added to each sample and a pass all the stages of the analysis.
Today there are a large number of devices for carrying out a polymerase chain reaction.These include Bio-Rad, Applied Biosystems, Cepheid and Roche.Each has advantages and disadvantages
It should be noted that the PCR - method of diagnosis used in the major scientific, research and diagnostic centers around the world, because it gives the possibility to carry out immediate interpretation of the results.