At present, the mechanism of polymerase chain reaction (PCR) has been developed in molecular diagnostics , which has become the impetus for the development of a large number of methods for diagnosing diseases based on the determination of genetic material. For real-time PCR, a fragment of the gene (in particular nucleic acid) of the pathogen that is not found in other individuals is used.
The principle of PCR is to multiply a specific DNA fragment of the pathogen millions of times using DNA polymerase in a special apparatus called a thermal cycler, which ensures cyclic repetition of the temperature regime according to a given program.
Diagnosis of PCR makes it possible to repeat a series of amplifications, while maintaining the activity of the enzyme. When amplification proceeds without hindrance, in one cycle the number of copies of a certain section of the pathogen DNA doubles, after thirty cycles - one hundred and ten times. That is why PCR is used in diagnostics to determine sufficiently small pathogenic organisms, the identification of which is impossible without it.
It should be noted that RNA cannot be a matrix for real-time PCR. Therefore, in order to detect viral RNA, before amplification, it is necessary to obtain DNA corresponding to its corresponding fragment. The polymerase chain reaction makes it possible to measure the DNA and RNA of the pathogen. The information obtained in this way is used to monitor the effectiveness of the therapy used, as well as to evaluate the clinical prognosis.
In addition, real-time PCR does not require additional actions regarding the decoding of the DNA or amplicons under investigation, which serve as a barrier to making a correct diagnosis and lead to false results. This approach allows not to use the electrophoresis stage, which leads to a decrease in the risk of infection by products of polymerase chain reaction. Also, due to the decrease in the number of manipulations with the pathogen, the time of the study is reduced, its process is simplified, the likelihood of errors is reduced. PCR also allows you to determine the initial number of copies of the DNA of the pathogen in a clinical sample, which helps to identify periods of exacerbation of diseases and take timely measures to cure the patient.
To date, there are a large number of methods for fixing amplification in real time. Most often, various techniques are used that use fluorogenic probes correlating with amplicon, the radiation intensity of which varies depending on the accumulation of the amplicon and is recorded in real time using a special device called an amplifier with an optical module. This provides a high level of sensitivity of the analysis. In this case, the PCR product can be detected along with a huge number of by-products, while when using electrophoresis it is often not noticed.
In real-time PCR, positive, control, and negative samples are used. Thus, the positive and negative samples during the study are analyzed as separate samples, a control sample is added to each such sample and goes through all stages of the analysis.
To date, there are a large number of devices for conducting polymerase chain reaction. These include Bio-Rad, Applied Biosystems, Cepheid or Roche. Each of them has advantages and disadvantages.
It should be noted that PCR is a diagnostic method used in large scientific, research and diagnostic centers of various countries of the world, since it makes it possible to conduct an instant interpretation of the results.