PCR, or polymerase chain reaction, is a sophisticated laboratory technique that is widely used in medicine and other scientific fields. PCR diagnostics at one time became a real breakthrough in science.
Description
Quantitative PCR is a type of real-time PCR.
This is understood as a variant of PCR, in which the kinetics of the reaction is constantly recorded, which makes it possible to qualitatively evaluate the content of specific DNA nucleotide sequences in a complex molecular mixture.
Quantitative PCR method will be discussed in more detail below.
The essence of the analysis
Cultural and serological methods are especially often used to diagnose infections. Under the first, antibodies to an infectious pathogen in the blood serum are determined. In the second, biological material taken from a sick person is used to sow a specific medium that is favorable for the growth of pathogen colonies. Diagnostics in bothcase may last days or weeks.
PCR examination can be carried out with different biological materials that are obtained from a sick person. Blood and other pathological, physiological and biological media and fluids act as samples. You can do PCR feces or urine.
Atypical and viral infections are most often diagnosed by quantitative PCR, since they may not be easy to diagnose due to the specific nature of the pathological process provoked by them. To determine such infections, it takes time, during which antibodies begin to be produced in the body, determined by serological methods. But this is unacceptable in some cases.
Method of carrying out
PCR method - laboratory detection of infection in samples isolated from the patient (in vitro).
To conduct a polymerase chain reaction, a set of special reagents is required.
The test material is introduced into test tubes with reagents. The test tubes are placed in a special apparatus - a PCR amplifier, which is designed to amplify (increase the number) of the desired RNA or DNA fragments, the PCR amplifier operates in a cyclic mode. In any cycle, if the samples contain the RNA or DNA sequence of the pathogen, then more and more copies of particles of these nucleic acids accumulate in the solution. You can determine the presence of the pathogen, and its amount in the samples.
Types of PCR
Qualitative analysis by PCR allows you to get the following result:
- negative when the pathogen of interest is not found in the samples;
- positive if sequences are found in the samples that are characteristic of a particular pathogen.
With a positive PCR result, we can speak with 95% accuracy about the presence of a diagnosed infection. At the same time, the accuracy of PCR sets used for diagnostic purposes reaches 100%.
Usually 5% of incorrect results are determined by the human factor. For example, violations of the technique of analysis and storage of reagents can significantly reduce the accuracy of the study.
Quantitative PCR defines the concept of viral load. It is possible to determine how many sets of pathogen DNA were in the samples taken from the patient.
The severity of the infection is directly proportional to the increase in their number. You can also determine the success of therapy to reduce the viral load.
Submission for PCR biomaterial
Quantitative PCR is carried out in the clinic, mainly in the morning. During a visit to the doctor, the patient will be told what needs to be taken: scraping, smears, urine or blood. PCR can detect pathogens regardless of the level of contamination of the material.
For a positive analysis, in theory, the presence of only one pathogen in the samples is required. In practice, they try to create even more favorable conditions. There are some recommendations for this:
- if they take a scraping or swab from the genitalsorgans, you need to refrain from intimate intercourse three days before the study;
- you can not douche with antibacterial drugs or wash yourself before the analysis;
- three hours before taking a smear from the urethra, you need to be patient and not empty your intestines.
Don't follow these rules when donating blood.
How are the results of quantitative PCR analysis interpreted?
Quantitative evaluation of the results obtained
In a number of clinical situations, the problem of the effectiveness of the treatment and / or the dynamics of the pathology process appears. Such questions are especially relevant when examining people with chronic infections (human immunodeficiency virus, hepatitis B and C). When diagnosing, they are based on the fact that the accumulation of amplicons (PCR products) is proportional to the presence of copies of the desired gene in the analyzed sample.
Of course, taking into account the results of quantitative PCR is done by gel electrophoresis with the study of the intensity of special PCR signals. Also, a prerequisite for a correct quantitative assessment of the results obtained is positive reliable control samples containing a known number of copies of the desired gene (for example, one thousand copies of the hepatitis C gene for one PCR reaction). Several serial dilutions of the quantitative control allow calibration curves to be generated and are used to evaluate the content of clinical genocopies in clinical specimens.
In the definition of quantitative PCR, a key achievement has been the creationfluorescent DNA probes that are added to the reaction mixture simultaneously with simple primers and allow you to track the PCR process in time, that is, real-time PCR.
The TaqMan methodology refers to the synthesis of fluorescent DNA probes that are specific to the middle part of the amplicon and have two labels at the ends. One of them is a fluorescent label, the second is a quencher molecule for this fluorescence.
A special device, which is a hybrid of a fluorimeter and a thermoblock-amplifier, makes constant measurements of fluorescence in each test tube (real-time PCR principle). After 20-40 cycles of PCR, individual curves will be obtained for each sample. The number of copies of the desired gene that is contained in the test sample can be calculated from calibration curves with control samples.
Also, an important feature of the implementation of PCR by the fluorescent method is that there is no need to open tubes with a PCR mixture when taking into account the results. This reduces the possibility of room contamination with amplification products, and there is no need to allocate special work areas for electrophoresis.
What does a quantitative PCR transcript show?
What is being discovered?
Through a method such as quantitative PCR, qualitative changes in genes are found: insertions, deletions and point mutations. But with some hereditary disorders, the corresponding symptoms appear in response to a change in the content of certain genes.
ThanksQuantitative analysis gives a numerical result, most often in IU / ml. This means that in one milliliter of the test sample, a certain number of copies of the RNA or DNA of the pathogen, measured in international units, was found.
Depending on the magnitude, the severity of the infection is diagnosed. Blood is usually tested to determine viral load, as viruses move freely in the blood during illness.
Features
Quantitative PCR in a blood test has two features.
- The analysis is carried out in the presence of primers or deoxyribonucleoside triphosphates, which are labeled with a fluorescent or radioactive label to accurately determine the content of the formed PCR product.
- In the PCR process, end the reaction early before there are too many PCR products. This is due to the fact that at the final stage of saturation, when there are a lot of PCR products, both the enzyme itself and the substrates act as the limiting links of this reaction. The end of the next PCR cycle under such conditions is no longer characterized by a doubling of the number of PCR products, insignificant quantitative differences between different samples are leveled, which are quite clearly defined in the early stages after passing through a series of reaction cycles.
Cost of PCR diagnostics
PCR research has a cost, which depends on what kind of infection will be tested, on the analysis methodology and the test material. To determine one infection, you will have to give within200-800 rubles. A fee for taking biomaterial is also added - approximately 400 rubles.
