PCR, or polymerase chain reaction, is a complex laboratory method that is widely used in medicine and other scientific fields. PCR diagnostics at one time was a real breakthrough in science.
Description
Quantitative PCR is a type of PCR that is performed in real time.
This is understood as a variant of PCR, in which the kinetics of the reaction is constantly recorded, which allows a qualitative assessment of the content of specific nucleotide DNA sequences in a complex molecular mixture.
The quantitative PCR method will be considered 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 blood serum are defined. In the second, biological material taken from a sick person is used to inoculate a specific medium that is favorable for the growth of pathogen colonies. Diagnostics in either case can last days or weeks.
PCR examination can be carried out with various biological materials that are received 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, because they may not lend themselves to simple diagnosis due to the specifics of the pathological process they provoke. To determine such infections, time is required during which antibodies determined by serological methods begin to be produced in the body. But this is unacceptable in a number of cases.
Way of conducting
The method of PCR is the laboratory determination of infection in samples isolated from the patient (in vitro).
To carry out the polymerase chain reaction, a set of special reagents is required.
The test material is introduced into test tubes with reagents. The tubes are placed in a special apparatus - 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 a sequence of RNA or DNA of the pathogen, then in the solution more and more copies of the particles of these nucleic acids accumulate. It is possible to determine the presence of the pathogen, and its amount in the samples.
Types of PCR
Qualitative analysis by PCR allows you to get this result:
- negatively when the desired pathogen is not found in the samples;
- positively 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 the PCR sets used by the diagnostic target reaches 100%.
Usually 5% of incorrect results are determined by the human factor. For example, violations of the analysis technique and reagent storage rules can significantly reduce the accuracy of the study.
Quantitative PCR defines the concept of viral load. In this case, one can determine how many sets of DNA of the pathogen 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 viral load.
Submission for PCR biomaterial
Quantitative PCR is performed at the clinic, mainly in the morning. During the visit to the doctor they will tell the patient that it is necessary to take: scraping, ointments, urine or blood. PCR can identify pathogens regardless of the level of contamination of the material.
A positive analysis in theory requires the presence of only one pathogen in the samples. In practice, they try to create even more favorable conditions. There are some recommendations for this:
- if they pass a scraping or smear from the genitals, you must refrain from intimate communication three days before the study;
- You can not douche with antibacterial drugs or wash before analysis;
- three hours before taking a smear from the urethra, you need to tolerate and do not empty the intestines.
When donating blood to a patient, such rules can not be followed.
How are the results of quantitative PCR analysis decrypted?
Quantification of the results
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). In the diagnosis, 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, the results of quantitative PCR are taken into account 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 a reliable reliable control sample containing a known number of copies of the desired gene (for example, one copy of the hepatitis C gene per PCR reaction). Several consecutive dilutions of quantitative control allow you to create calibration curves, and they are used to evaluate the content in clinical samples of genocopies.
In determining quantitative PCR, a key achievement was the creation of fluorescent DNA probes, which are added to the reaction mixture simultaneously with simple primers and allow monitoring the PCR process, i.e. real-time PCR, in time.
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 fluorescence label, the second is a quencher molecule of this fluorescence.
A special device, which is a hybrid of a fluorimeter and a thermoblock-amplifier, makes continuous measurements in each fluorescence tube (the principle of real-time PCR). After 20-40 PCR cycles, individual curves will be obtained for each sample. The number of copies of the desired gene, which is contained in the test sample, can be calculated from calibration curves with control samples.
Another important feature of PCR using the fluorescence method is that tubes with a PCR mixture do not need to be opened when taking into account the results. Due to this, the possibility of infection with the products of amplification of the premises is reduced, and the need for the allocation of special work areas for electrophoresis is eliminated.
What does the decoding of quantitative PCR show?
What is being discovered?
Using 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.
Due to quantitative analysis, a numerical result is obtained, most often in IU / ml. This means that in one milliliter of the test sample, a certain number of copies of RNA or pathogen DNA was found, measured in international units.
Depending on the size, the severity of the infection is diagnosed. To determine the viral load, blood is usually examined, since viruses with the disease move freely in the blood.
Features
Quantitative PCR in a blood test has two characteristics.
- 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 resulting PCR product.
- In the PCR process, the reaction should be completed early, before too many PCR products appear. This is due to the fact that at the final stage of saturation, when there are a lot of PCR products, both the enzyme and the substrates act as limiting links in 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, minor quantitative differences between the various samples are leveled out, which are quite clearly defined in the early stages after passing through a series of reaction cycles.
The cost of PCR diagnostics
PCR testing has a cost, which depends on which infection will be checked, on the analysis technique and the material being studied. To determine one infection, you will have to pay within 200-800 rubles. A fee for taking biomaterial is also added - about 400 rubles.