PCR doctor. PCR: a method for diagnosing genital infections

PCR(polymerase chain reaction) - an analysis method based on obtaining a reliable result from a minimum number of DNA fragments of the material under study, confirming that this nucleic acid belongs to a certain type of living organism (bacteria, virus, protozoa, fungus).

The inventor of the PCR method in 1983 received the Nobel Prize for the most accurate and highly informative method for diagnosing infectious diseases, for 100% reliability of the method.

The basis of the PCR method

In the course of the research, the DNA fragment is duplicated in an artificially created environment, outside the body. As a result of the analysis, under the influence of specific enzymes, DNA molecules increase to the amount required for identification using microscopic studies.

The program copies only those DNA molecules that are in the analyzed material. This feature of the method allows not only to determine the types of infection, it is also used to establish paternity, in genetic engineering.

The PCR method allows you to identify hidden infections, and infections, whose pathogens have a long growth period, which makes them invisible during bacteriological culture.

Application of the PCR method

The PCR method is possible to identify any type of infectious agent. Many microorganisms acquire the L-form at the time of exposure to antibacterial drugs and remain hidden from the microscope and immunological analyzes.

PCR is especially widely used to diagnose latent or sluggish inflammatory diseases, if the infection cannot be isolated in another way. First of all, this applies to the following sexually transmitted infections:

• hepatitis C and B,
• papillomavirus infection,
• gardnerellosis,
• herpes.

In therapeutic activities, PCR analyzes are used to diagnose:

• pneumonia,
• pleurisy, difficult to treat,
• for the isolation of Helicobacteria in chronic gastritis with a tendency to erosion,
• to identify a tendency to oncological diseases - the detection of oncoviruses.

The material for PCR analysis is:

• blood,
• , cervix, vagina,
• secret of glands, saliva, phlegm,
• discharge from erosions and ulcers, others.

Where to get a PCR examination

The PCR method is a complex sequential chain of many stages and cycles. For the analysis, specific chemical compounds for the media and enzymes are used. The analysis should be performed by a laboratory assistant who has undergone special training for laboratory research on high-precision equipment using computer technology.

Help desk "Your Doctor" has information about private clinics that have in their arsenal laboratory equipment and reagents that meet the requirements for material analysis by PCR. All manipulations for the collection of blood or other material are carried out in these institutions using sterile instruments and materials, in accordance with the requirements of the Ministry of Health of the Russian Federation to combat the spread of infectious diseases.

In many clinics of the Reference "Your Doctor" vacuum systems are used for taking blood, which completely excludes the theoretically possible error as a result of the examination, and significantly increases the objectivity of the analysis.

Publication date: 2019-06-12

This article is posted for educational purposes only and does not constitute scientific material or professional medical advice.

PCR analysis is a high-precision diagnostic study that allows you to detect causative agents of infectious diseases by their genetic material (RNA, DNA) in a sample of biological fluid taken from a person.

The polymerase chain reaction makes it possible to diagnose a wide range of diseases of viral etiology, including those present in the human body for a long time, proceeding latently. Most often, PCR analysis is prescribed in gynecological and urological practice.

Basic indications

An analysis for PCR is prescribed if the following pathologies are suspected:

• viral hepatitis (A, B, C);
• cytomegalovirus infection;
• gastrointestinal infections (enteroviruses);
• herpes zoster (shingles);
• Filatov's disease (monocytic tonsillitis);
• neurellosis (neonatal granulomatosis).

The ability to use not only venous blood as a biomaterial significantly expands the list of pathologies diagnosed by the PCR method. This list includes:

• bacterial vaginosis;
• salmonellosis;
• diphtheria;
• various forms of tuberculosis;
• human papillomavirus;
• STIs;
• mycoplasma hominis;
• ureaplasma urealiticum.

How to prepare for the study

Preparation for diagnostics by PCR method depends on what kind of biological material will be examined. When donating blood 3 days before, the intake of alcohol, blood thinners is excluded, a certain diet is recommended. When taking a smear from the urethra and vagina in 3-5 days, intimacy is excluded, and douching cannot be done at this time. Smears are not given during menstruation, before it and within 5 days after it ends.

Analysis features

Biological material for PCR research can be blood, saliva, smears. The choice depends on the type of virus or infectious pathology. If you suspect a sexually transmitted disease, morning urine and scraping from the mucous membrane of the vaginal wall are given. For herpes, hepatitis, HIV - venous blood, in a volume of at least 5 ml. In case of damage to the central nervous system, cerebrospinal fluid serves as a biomaterial.

PCR is used in the diagnosis of intrauterine fetal pathologies (material - placental tissue), with pulmonary-type infections (fluid from the pleural cavity or sputum is examined).

There are several methods for performing PCR analysis:

• diagnostics in real time;
• determination of the amino acid and nucleotide sequence of DNA;
• DNA sequencing method;
• nanofluidics.

Most often, real-time research is used, it belongs to the most informative methods, gives the lowest percentage of false-positive results. The analysis is carried out within a day.

In the conclusion, one of the variants of the research result is indicated - positive (confirms the presence of a pathological process) or negative (indicates its absence).

The polymerase chain reaction (PCR, PCR), discovered by the American Carrie Mullis in 1983, and the DNA detection method developed on its basis, brought the scientist the Nobel Prize in Chemistry in 1993. Today it is one of the most accurate methods in laboratory diagnostics of infectious diseases. It allows you to discover what was hidden before the advent of PCR.

Why has such a wonderful method not supplanted other types of laboratory diagnostics, but is used on a par with them, and in some ways even loses to them?

There are different methods for detecting infections in biological fluids of patients.

• Microscopic methods.

Using a microscope, a laboratory assistant looks for pathogenic microorganisms in a specially stained smear - a sample of discharge from the urethra or from the wall of the mucous membrane. Cheap, simple, but uninformative. Thus, it is possible to discern Trichomonas, gonococci, chlamydia, ureaplasma and gardnerella. And that's all.

• Cultural methods (crops).

The same smear is applied to a nutrient medium in a Petri dish and see what grows. All microorganisms form different colonies - in color, shape, size and consistency. It is for these colonies that it is determined: who was in the smear. Also, in general, inexpensive, but long, growing of microorganisms can last from 3 to 8 weeks - during this time the patient's condition can deteriorate significantly. And there is one more nuance: only what was in the smear and in a certain amount will grow. If there were too few pathogenic bacteria, then it will not be possible to identify them.

• Blood tests.

Any pathogens after contact with the human body leave their immune traces in his blood: antigens or antibodies. The method is not the cheapest, but fast. But here it should be borne in mind that up to a certain point the infection may already be in the body, and antibodies and antigens have not yet been detected: too little time has passed or the concentration of pathogenic microorganisms is too low. In addition, this method does not allow distinguishing between the types of individual microorganisms, which are far from always dangerous to humans.

So, all methods have their drawbacks. Among the key ones are the following:

• Low sensitivity, that is, a certain minimum number of microorganisms in the sample is required to be detected.
• Low specificity, that is, if two different types of bacteria (one dangerous, the other not) have the same proteins on the cell surface, then not all analyzes allow you to find out which of these species is in the body.

Both of these problems can be solved by the PCR method.

The method is based on the fact that every living organism (micro or macro) has a unique DNA (or RNA - if we are talking about some viruses). It is the best marker of the presence of a foreign microorganism in the human body that one can imagine. Even if trace amounts of DNA are present in the sample, it means that the body has already encountered this infection in one way or another. How can we “see” these trace amounts? They need to be “multiplied” first.

The PCR method consists of the following steps.

• Stage 1. Sample preparation, i.e. DNA extraction.

Suppose we are looking for a specific bacteria in the sample, for example, chlamydia (Chlamydia trachomatis - the causative agent of chlamydia). At this stage, we don't know if the sample contains her DNA. The sample is specially processed to concentrate the DNA, if present, in a minimum volume of PCR liquid.

• Stage 2. Amplification (multiplication) of DNA using PCR.

First, double-stranded DNA is "unwound" by increasing the temperature and single-stranded strands are obtained. After that, special short nucleic acid fragments - primers - are added to the solution. They will be the seed for the synthesis of the second strand of DNA on a single-stranded "base". So, there is a base, there is a seed, it remains to add nucleotides, from which the second chain will be completed, and a device that will be engaged in this construction. The enzyme DNA polymerase acts as such a device, and the process of completing single-stranded DNA to double-stranded is a polymerase chain reaction.

At the beginning of the cycle, we had two single-stranded DNAs, at the end we get two double-stranded DNAs. And this cycle is repeated many times until the amount of DNA is sufficient for laboratory analysis. For DNA multiplication, special devices are used - amplifiers. At this stage, the researchers also still do not know what kind of DNA they amplified and whether there is chlamydia in the DNA sample.

It should also be added that there are many varieties of the PCR method, which are used depending on the task set before the researcher.

• Stage 3. Detection (definition) of DNA.

At this stage, it is just the finding out whether there is DNA of the desired bacterium in the sample. Previously, the electrophoresis method was used for this, and later scientists began to use DNA probes - special labeled DNA fragments that interact only with a specific region of the analyzed DNA and thus allow the determination of specific nucleotide sequences. Finally, for express diagnostics, fluorescent markers are now used, which make it possible to determine the result of PCR directly in a reaction tube. If there was chlamydia DNA in the sample, the laboratory assistant will find out about it.

What PCR can do

The PCR method allows you to identify pathogenic microorganisms, as well as to analyze for their resistance (resistance) to drugs. In this respect, in comparison with culture and other methods, PCR is much faster and more accurate. PCR methods are increasingly being used in medical practice. For example, now with its help, in some cases, it is possible to determine the presence (or absence) of genetic markers of resistance.

PCR advantages:

• Sensitivity.

To detect the presence of a pathogenic microorganism in a patient's body, even one copy of his DNA is enough. 1 microorganism per sample - this is enough for PCR, a similar approach is impossible for other diagnostic methods.

• Specificity.

As mentioned earlier, the uniqueness of the DNA of each type of microorganism makes it possible to detect them even in the presence of large volumes of foreign, unnecessary, “ballast” DNA that interferes with the analysis.

• Analysis speed.

The whole process of diagnostics by PCR method takes no more than 1 working day.

• Performance.

PCR diagnostics, today, is a fully automated process.

What PCR can't

The PCR method is a truly ingenious invention of molecular biology of the 20th century. But sometimes it is so good that it cannot be diagnosed.

Disadvantages of PCR:

• Specificity.

She's stunningly tall, but too narrow. When inoculated in a Petri dish, everything that was in the sample will grow. And when carrying out PCR, you can find only what you were looking for, what you used the DNA probes for. You can not carry out PCR analysis on chlamydia and accidentally detect hepatitis.

• Sensitivity.

There are many microorganisms that doctors consider opportunistic. They are part of the normal human microflora as long as their concentration does not exceed certain limits. PCR will certainly show their presence, but this does not mean that the person is sick. These microorganisms include mycoplasma, ureaplasma, gardnerella, etc.

• Contamination problem.

Contamination is contamination of a sample with DNA samples from the outside. PCR diagnostics should be carried out in specially trained laboratories and under sterile conditions. Even one copy of "external" DNA from another sample, laboratory air, or laboratory technician's gloves can lead to false positives - with the appropriate primers.

• Influence of other sample components on PCR.

The sensitivity of PCR can be significantly reduced by the influence of other substances. For example, some drugs (anticoagulants) suppress the amplification reaction. To identify such situations, there are special control methods. But the risk of getting false negative results still remains.

• Price.

This is an expensive method. The equipment itself and the reagents for it, as well as the training of laboratory technicians who know how to handle them, are very expensive for clinics and laboratories. Such equipment is not produced in Russia, and the use of domestic reagents impairs the quality of the analysis.

conclusions

• PCR is an excellent modern and expensive method for diagnosing pathogens of various infectious diseases.
• A PCR method is not always required for diagnostics. If the doctor does not consider it necessary to send you for analysis, trust his opinion. With self-medication and self-delivery of tests for research by PCR in a network laboratory, there is a high risk that you will be treated for years for a non-existent disease.
• If the laboratory gives you a result that you doubt, retake the analysis in another laboratory. The risks of false positive and false negative results have not been canceled - despite all the modernity and manufacturability of the method.

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Most people get tested for infections by PCR at least once in their life. But few people know what it is and what the essence of the study is.

Diagnosis of infections by PCR is based on the detection of DNA or RNA of a pathogenic microorganism. It can be detected in any invasive or non-invasive clinical material. It reveals the genetic material of the pathogen, which is copied many times. A certain fragment of the genetic code that corresponds to the standard is determined.

If present in clinical material, the result will be positive.

Advantages of PCR diagnostics of genital infections:

• high accuracy and information content;
• minimal risk of a false positive or false negative result;
• with the help of PCR, the diagnosis of infections can be carried out in any clinical material;
• almost any pathogen can be detected;
• the research is carried out quickly - the results can be obtained within a few hours after the analysis.

Currently, PCR diagnostics of infections is one of the main methods for detecting most sexually transmitted diseases.

Quantification of PCR infection

Most often, doctors prescribe high-quality PCR tests for infections. That is, the result can be either positive or negative. But in some cases, quantitative research is required.

It differs in that, in addition to the presence of the pathogen in the structures of the genitourinary system, its concentration in the clinical material is determined.

Quantitative PCR for infections is necessary in such cases:

• when diagnosing infections that are considered opportunistic (in this case, they can cause inflammatory processes only with an excessive increase in the bacterial population);
• to assess the dynamics of the course of the pathological process and the effectiveness of therapy;
• to assess the prognosis of the disease, determine the risk of complications, and select adequate therapy.

Does it hurt to take a smear analysis of PCR infection

Most tests are painless or cause minimal discomfort to the patient. By itself, PCR cannot cause pain in any way, because it is carried out in the laboratory, away from the patient.

Only the procedure for collecting clinical material can be unpleasant. And it can be different. Obviously, if urine or ejaculate is taken for analysis, then the analysis will not be painful. At the same time, if the cerebrospinal fluid, scraping from the urethra, and prostate secretion are examined, then the sampling of the biomaterial will be more or less unpleasant. Most often, for the diagnosis of sexually transmitted infections, a scraping of urethral cells is taken. This procedure is usually unpleasant, but in our clinic it is painless.

What is the difference between smear and blood analysis of PCR infection

As already mentioned, different clinical materials can be used for diagnosis. In a smear from the urethra, those pathogens that are present in the structures of the urethra are determined. But they are not always there. Therefore, it is often necessary to examine another biomaterial, for example, blood.

Here are some infections that can be taken in a smear:

• analysis of PCR bacterial infections - gonorrhea, chlamydia, ureaplasmosis, mycoplasmosis;
• trichomoniasis;
• viruses - HPV, herpes;
• fungi - candida.

Here are some infections you can donate in your blood:

• analysis of PCR viral infections - HIV, viral hepatitis, herpes, cytomegalovirus;
• bacterial pathologies - syphilis (rare);
• any venereal pathologies in case of their generalization (gonorrhea, candidiasis).

In practice, most often, blood PCR is performed on suspicion of viral infections that may not be released into the external environment through the urethra.

Who needs PCR diagnosis of infection

The main indications for a PCR analysis for infections:

• control after treatment;
• the presence of symptoms of a sexually transmitted disease;
• a history of unprotected intercourse with a new partner;
• detection of infections in a regular partner;
• examination before pregnancy, IVF, surgery;
• diagnostics of infertility in order to determine its causes.

Some people are examined without indications, for prophylactic purposes.

This is because most sexually transmitted infections can be asymptomatic. However, they still spread in the population, cause complications and pose a threat to reproductive health. Therefore, every person who is sexually active, changing partners from time to time, should be screened for infections at regular intervals. It is advisable to be examined once a year. A standard PCR test for 12 infections will take a maximum of half an hour of your time. But it will allow you to timely identify dangerous diseases.

PCR analysis of infection - how to prepare for smear and blood tests

Before taking a smear, you need not to have sex for 2 days and not urinate for 2 hours.

There is no need to prepare for a blood test. Regardless of what clinical material you donate, you should not take antibiotics until the end of the diagnosis.

How to evaluate the result of a PCR test for infections

When carrying out PCR diagnostics of infections, the interpretation of the results is carried out only by a doctor. At first glance, it presents no difficulty.

Indeed, in the analysis form, most often the result is positive or negative. But it is important to take into account the symptoms in the complex, data from other analyzes, and anamnesis data. Sometimes the results of the analysis of PCR for infections are false positive. In other cases, the results of the PCR analysis for infections are false-negative.

In the first case, an infection is found that actually does not exist. In the second case, the pathogen present in the body is not detected. For the result to be reliable, it is important to understand when a positive PCR test for infections after infection. It is advisable to carry out the diagnosis no earlier than 2 weeks after unprotected sex.

PCR analysis for infections - which doctor prescribes

Diagnosis of infections by PCR is used in many branches of medicine. One of the main areas of application is venereology. Such tests are prescribed by a venereologist, less often by a gynecologist or urologist.

If you are looking for where to get a PCR test for infections, come to our clinic.

Our advantages:

• painless collection of clinical material;
• fast diagnostic tests;
• no queues
• high diagnostic accuracy;
• the possibility of obtaining a doctor's consultation and, if necessary, prescribing treatment.

The production time for PCR infection analysis usually does not exceed 1 day. In some cases, it is possible to get the result after a few hours. But the cost of urgent diagnostics is higher. Our clinic conducts research for individual diseases, as well as complex PCR tests for 12 infections.

The price of diagnostic procedures depends on a number of factors:

• urgency of diagnosis;
• used clinical material;
• the number of pathogens.

Our clinic works with several laboratories. This allows us to provide a wide range of diagnostic services and low research prices.

If you need to take PCR tests for infections, contact a competent venereologist.