PCR analysis for 5 infections. PCR analysis is the most accurate test for infections. The PCR method allows

For successful treatment the most important point is a quick and error-free diagnosis. IN Lately The list of methods and capabilities of diagnostic centers, clinics and hospitals in the field of diagnostics is constantly expanding. After the advent of a relatively new technique - PCR analysis, this method quickly gained adherents among medical workers, since it is often indispensable in identifying infectious diseases.

The scope of application of the method based on the polymerase chain reaction is not limited to medicine, but PCR diagnostics have become most widespread for identifying various diseases.

The undeniable advantages of PCR include the following:

Very high sensitivity. Usually they talk about sensitivity from 95 to 100%, but these are average data. This indicator differs for different microorganisms. So, for hepatitis C it is never lower than 97% (provided all requirements are met), and for determining ureaplasmosis - no less than 99%. This sensitivity is the highest at the moment, so the method has almost no competitors in terms of this characteristic.
Possibility of use at the earliest stages. At a time when other diagnostic methods are not yet able to identify the pathogen due to its small amount in the sample, PCR already produces the correct result. The presence of a pathogen can be detected even in the presence of a single specimen, and in some cases, only a fragment of the microbe. For this reason, the method is also used for chronic or latent pathologies, as well as in the incubation stage.
Wide range of results. The analysis is capable of identifying several pathogens at the same time; this does not require repeated manipulations. Sometimes it is possible to find pathogens that were not even expected during the initial diagnosis.
Versatility of samples. The sample can include samples of blood, saliva, sputum, hair, vaginal discharge, and cells of various tissues.
Speed of obtaining results. In most cases, the answer is received within 5-6 hours. For a culture assay approaching polymerase chain reaction in terms of accuracy, the same result can be obtained in a few days to a couple of weeks.
Quantitative determination of the pathogen. This parameter allows you to determine the titer of microbes in the material, and this property is often necessary to assess the effectiveness of the chosen treatment tactics.
Identification of the microorganism itself, and not determination of its reactions. Most analyzes are based on the response of the pathogen to chemical reagents, staining, etc. This diagnostic method allows you to detect the microbe directly, and not indirect signs, antibodies, etc.

Despite such a wide list of advantages, the method also has some disadvantages:

False detection of microorganisms may occur due to sample contamination from a previous sample. This occurs when the instruments or reagents involved in the analysis are contaminated. Even in the air there are fragments of microbes that can affect the results, so in such laboratories a biological filter with the maximum degree of air purification must be installed.
False negative result. Even with all the obvious manifestations of the disease, negative data are sometimes obtained. The reason for this is the wrong location for collecting the sample, so the laboratory personnel must have sufficient qualifications, knowledge and experience.
The data should be assessed exclusively by the attending physician, because interpreting the results of PCR analysis is a complex process. A positive response does not always mean illness. The fact is that after the disease has been successfully treated, fragments of the pathogen are present in the blood for a long time, so only a very qualified specialist can make a conclusion.
High cost of diagnostics using polymerase chain reaction. The price is due to expensive equipment and high requirements for laboratory conditions and personnel qualifications.

Mandatory conditions for obtaining accurate data in PCR laboratories:

correct mechanism for collecting samples and their correct transportation to the laboratory;
strict adherence to methods during analysis;
high sterility and maximum automation of the process;
use of disposable instruments;
internal laboratory control system to confirm the reliability of data.

Provided that all requirements are met, based on the data obtained using the PCR technique, it is possible not only to determine the disease, but to make a prognosis of its course and create a treatment algorithm.

The essence of the method

The polymerase chain reaction allows you to quickly copy and reproduce the DNA chains of the microbe that caused the disease. In order for the process to proceed quickly, special enzyme preparations are used that accelerate replication and construction of a chain of nucleotides.

After the procedure is completed, the presence of the pathogen in the sample increases millions of times, after which its detection and identification is not a problem.

The laboratory technician can compare the resulting sample with those presented in the database and accurately name it, as well as determine its original quantity.

In addition, the technique allows you to clone individual sections of a DNA chain, cause the necessary mutations in them, and combine and combine sections with each other.

Areas of application

PCR diagnostics are most widely used in medicine. With its help, you can identify viral, infectious, hereditary diseases, and this can be done not only in the active phase, but also immediately after infection.

Areas of medicine in which the PCR method is actively used for diagnostics:

urology and gynecology - to detect most sexually transmitted diseases (ureaplasmosis, herpes, syphilis, gonorrhea, gardnerellosis, trichomoniasis, chlamydia, HIV, mycoplasmosis, human papillomavirus, candidiasis, etc.);
pulmonology - for tuberculosis or pneumonia of a bacterial or viral nature;
gastroenterology - for helicobacteriosis;
in hematology - with cytomegalovirus, various blood oncoviruses;
in the field of infectious diseases - to detect diphtheria, salmonellosis, mononucleosis, listeriosis, hepatitis of viral origin, encephalitis;
in oncology - to determine breast cancer, leukemia, lymphoma.

This method is also used in neonatology, pediatrics, ophthalmology, neurology - in almost any branch of medicine it has proven to be necessary.

The video describes in detail which infections can be detected using the polymerase chain reaction.

Most sexually transmitted diseases are quickly and successfully diagnosed thanks to PCR diagnostics. In addition to identifying the pathogen, this method allows you to find out the specific strain of the microorganism, which makes it possible to select the most effective drug for treatment.

In addition to medicine, the PCR method is also used in other areas:

Forensics and Forensic Medicine - Helps identify a criminal by comparing the suspect's genetic material with samples found at the scene of the crime.
Establishing paternity - the multiplied genetic sets of the parent and child are compared, identical fragments are found, on the basis of which one can judge the degree of their relationship.
Science uses methodology during experiments using gene mutations, establishing the relationship of evolutionary branches of individual organisms, etc.

PCR diagnostics in venereology

Many infections that cause venereal diseases, in the initial stages are very similar in manifestations and symptoms, so it is quite difficult to differentiate them. At the same time, early identification of the infectious agent allows treatment to begin as early as possible and the treatment time to be reduced to a minimum, avoiding the development of possible complications.

Polymerase chain reaction is an effective mechanism for identifying the following groups of genital diseases:

bacterial infections (syphilis, donovanosis, gonorrhea, chlamydia, etc.);
viral pathologies (papillomatosis, condylomatosis, HIV, genital herpes, etc.);
fungi (thrush, balanoposthitis, etc.)

In dermatovenereology, the property of this technique to find infectious agents during periods of remission or during carriage, when a person can infect partners without having symptoms of the disease, is especially useful.

Progress of polymerase chain reaction analysis

PCR analysis occurs in 4 main stages:

preparing material for copying;
combining a DNA fragment and a primer;
copying the genetic set of the material being studied;
identification and evaluation of the resulting DNA set.

Stage 1

The sample is heated to a temperature of 94-98°C in a special device - a thermal cycler, which is programmed to maintain precise temperatures with low error. The set temperature is maintained for a minute or two. This is necessary to denature the DNA so that the double helix separates into two separate strands.

Stage 2

The temperature in the thermal cycler is reduced to 72-75°C so that the primer can recognize a section of the DNA chain similar to it and combine with it.

Primers are special reagents that are suitable only for one type of pathogen. If it is necessary to analyze several pathogens, then the required number of primers is added to the sample.

At this stage, the DNA replicates its template using a primer.

Laboratory capabilities are limited by the availability of primers for specific microorganisms, so the range of possibilities is determined by the availability of such samples.

Stage 3

In this segment, an increase in the genetic material of the pathogen occurs. An enzyme approaches the primer with the template and reproduces a new DNA strand identical to the one contained in the original strand.

When synthesis is complete, one full cycle of the polymerase chain reaction is completed. In time it lasts on average up to 3 minutes. During this time, the material doubles. A further increase in the number of chains occurs in geometric progression, and by the end of the analysis 50-80 cycles have been completed.

Stage 4

A stage that requires attentiveness and special care on the part of the staff.

Determination of the microorganism occurs using electrophoresis or staining marks. When using electrophoresis, the determining factor is the length of DNA fragments, and when using labels, the development of color during the enzymatic reaction.

Types of PCR tests

There are many variations of such PCR diagnostics. The choice of a particular method depends on many factors: the presumptive diagnosis, the capabilities of available laboratories, the method of collecting material, etc.

The most commonly used types of PCR are:

nested;
stepped;
with hot start;
virtual;
asymmetrical;
in real time, etc.

Real-time PCR is predominantly used, since the method is characterized by the most accurate results and maximum data processing speed. After using it, the conclusion is ready in an hour or two.

The method is widely used in pediatrics to detect viruses and blood infections, genetic diseases, and TORCH infections. Neonatologists prescribe such diagnostics in problematic cases literally from the first days of life. Sometimes such diagnostics make it possible to detect infection of intrauterine fluids, which constitute a danger to the development of the fetus during the period of active formation.

If a doctor issues a referral for an examination using polymerase chain reaction, then he must clarify what kind of biomaterial needs to be provided to the patient and how to prepare for sample collection.

Blood analysis

Blood is taken as a biomaterial for testing if HIV, viral hepatitis, toxoplasma, genital herpes and cytomegaly are suspected. For most of these pathogens, before the use of this method, diagnosis was based on the detection of antibodies to the pathogen, which greatly delayed the time of detection of microbes during the period when antibodies had not yet been produced.

Before donating blood for diagnosis in this case, no special preparations are needed, but it is better if the sample is taken on an empty stomach. The only circumstance that can seriously distort the results is the use of antibiotics, so the test should be preceded by a period of several days or weeks after taking such medications, depending on the type of antibiotic.

In the most modern clinics, vacuum systems have recently been used to collect blood for PCR, which minimally damage the skin and minimize the likelihood of contaminants from the air, instruments or the hands of laboratory technicians entering the sample.

Smear analysis

It is used both in gynecology and venereology to determine sexually transmitted diseases. Smear PCR is often prescribed during pregnancy to identify hidden factors that can negatively affect the formation of the fetus or the course of pregnancy.

Preparation for the selection of material consists of completely eliminating sexual intercourse several days before the procedure. Douching with potassium permanganate or herbal infusions is also prohibited. During the critical days and 2 days after them, a smear is not taken for PCR analysis.

Biomaterial is collected in the laboratory from the vagina, cervix, cervical canal or urethra.

PCR analysis of a smear allows you to identify a pathogen that is resistant to a certain type of antibiotic. In addition, indications for this type of diagnostic examination are:

complicated pregnancy for no apparent reason;
acute course of venereal disease;
suspected chronic course of a venereal disease;
identifying the causes of infertility or spontaneous abortions.

Stool analysis

A stool test using polymerase chain reaction is prescribed by a specialist to determine the presence of infection. Preparation for collecting a stool sample for PCR is as follows:

eliminate laxatives in the form of any suppositories, oils or oral medications for several days;
Do not consume medications or foods that give the stool a distinct color the day before.

Material for PCR determination is best delivered in a special container for stool with a stick. The interior of the container is sterile and should not be wiped or washed. When collecting a sample, it is important to ensure that you do not touch the inner surface of the receiver, and there should be no traces of urine in the sample. Before the selection, the use of enemas is prohibited.

Analysis of urine

This PCR study allows you to extract a DNA sample of a virus or bacteria. It is better to collect urine in the morning on an empty stomach. It is best if this happens directly in the laboratory.

Mandatory preparation conditions for obtaining urine test results in a polymerase chain reaction laboratory:

wait 3 weeks after taking antibiotics;
do not drink or eat before collecting fluids;
prohibition of vaginal sex one day before taking the sample;
You need to place the first portion of material in the container after waking up.

Interpretation of results

The diagnostic results are deciphered by the specialist who referred the patient for the PCR test. The protocol issued by the laboratory may include a negative or positive PCR result:

Negative - a case in which no fragments of the causative agent of the alleged disease were found, that is, the person is healthy and has not suffered from this disease recently.
Positive - RNA or DNA of a pathogen is detected in the sample and the person needs treatment.

You should not interpret the data obtained on your own, since age norms can differ significantly, and different titers indicate completely different stages of the disease and approaches to therapy.

Armed with the results of PCR diagnostics, it will be easy for the doctor to determine the list of necessary treatment measures.

The accuracy of PCR diagnostics has made this technique indispensable in diagnosing a huge list of diseases and has made it possible to more effectively identify and eliminate pathogenic processes occurring in the human body.

Polymerase chain reaction (PCR) is a high-precision method in the field of diagnosing hereditary pathologies, infections, viral diseases at any stage (acute or chronic), as well as - at an early stage - before obvious manifestations of the disease by identifying pathogens based on their DNA, RNA , which are genetic material, in samples obtained from the patient. And today we will talk about the essence, diagnostic stages and principles of polymerase chain reaction (PCR) methods, as well as its cost.

What is polymerase chain reaction

The basis of the analysis is amplification (doubling) - the creation of many copies from a short section of DNA (deoxyribonucleic acid), which represents the human genetic complex. The study requires a very small amount of physiological substance (sputum, feces, epithelial scrapings, prostate juice, blood, sperm, amniotic fluid, mucus, placental tissue, urine, saliva, pleural fluid, cerebrospinal fluid). In this case, for example, even a single harmful microbe can be detected in the patient’s genitourinary tract.

The PCR (polymerase chain reaction) technique was developed by the American scientist K. Mullis, who received the Nobel Prize in 1993.

Actively used:

in the early diagnosis of infections, genetic;
in forensic medical examination when there is an extremely small amount of DNA available for examination;
V veterinary medicine, pharmaceuticals, biology, molecular genetics;
for identification of a person by DNA, confirmation of paternity;
in paleontology, anthropology, ecology (when monitoring the quality of products, environmental factors).

This video will tell you in detail what a polymerase chain reaction is:

Who is it prescribed to?

Polymerase chain reaction in the diagnosis of infectious diseases is one of the most reliable methods of particular accuracy and reliability. For example, the reliability of the PCR analysis for chlamydia and many other pathogens is close to 100% (absolute). Most often, the polymerase chain reaction procedure is prescribed to patients who have difficulty identifying a specific pathogen during diagnosis.

Laboratory PCR test is used:

to detect pathogens, causing infection urinary and genital organs that are difficult to identify using culture or immunological methods;
for re-diagnosis of HIV at the initial stage in case of a positive but questionable result of the initial test (for example, in newborns from AIDS-infected parents);
to identify cancer at an early stage (study of oncogene mutations) and individually adjust the treatment regimen for a particular patient;
for the purpose of early detection and potential treatment of hereditary pathologies.

Thus, future parents take a test to find out whether they are carriers of a genetic pathology; in children, PCR determines the likelihood of exposure to a disease transmitted by inheritance.

to detect fetal pathologies in the early stages of gestation (individual cells of the growing embryo are examined for the presence of possible mutations);
in patients before organ transplantation - for “tissue typing” (determining tissue compatibility);
to identify dangerous pathogenic organisms in donor blood;
in newborns - to identify hidden infections;
to evaluate the results of antiviral and antimicrobial treatment.

Why undergo such a procedure?

Since PCR is a highly effective diagnostic method, giving almost 100% results, the procedure is used:

to confirm or exclude the final diagnosis;
rapid assessment of the effectiveness of therapy.

In many cases, PCR is the only possible test for detecting a developing disease if other bacteriological, immunological and virological diagnostic methods are useless.

Viruses are detected using a PCR procedure immediately after infection and before signs of illness appear. Early detection of the virus allows prompt treatment.
The so-called “viral load” (or the number of viruses in the body) is also determined by DNA analysis using a quantitative method.
Specific pathogens (eg, Koch's tuberculosis bacillus) are difficult and take too long to culture. PCR testing allows rapid detection of minimal pathogens (live and dead) in samples convenient for testing.

Detailed pathogen DNA analysis is used:

to determine its sensitivity to specific types of antibiotics, which allows immediate treatment;
to control the spread of epidemics among domestic and wild animals;
to identify and track new infectious microbial species and pathogen subtypes that have fueled previous epidemics.

Types of diagnostics

Standard method

Polymerase chain reaction analysis is carried out on the basis of multiple amplification (doubling) of a specific fragment of DNA and RNA using special primer enzymes. As a result of the copying chain, a sufficient amount of material is obtained for research.

During the procedure, only the desired fragment (corresponding to the specified specific conditions) is copied and if it is actually present in the sample.

This detailed video with useful diagrams explains how PCR works:

Other methods

Real-time PCR. In this type of research, the process of identifying a given DNA fragment starts after each cycle, and not after completing the entire chain of 30 - 40 cycles. This type of research allows you to obtain information about the amount of a pathogen (virus or microbe) in the body, that is, carry out a quantitative analysis.
RT-PCR (reverse transcription mode). This test is used to look for single-stranded RNA to detect viruses whose genetic base is RNA (for example, hepatitis C virus, immunodeficiency virus). In this study, a special enzyme is used - reverse transcriptase and a specific primer and single-stranded DNA is built on the basis of RNA. Then the second DNA strand is recovered from this strand and the standard procedure is performed.

Indications for testing

The PCR procedure is used in the clinic of infectious diseases, neonatology, obstetrics, pediatrics, urology, gynecology, venereology, neurology, nephrology, and ophthalmology.

Indications for testing:

determining the risk of developing genetic abnormalities in a child with the likelihood of hereditary pathologies;
diagnosing both parents when planning pregnancy or in serious condition mothers during ongoing pregnancy;
difficulties with conception, identifying the causes of infertility;
suspicion of sexually transmitted infections in the acute stage and with symptoms of their transition to chronic;
detection of causes of inflammatory processes of unknown origin;
unprotected casual and regular sexual contacts;
determining the sensitivity of a pathogenic microorganism to specific antibiotics;
patients with suspected latent infection to detect pathogens before the development of obvious symptoms (preclinical diagnosis);
patients to confirm recovery after illness (retrospective diagnosis);:

Diagnostics is also used if it is necessary to accurately identify the following pathogens::

hepatitis viruses (A B C G), human immunodeficiency, cytomegalovirus;
Vibrio cholerae;
herpes simplex virus, herpetiform species;
retro - adeno - and rhinoviruses;
rubella, Epstein-Barr, varicella (Zoster) viruses;
parvo and picornoviruses;
bacterium Helicobacter pylori;
Legionella, pathogenic types of Escherichia coli;
Staphylococcus aureus;
pathogen;
clostridia, diphtheria and hemophilus influenzae;

It is also used to determine infections:

Infectious mononucleosis;
borreliosis, listeriosis, tick-borne encephalitis;
candidiasis caused by Candida fungi;
sexually transmitted infections – trichomoniasis, ureaplasmosis, treponema pallidum, gardnerellosis, gonorrhea, mycoplasmosis, chlamydia;
tuberculosis.

Contraindications for

Since the procedure is not carried out with the patient, without any impact on the body, but with biological material taken for research, there are no contraindications for PCR due to the absence of potential danger.

However, biomaterial is not collected from the cervical canal of the uterus after the colposcopy procedure. Submission of smears and scrapings for analysis is allowed only 4–6 days after the end of menstruation and the complete cessation of discharge.

Is the method safe?

None Negative influence on a patient during an isolated study of his biomaterial in laboratory conditions is impossible.

Preparation for the procedure (submission of biological substances for analysis)

Any biological fluid, tissue, or body secretions serve as a sample for PCR analysis, which detects the DNA of a foreign pathogen. The test substance is taken in the form of taking blood from a vein, scraping from the larynx, nasal cavity, urethra, pleural cavity, cervix.

Before the diagnostic procedure, the doctor explains to the patient what material will be collected:

When examining for sexually transmitted infections, secretions from the genital organs, urine, and a smear from the urethra are collected.
When analyzing for herpetic infections, cytomegalovirus, mononucleosis, urine and a throat swab are taken for analysis; for hepatitis, toxoplasmosis, blood from a vein is taken.
For diagnostic purposes various types Cerebrospinal fluid is collected.
In pulmonology, samples for analysis are sputum and pleural fluid.
When conducting a study of possible intrauterine infections during pregnancy, amniotic fluid and placental cells are used for analysis.

The reliability and accuracy of the analysis depends on the sterility of the conditions when taking the material. Since PCR testing is highly sensitive, any contamination of the test substance can distort the result.

Competent preparation for the delivery of biomaterial does not present any difficulties for patients. There are certain recommendations:

when analyzing for sexually transmitted infections:
- exclude intimate contacts 72 hours before submitting the material;
- stop using any vaginal products 3 days before;
- from the evening of the previous day, do not carry out hygiene of the area being examined;
- exclude urination 3–4 hours before taking a sample from the urethra;
stop taking antibiotics a month before testing for infections;
blood is donated in the morning before eating and drinking;
The first morning urine sample is collected in a sterile container after a thorough intimate toilet.

Read below about how diagnostics are carried out using the polymerase chain reaction method.

How does the procedure work?

By doing PCR studies over and over again in the reactor (amplifier or thermal cycler) certain cycles are repeated:

The first step is denaturation. Saliva, blood, biopsy material, gynecological samples, sputum, in which the presence of DNA (or RNA) of a pathogen is suspected, is placed in an amplifier, where the material is heated and the DNA is split into two separate chains.
The second step is annealing or slight cooling of the material and adding primers to it that can recognize the desired regions in the DNA molecule and bind to them.
The third step is elongation– occurs after 2 primers are attached to each of the DNA strands. During the process, the DNA fragment of the pathogen is completed, and its copy is formed.

These cycles are repeated like a “chain reaction,” each time leading to doubling of copies of a specific DNA fragment (for example, the segment where a specific virus is programmed). Within a few hours, many copies of the DNA fragment are formed, and their presence in the sample is detected. After this, the results are analyzed and compared with data from a database of various types of pathogens to determine the type of infection.

Read below about decoding the results and conclusion based on the PCR reaction.

Decoding the results

The final result of the study is issued 1 – 2 days after the submission of biological material. Often - already on the first day after the analysis.

Qualitative analysis

Negative the result means that no traces of infectious agents were found in the substance submitted for testing.
Positive the result means the detection of pathogenic viruses or bacteria in a biological sample with a very high degree of accuracy at the time of submission of the material.

If the result is positive, but no signs of increased infection are detected, this state of the body is called asymptomatic “healthy carriage.” Most often observed when taking biomaterial from a certain place (cervical canal, urethra, oral cavity) in viral diseases. In this case, treatment is not required, but constant medical supervision is required, since there is a possibility of:

spread of the virus from carriers and infection of healthy people;
activation of the process and transition of the disease to a chronic form.

However, if the blood test is positive, this indicates that the infection has struck the body, and this is no longer a carrier state, but a pathology that requires immediate specific therapy.

Quantitative Analysis

The quantitative result is determined by a specialist specifically for certain type infections. Based on it, it is possible to assess the degree of development and stage of the disease, which makes it possible to promptly prescribe the correct treatment.

average cost

Prices for polymerase chain reaction are determined by: the type of research, the difficulty of identifying the pathogen, the difficulty of collecting biological material, the type of analysis (qualitative or quantitative), and the price level in the laboratory.

On the other hand, when studying PCR, it is possible to identify several pathogens at once when collecting one type of material for analysis. This allows you to save on other laboratory tests.

Approximate cost of PCR analysis in rubles:

gonococcus, gardnerella, trichomonas vaginalis – from 180
chlamydia trachomatis – from 190
papillomavirus – from 380 to 500
biocenosis of the urogenital tract in women (quantitative and qualitative assessment of microflora) – from 800.

Even more useful information regarding the PCR study is contained in the video below:

The polymerase chain reaction has been known for 30 years. It is widely used in many fields, from archeology to genetics. It is the PCR method that helps establish paternity, but it is most often used to identify various infectious diseases in the human body. What is PCR analysis?

This is nothing more than an imitation of the process of DNA reproduction, as a result of which a huge number of identical molecules are obtained from a tiny fragment of a DNA molecule over a period of time (usually several hours), which can already be studied. Does this remind you of anything? Very similar to cloning. It is the polymerase chain reaction that is the basis of cloning and is used in the reproduction of tissues in the laboratory. Well, in medicine, PCR analysis is used to identify bacteria (mycoplasmas, , and pathogens), viruses (and papillomavirus), fungi (pathogens) and protozoa (Trichomonas) in the human body.

This method of molecular diagnostics of infectious pathogens is the most sensitive. Allowing the detection of even a single DNA fragment in a sample, the PCR method has virtually no sensitivity limit. And in most cases, its result shows 100% accuracy, especially when diagnosing bacterial infections. It never gives false positive results.

It is also universal, as it allows you to simultaneously identify several microorganisms in one sample, while bacteriological methods use different ways cultivation for different groups of pathogens. In addition, PCR significantly simplifies the task of researchers, reducing the time to conduct the test to 24 hours and simplifying the requirements for transporting samples. If the material for bacteriological and virological methods must be alive, then for the PCR method anything that contains DNA, even dead cells, is suitable.

PCR analysis for infections is extremely important because most of them, affecting the human body, develop asymptomatically or have similar symptoms. For example, the PCR method is widely used to identify the causes of liver diseases, including A, B, C, D, E, as well as the herpes simplex virus. To combat each virus, it is necessary to use completely different treatment tactics. Therefore, to prescribe effective therapy, the doctor needs to accurately identify the pathogen. And if prolonged research is carried out, the infection can become chronic, causing complications that are especially dangerous during pregnancy or other concomitant diseases. And it is the PCR method that is the main assistant in quick and accurate diagnosis.

But here you need to understand that, being almost the only method for identifying some microorganisms (for example, mycoplasma), PCR may be completely unsuitable for others (staphylococci, streptococci, etc.) Therefore, a specialist must choose diagnostic methods and prescribe treatment. It’s not worth going to the laboratory on your own and doing all the tests in a row. They may turn out to be completely useless. And in private clinics, at your request and for your money, they will conduct any tests for you, without even asking why you need them.

How to take a PCR test

To conduct PCR studies, biological fluids and secretions are used human body, which may contain microbes and their fragments. These include: blood, saliva, sputum and urine. In addition, scrapings of epithelial cells of the mucous membrane of the urethra and cervical canal are taken for PCR analysis. For example, to detect the HIV and hepatitis virus, blood is taken from a vein. To diagnose genital infections, genital discharge and a smear from the cervix or urethra are analyzed. To diagnose infectious mononucleosis, a throat swab is used.

Preparation for PCR analysis

Before taking the test, you should consult your doctor and carefully follow his recommendations. Ask the doctor what materials will be taken for the study. A PCR blood test is taken on an empty stomach. But before taking a PCR test in the form of scrapings of epithelial cells, doctors recommend that patients drink beer in the evening, go to the bathhouse and have sex. It sounds strange, but all this activates the infection in the genitals, which can occur in a very mild form, and helps to identify it.

Deciphering the PCR analysis

The result of the PCR test can be positive or negative. This is always noted in the document, which must be issued by the laboratory. A positive result means that DNA of the infection was detected in the biological material donated by the patient. A negative result means there is no infection in the human body.

Since the PCR method is highly sensitive, it should be used to monitor the effectiveness of treatment very carefully and only several weeks after the therapy. After all, even a DNA fragment of a dead pathogen will give a positive PCR test result.

Polymerase chain reaction helps doctors diagnose infections that occur in the human body in a latent form, preventing their development and severe damage internal organs. Carefully monitor your health and at the first suspicion of an infection, consult a doctor.

In most cases, patients have to face a situation where PCR diagnostics are required to examine or determine the disease. It is important to understand what it is and why it is needed.

The polymerase chain reaction (PCR) method is a test that helps identify many diseases in initial stages development. Its essence is to find the DNA or RNA of the pathogen present in the body. PCR allows you to determine the pathogen in cases where other types of research are ineffective.

Popularity this method is simplicity and breadth of use. It is used by clinicians to diagnose various diseases. Allows you to quickly conduct complex quantitative and genomic studies.

One of the most important medical applications of the classical PCR method is the detection of microorganisms that are capable of causing pathological conditions.

The essence of PCR analysis

PCR diagnostics (what it is is explained by molecular biology) is a molecular cloning method used to analyze a short sequence of DNA that is present in minimal quantities in samples containing any genetic material. This method is often called a "DNA copying machine."

PCR diagnostics are carried out in vitro (outside the body) in the laboratory and are based on the natural process of DNA doubling before it divides. In addition to the ability to detect a virus, bacteria or other microorganisms in a sample, PCR allows you to identify their quantity.

For diseases such as hepatitis C virus or human immunodeficiency virus (HIV), viral load is a good indicator of where the disease is and how effective therapy is.

Armed with this information, doctors can determine when to begin treatment and monitor the process. This allows us to create an individual approach for each patient.

PCR diagnostics includes three stages, in each of which the amount of target DNA increases significantly. As a result of this reaction, more than one billion copies of the original or “target” DNA are obtained, which makes it possible to assess the presence of the virus in the body. This is the main property of the PCR method.

Advantages and disadvantages of the method

PCR diagnostics (what it is is described above) was described in detail by Keri Mullis in 1983. Since then, the method has been extremely popular in laboratory practice.

The main advantages of this type of research are:

versatility;
speed of results;
high probability of obtaining reliable results;
specificity;
variety of diseases that can be identified.

The versatility of the PCR method lies in the possibility of using any human physiological materials. Even one hair is enough for analysis.

The data processing process, thanks to automation, can take several hours. Specificity consists in the isolation of a certain DNA fragment characteristic of a particular pathogen. Using PCR analysis, many diseases can be diagnosed, both in the acute stage and in the latent stage.

But, despite the huge number of advantages, the PCR diagnostic method also has disadvantages:

probability of false positive results;
high sensitivity;
variability of microorganisms.

The main disadvantage of the method is the inability of PCR to distinguish living microorganisms from dead ones. As a rule, this refers to a situation where the success of the treatment should be assessed. In this case, if the disease has been treated, then the dead cells remain in the human body for some time and for its complete renewal it takes some time from 4 to 6 weeks.

High sensitivity is an advantage and at the same time a disadvantage. Some microorganisms are present in small quantities in the body. Therefore, in this case, this diagnostic method is not informative, and it is recommended to use other types of research.

Any microorganisms can mutate. And even though the amplification process uses a specific fragment that is least susceptible to changes, it can still acquire mutations and become invisible to the test system. This situation explains the fact that different laboratory testing systems can show completely different results.

In what areas is the method used?

PCR testing can be used in clinical practice to identify various diseases. Widely used in gynecology, urology, gastroenterology, oncology, etc. In addition, it is effectively used in forensic science to establish identity and establish paternity.

This type of study is used at the planning stage and during pregnancy. Analysis for TORCH infections is carried out in order to detect the presence of infection in a woman’s body. Diseases caused by one of the infections can be mild for a woman, but cause serious congenital defects in the fetus, including its death.

Also, the PCR diagnostic method helps to determine a person’s genetic predisposition to developing certain diseases in the future.

What infections can be detected by PCR diagnostics?

The PCR method helps identify viruses, bacteria and fungi.

The most common diseases that can be diagnosed using it:

What biological materials are being studied?

Taking into account the disease, symptoms and goals of the study, PCR diagnostics can use any physiological fluid or tissue particle.

It could be:

blood;
saliva;
sputum;
urine;
excreta;
oropharyngeal swab;
a piece of affected tissue;
synovial fluid;
scraping from the vagina in women or from the urethra in men;
ejaculate;
hair and others.

For diseases of the genitourinary system, urine or a smear from the vagina in women or from the urethra in men is taken. Venous blood is donated for the diagnosis of diseases such as HIV, hepatitis various types, TORCH infections.

In order to exclude or confirm oncology, during surgical interventions pieces of tissue are removed for biopsy.

How to prepare for analysis

Features of preparation for research depend on the biomaterial that is submitted. You should also remember that it is necessary to avoid taking antibiotics and other medications. To monitor the effectiveness of treatment, an interval after taking the drugs should be maintained for at least 4 weeks.

Blood is donated in the morning on an empty stomach. Urine and feces must be placed in a designated, completely sterile container. At the same time, before collecting urine, it is necessary to carry out hygiene of the external genitalia.

If you are having a smear test, you must:

do not urinate for 3 hours;
It is not recommended to wash and douche the day before (for women);
stop sexual activity one day before;
Women should have a smear test mid-cycle.

Stages of PCR research

Before starting PCR, DNA must be extracted from the sample. DNA extraction is a multi-step process that can be performed manually or instrumentally.

Once the sample is prepared, the three-step PCR process begins:

separation of target DNA (denaturation);
amplification (reproduction) of DNA fragments;
detection (detection) of amplified DNA fragments.

The first stage of PCR is called denaturation. Its essence is that a test tube with DNA samples is heated to high temperatures(approximately 90 degrees). Due to high temperatures, weak bonds between nucleotides that form the code of molecules are destroyed. This results in the splitting of double-stranded DNA or RNA into two separate strands.

For the “purity” of the study, a special solution is added to the biomaterial under study, which dissolves organic substances. The duration of this stage depends on the type of material being tested and the infectious agent.

PCR diagnostics (what it is is described above) at the second stage reproduces DNA fragments based on the DNA of the infection itself. PCR does not copy all the nucleic acid in a sample. It only copies a special sequence genetic code, short fragment (primer).

In the second stage, the test tube is cooled. The entire PCR process is automated using a machine called a thermostat or cycler and can be completed within a few hours. The PCR machine is programmed to change the temperature of the reaction mixture every few minutes. This occurs so that short fragments can bind to the single-stranded template.

Amplification occurs in 30-40 cycles. During this period, the laboratory doctor copies the DNA fragment and increases its quantity using the chain reaction method. Thus, with each cycle, the number of copies increases to several millions or even billions.

In the third phase of the study, the presence of DNA of viruses and bacteria is detected. This is done by distributing the resulting mixture and adding a special solution to it. This liquid allows DNA fragments to acquire a certain color under the influence of ultraviolet rays. This is an indicator that pathogenic microbes are present in the human body.

PCR during pregnancy

PCR diagnostics (what it is is described above) can be prescribed for the first time during pregnancy, as it is a mandatory procedure. This method helps to identify hidden infections and the risk of developing defects in the fetus.

To carry out the analysis use:

There are infectious diseases that can negatively affect the development of the fetus, causing defects, causing premature birth and miscarriages. They are especially dangerous in the first trimester of pregnancy and, in most cases, are a medical indication for termination of pregnancy. These infections are collectively called the TORCH complex.

PCR analysis helps determine the presence or absence of infection in the body of a pregnant woman. Often performed at the planning stage of pregnancy or in the first trimester. If the result is positive, a repeat test is required. And only after confirmation of the results, appropriate treatment is prescribed.

PCR for HIV diagnosis

The PCR method for diagnosing HIV infection is an expensive test and is used according to special indications. These include donation, and it can also be prescribed to patients injured in a car accident with people carrying HIV.

PCR helps determine the presence of infection on early stages before the body produces antibodies to HIV. Result with high percentage The study can show the likelihood within 5-14 days after the virus enters.

In other cases, the presence of a virus in the human body helps to establish an enzyme-linked immunosorbent assay (ELISA). It detects antibodies to HIV in a person’s blood, which begin to be produced 1-3 months after infection. Quantitative determination and analysis of microorganisms is necessary to monitor the treatment of patients.

PCR for diagnosing hepatitis

Viral hepatitis C is a serious disease that can lead to the development of cirrhosis of the liver. During the incubation period, which can be up to 12 months, the virus does not manifest itself in any way, the patient is practically not contagious, and the virus cannot be detected by a routine blood test.

Most often, if not treated in a timely manner, the disease becomes chronic and threatens with multiple complications.

When infected with hepatitis B, the incubation period is not so long and the first symptoms can be observed after 4 weeks. It is during this period that ELISA can show the presence of the virus in the body.

The PCR method allows:

identify the disease at the stage of incubation development;
resolve questionable analyzes of other studies;
evaluate the effectiveness of antiviral therapy.

The absence of hepatitis DNA virus after treatment is a sign of its effectiveness.

Test for tuberculosis

The development of tuberculosis is provoked by Koch's bacillus, which can enter the human body through airborne droplets. The disease has two forms: hidden and open. In the first case, the infected person is not a carrier of the infection and mycobacteria in the sputum are not detected even using PCR. This form is still dangerous, since it is capable of entering the open stage.

The open (active) stage poses a danger to others even if the rules of personal hygiene are strictly observed. Fluorography, tomography and x-rays are used to diagnose the disease. chest. Also for this purpose, a tuberculated skin test (Mantoux test) is used. But most effective method research is PCR.

The following samples are used as test samples:

blood;
sputum;
bronchial fluid.

PCR for diagnosing HPV

Diagnosis for HPV is carried out in the presence of symptoms of the disease such as papillomas and warts in the genital area. Also, the study is prescribed at the stage of pregnancy planning, in case of infertility, miscarriages, as well as in the situation of infection of one of the partners, since one of the mechanisms of transmission of the virus is sexual.

The most dangerous thing is not the presence of the virus in the body, but its type. Some of its types (16 and 18) are highly oncogenic and can cause cervical cancer in women. The PCR method can be prescribed in conjunction with other types of research. For analysis, a smear from the genital organs is most often used. Blood, urine and vaginal discharge are used extremely rarely as biomaterials.

For herpes

This test diagnoses genital infection with herpes simplex virus types 1 and 2 (HSV1/HSV2). If the HSV2 result is positive, you should be tested for HIV and other STDs. The analysis requires the patient's venous blood.
Urine, secretions, and rash contents may also be examined if symptoms are present.

Comprehensive PCR examination

If indicated, or for the purpose of disease prevention, a set of PCR tests may be prescribed. Most often, the initiator is the patient himself; there are cases when a comprehensive examination is prescribed to prevent outbreaks of certain diseases in a particular region.

PCR-12 is a comprehensive study that covers the following infections:

Hepatitis.
Herpes.
STD.
Tuberculosis.
Encephalitis.
Candidiasis.
Helicobacter pylori infection.
Cytomegalovirus.
Mononucleosis.
Oncoviruses.
Listeriosis.

Explanation of PCR analysis

After analysis using the PCR method, a positive or negative result can be announced.

The results of PCR diagnostics look like this. The doctor will tell you what it is and how to decipher it

Accuracy of PCR diagnostics

In most cases, PCR diagnostics provide highly accurate results. But in some cases, additional research methods are required to confirm the correct diagnosis.

What could cause a false result?

False-positive results can result from non-compliance with the rules for collecting the analysis and conducting the research procedure itself.

The patient himself may be the culprit of false information in case of inadequate preparation for the delivery of biomaterial. The equipment of the laboratory and the professionalism of the doctor can directly influence the results of the study.

Where to get PCR diagnostics

PCR diagnostics are carried out in private clinics and laboratories on doctor’s orders or on the initiative of the patient himself for the purpose of prevention.
Preference should be given to clinics and laboratories with modern equipment that have been on the medical market for several years and have proven themselves.

PCR analysis: cost and timing

This diagnostic method is expensive. Its price ranges from 200 to 500 rubles for each type. A comprehensive examination for the presence of several infections can save significantly compared to paying for each individual examination.

The duration of the study depends on the type of infection and can take from 1 to 5 days. Also, these indicators depend on the equipment of the laboratory and the qualifications of the medical worker.

PCR diagnostics (what it is is described in detail in this article) is the most accurate and fastest research method. It is one of the most important discoveries in the 20th century. in medecine.

Thanks to this method, complete cure for many diseases became possible. But we should not forget that PCR also has some disadvantages, and its effectiveness is sometimes increased together with the use of other research methods.

Article format: Vladimir the Great

Video about PCR diagnostics

About PCR accuracy:

IN modern medicine PCR analysis of biological fluids of the body is highly accurate and informative. Using this analysis, the presence of viral and microbial particles in the body is detected, even if the disease is hidden and does not give any symptoms.

What does it mean to prescribe a PCR test for you? This abbreviation stands for polymerase chain reaction method - this is a special way of carrying out laboratory research. With this method, biological material obtained from the patient is placed in a special apparatus. A set of reagent enzymes is added to the test medium, which helps reproduce the genetic material of the pathogen (virus or microbe). The reaction produces a large number of copies of DNA or RNA, allowing identification by comparison with a database of both viral and microbial infections.

What does the PCR test result mean? If there is a causative agent of any infection in the human body, even a hidden one, this analysis will reveal not only its presence, but also in what quantity this infection is present in the body.

To test for infections using the PCR method, you can examine all biological fluids of the body - blood, urine, saliva, genital secretions, mucus of the throat and nose. The analysis requires very little material, since if pathogens are present, by reproducing their copies, the concentration of genetic information is brought to such a level that will identify the microbe or virus and determine its type. The accuracy of PCR analysis is very high; with the help of this analysis, today it is possible to determine almost all widespread viral, microbial and other infections.

But what does PCR analysis most often reveal? The list of infections that can be detected by this method includes infectious, including socially dangerous infections such as tuberculosis or hepatitis B and C. The same method can detect HIV infection, chlamydial and ureaplasma infections, mycoplasmosis of both the respiratory system and genital Using this analysis, thrush, bacterial vaginosis, and trichomoniasis are detected. The capabilities of this method make it possible to identify hidden infections - different types of herpes, HPV (papillomavirus), as well as identify a special microbe responsible for the development of stomach ulcers - Helicobatera.

Today, you can take a PCR test in almost any private or public laboratory. These types of tests are performed on everyone, children and adults. Depending on the purpose of the analysis, different biological material is analyzed depending on gender.

Thus, PCR analysis in men is carried out on blood or urine, urethral secretions, mucus from the throat or nose, prostate juice or even sperm. PCR analysis in women is possible in blood and urine, vaginal secretions, urethral discharge, nasal and pharyngeal mucus.

PCR analysis is very often used during pregnancy; with its help, hidden infections are detected in vaginal secretions or blood, requiring special monitoring during this period, and sometimes adequate treatment.

How is PCR taken?

An infection test using this method is prescribed by a doctor; often the material is collected immediately in the doctor’s office - a urologist or gynecologist. How is PCR analysis done?

The material being studied is mixed with reagents in the laboratory and the reaction is waited for. When pathogens are present, copies of their DNA or RNA multiply. The device compares the identified fragments of genetic material with databases and accurately identifies the infectious agent. If necessary, the amount of the pathogen in certain body fluids is also indicated. Typically, the study lasts no more than 1-2 days; if necessary, express tests are performed.

Where do you get PCR analysis from? This will depend on what types of pathogen are suspected. If it is HIV or hepatitis, blood is taken, if it is sexually transmitted infections, a smear is taken from the urethra or vagina. If mononucleosis or herpes is suspected, a throat swab is taken. Urine, cerebrospinal fluid, discharge from wounds, sputum, etc. can also be used.

PCR blood test

For accurate results and diagnosis of infections, it is important to donate blood morning hours on an empty stomach. A blood test using PCR can identify pathogens dangerous diseases- HIV, hepatitis, syphilis. During periods of exacerbation and activity of the virus, herpes viruses, papillomas and some others can be detected in the blood.

PCR analysis: preparation

For the absolute accuracy of the analysis, proper preparation for it is important. The easiest way to prepare for a blood test is that there are no restrictions on diet or activity; blood must be taken in the morning, on an empty stomach. A urine test is taken after washing the genitals, from the middle portion into a special sterile container. PCR analysis from the genital tract deserves special attention, so it's important to know how to prepare. One day before the examination, sex is prohibited; it is recommended not to urinate before the examination. During menstruation in women, the date of analysis is postponed by 3-5 days from the moment of the last discharge.

How long does it take to perform a PCR test?

Depending on the laboratory’s capabilities, the analysis can be prepared from several hours to several days. On average, how many days does a PCR test take? Usually this is one or two days. At emergency situation the analysis can be performed on the same day, in a few hours.