POLYMERASE CHAIN REACTION (PCR): METHOD DESCRIPTION

Polymerase chain reaction (PCR) is the creation of multiple copies of a short section of human DNA. A small amount of physiological material is necessary for research and obtaining results: sputum, blood, amniotic fluid, saliva, urine, etc.

What is a polymerase chain reaction?

The PCR method was developed by the American scientist Melison, who in 1998 received the Nobel Prize for his development. Actively used:

with the early diagnosis of infections, oncological pathologies;
in a forensic examination for the study of an extremely small amount of DNA;
in veterinary medicine, biology, molecular genetics;
for personal identification by DNA;
to confirm paternity;
in ecology, for example, when trackingInd the quality of the product, in anthropology.

Who is prescribed PCR?

Polymerase chain reaction in the diagnosis of infectious pathologies is one of the most reliable methods that gives the most accurate and reliable analysis. For example, the reliability of the analysis on chlamydia is close to 100%. Most often, this diagnostic measure is prescribed to patients who, during the test, have difficulty identifying a particular pathogen.

PCR laboratory tests use:

To search for pathogenic organisms that cause infection of the urinary and genital tract, it is difficult to identify when applying crops or method of immunology.
For re-analysis of HIV at the initial stage with a positive, but questionable result of the initial analysis, for example, in newborns from infected parents.
To establish oncological pathology at an early stage and for individual correction of the treatment regimen in patients.
For early detection and potential treatment of hereditary pathologies. So, many future parents are testing to find out if they are carriers of genetic diseases. In children, this method determines the probability of exposure to diseases that are inherited.

Also, this technique is used:

for the detection of fetal pathology in early pregnancy;
in patients before surgery for organ transplantation - necessary to determine the compatibility of tissues
to detect dangerous pathogens in the blood of the donor;
in newborns to detect hidden infections;
to evaluate the results of antiviral or antimicrobial treatment.

Why go through this procedure?

Since PCR is considered to be a highly effective diagnostic method that gives almost 100% results, this procedure is used:

To confirm or exclude the diagnosis.
For a quick assessment of the effectiveness of the treatment.
In many cases, this method is considered the only possible way to detect developing pathology, if other bacteriological, virological techniques have proved to be useless.
Viruses using the PCR procedure are detected immediately after infection and before the onset of the main symptoms.
Early detection of microorganisms will allow prompt treatment.

In addition, PCR analysis will allow you to quickly detect a small number of microorganisms in living or dead samples. Therefore, this technique is used to search for a specific pathogenic organism, for example, a tubercle bacillus.

In addition, PCR analysis is used:

to determine the sensitivity of DNA to specific types of antibiotics, which will allow as quickly as possible to begin treatment;
to control the spread of epidemics;
to identify and track new types of microbes, pathogens.

The main types of PCR

Standard method

PCR analysis is carried out on the basis of multiple doubling of a specific DNA fragment and RNA using the primer enzymes. Thanks to this, the researcher receives the required amount of material.

Real-time PCR

In this type of research, the process of identifying a given fragment is started after passing through each cycle. This type of research allows to obtain information on the number of viruses and microorganisms in the body, that is, to carry out a quantitative analysis.

Reverse Transcription Mode from PCR

This analysis is used to search for single-stranded RNA for the further detection of viruses whose genetic base is named RNA. For example, this is the hepatitis C virus, immunodeficiency, and so on. In conducting this study, a special enzyme is used, thanks to which single-stranded DNA is built. The system then restores the second DNA strand and performs the traditional procedure.

Indications for

The PCR procedure is used in the clinics of infectious diseases, obstetrics, urology, gynecology and nephrology, pediatrics, and so on. The most common indications for assignment analysis are as follows:

to determine the risk of developing genetic abnormalities in a child in the presence of hereditary pathologies;
to diagnose both parents when planning a pregnancy or when the mother is in a difficult condition while carrying a child;
with the difficulties of conception to identify the causes of infertility;
in case of suspected sexual infections in the acute stage or at risk of their transition to the chronic stage;
to detect the causes of inflammatory processes of unknown origin;
with unprotected casual sex;
to determine the sensitivity of a pathogenic microorganism to a specific drug;
suspected latent infection to detect pathogens without obvious symptoms;
to confirm recovery from an illness, that is, a retrospective diagnosis.

Also, PCR is used when it is necessary to accurately identify such pathogens as: hepatitis viruses, human immunodeficiency, cytomegalovirus, herpes simplex virus, rubella virus, papilloma virus.

There, this method is used to determine infections: infectious mononucleosis, tick-borne encephalitis, candidiasis, which is caused by candida fungi, genital infections, and tuberculosis.

If contraindications and safe method?

Since this procedure is carried out without any impact on the body, and only biological material is used, which is taken for research, there are no contraindications due to the absence of potential danger to the body. But for example, the biomaterial sampling of the cervical canal of the uterus is not carried out after a colposcopy. Delivery smear, scrapings are allowed only 4-7 days after the end of menstruation and the complete cessation of discharge.

Features of preparation for the procedure

As a sample for PCR analysis, in which the DNA of a foreign pathogen is detected, either a biological fluid or tissue is used. The intake of the test substance takes place in the form of blood sampling from a vein, a scraping from the nasal cavity, larynx, cervix, urethra. Before the diagnostic procedure, the doctor must explain to the patient what material will be taken:

in the analysis of herpes infections, mononucleosis take an analysis of urine, as well as a smear from the pharynx;
when testing for hepatitis or toxoplasmosis, blood is taken from a vein;
in order to diagnose various types of CNS damage, the cerebrospinal fluid is taken;
in pulmonology, samples for PCR analysis are pleural fluid and sputum;
if it carries out a study of intrauterine infections during pregnancy, amniotic fluid, placental cells are used for analysis.

The reliability and accuracy of the result depends on the sterility and conditions of taking the biomaterial. Since PCR studies are highly sensitive, any contamination of the substance taken may distort the results. Competent preparation for the delivery of the biomaterial does not pose any difficulties for the patient.

But there are a number of some recommendations:

when analyzing sexually transmitted infections, it is necessary to exclude intimate contacts three days before the delivery of the material;
72 hours should stop the use of any vaginal preparations;
from the evening of the previous day, no hygiene of the studied areas can be performed
3-4 hours before taking a sample from the urethra, urination should be excluded;
a month before taking the tests you need to stop taking antibiotics;
blood is given in the morning on an empty stomach before eating and drinking;
The morning urine is collected in a sterile container after going to the toilet.

How is PCR?

When performing research in a special reactor, a certain cycle repeats:

First step. Denaturation. Saliva, blood, gynecological tests and other substances are placed in the applicator, where the material is heated and the DNA is split into two chains.
The second step. Slight cooling of the taken substance, adding to it special components that recognize the necessary parts of the DTC molecule and bind to it.
3 step. Elongation. During the process, a fragment of DTC of the pathogenic organism is completed, a copy of it is formed. This cycle is repeated, which doubles the copy of the specific DNA fragment.

In a couple of hours, many copies of this fragment are formed, their presence in the samples is revealed. Then the results are compared with the data bases of various types of pathogens. This allows you to determine the type of infection.

Features of decoding analysis?

The final result of the study will be given to the patient several days after the biological analysis.

Qualitative analysis

A negative result in this case means that no infectious agent was detected in the fluid that was submitted for testing. A positive result indicates the detection of pathogenic viruses or bacteria in a biological sample. If the result was positive, but at the same time the person has no signs of activation of the infection, such a condition of the body is called asymptomatic healthy carrier.

Most often, this is observed when taking the biomaterial in viral pathologies. In this case, treatment is not required, but it requires constant medical supervision. Since under such conditions there is a probability:

the spread of the virus from the carrier to healthy people;
activation of the process and the transition of the pathology to the chronic form.

Quantitative analysis

The quantitative result is determined by a specialist specifically for a particular type of infection. Based on the results obtained, it is possible to assess the degree of development, the stage of pathology, which makes it possible to prescribe treatment as quickly as possible.