PCR test

Methodology, advantages and disadvantages of the analysis

Polymerase chain reaction – what it is, what diseases it detects, what are the advantages and disadvantages of the analysis and what its results mean – in the RIA Novosti material.

PCR analysis

Polymerase chain reaction (PCR) is a method of molecular genetic diagnostics that allows detecting various infectious diseases in the human body. It is based on a multiple increase in small concentrations of RNA and DNA fragments. The polymerase chain reaction was invented in 1983 by American biochemist Cary Mullis. He wanted to ensure that it was possible to create multiple copies of DNA due to the enzyme DNA polymerase, which is involved in its replication. For this, Mullis received the Nobel Prize in Chemistry. It is known that in the early 1970s a similar method was presented to other scientists, but the idea was never realized. Later, the analysis was significantly improved and is widely used in biological, biochemical and medical practice.

How PCR works

Sophisticated, modern equipment is needed to carry out the analysis.

"A special device is used, it is called an amplifier, in which a virus particle or something else containing RNA or DNA is placed," a candidate of medical sciences, a clinical pharmacologist, told RIA Novosti Andrey Kondrakhin. – The fact is that RNA and DNA are small enough to identify them and understand whether there are harmful microorganisms in the human body, so they do a PCR test. A particle of the virus is put into a large machine, but it does not recognize it, then special components are added, primers that allow you to make a large number of these RNA chains. There are so many of them that the machine begins to "see" them. Even if there is at least the smallest RNA of a virus, for example, a coronavirus, this allows you to create many copies of the infection, which helps to determine whether it is present in the body or not. The method gives the result of almost one hundred percent accuracy. There is a separate primer for determining COVID-19. There is a large number of nucleotides from which the future RNA is built. It is first destroyed, and then rebuilt. The reaction is called a chain reaction because it happens very quickly. As soon as there are a lot of copies, the reading begins immediately."

Application of PCR

The PCR test detects the presence of pathogens of viral diseases when it cannot be done by other methods. For example, immunological, bacteriological and microscopic studies. The time factor also plays a significant role, because the analysis is done quite quickly compared to others.

"The method is used not only for viruses, but also for DNA particles, as well as bacteria," the specialist explained. "For example, we can determine which type of bacterium belongs to, for example, gram–negative or gram-positive."

Polymerase chain reaction is used in criminology when you need to do a DNA analysis from a crime scene, it is then compared with the genetic material of the suspect. It is also used to establish paternity or motherhood, to find viruses and bacteria in the human body, to detect allergies to certain drugs or their toxicity.

Conducting PCR

The analysis requires DNA or RNA matrices, sections of which need to be copied, two primers with the necessary enzymes, a thermally stable DNA polymerase, as well as some other components. The PCR method detects any DNA or RNA of a living or dead pathogen. To determine them, special primers are used, which contain degenerate positions of RNA or DNA.

Preparation of the material

This stage is extremely important for PCR analysis, because most errors, up to 70% of cases, are made during sample preparation, which affects the final result of the analysis. To avoid this, you need to properly pick up the material for testing and prepare it. Therefore, many laboratories use vacuum systems when taking blood, which injure a person less and prevent the material for analysis from coming into contact with the environment or personnel.

What biological materials are being investigated

Various biomaterials are usually used in the study:

– Urine. Such an analysis makes it possible to identify infections of the genitourinary tract in men and urinary organs in women.

– Sputum. It is used to detect tuberculosis and some other lung diseases.

– Biological fluids – amniotic, spinal, articular, etc.

– Scrapings from mucous membranes, for example, the nose or oropharynx. It is mainly used to detect sexually transmitted diseases, as well as coronavirus.

– Blood, serum, plasma. They are used to determine CMV (cytomegalovirus) viruses, HIV, hepatitis B, C, D, G, herpes, as well as human gene studies.

– Biopsies. They are taken by biopsy.

Advantages and disadvantages of the method

PCR differs from other studies in that it can detect several different pathogens at the same time. For him, you need to pass only one analysis, no additional tests are required.

"The advantage of the method is that it is possible to determine the pathogen in a small amount, just in a meager amount," explained the clinical pharmacologist. – Thanks to this, it is possible to make a diagnosis faster, because we know exactly which microorganism is beginning to develop. In the test, the concentration of microorganisms detected in DNA and RNA is written, if a smear is taken from the nose or mouth, as well as to which class they belong. That is, there is a clear understanding of what type the pathogen belongs to and what its quantity is. A little, a lot, or it is not in the body at all."

According to Andrey Kondrakhin, one of the disadvantages of the test is the difficulty in taking the material:

"The very first drawback of the method is that absolute cleanliness in the room is necessary, closer to sterility, because even the smallest foreign objects can get the wrong result due to the fact that the sensitivity of the device is very high. An increase in the concentration of these particles will immediately begin, which the machine can read. Secondly, in order for the reaction to go, special materials, primers are needed. Each reaction has its own primers – pieces from which new RNAs will be created. The test is not fast – it can take about a day in time. In addition, we need trained, highly professional staff. The devices themselves are quite bulky, although now there are smaller installations. But the bigger the machine, the more it can read. This is not a system for mobile use."

Contamination of the test material can also occur if mistakes were made during its transportation, due to the unsuitability of reagents, blood, epithelial cells, and a large amount of mucus getting into them.

Types of PCR

There are several types of PCR test:

– PCR with reverse transcription. It is used for RNA amplification.

– Inverted PCR is used if there is a small section of a DNA or RNA sequence for analysis.

– Nested PCR is needed to reduce adverse reactions, for this purpose two primers are placed in the amplifier.

– Quantitative PCR is used to monitor the change in the chain reaction.

– Step-by-step PCR reduces the binding of primers.

In addition, there is asymmetric PCR and group-specific PCR.

"There is a real–time PCR technique," said Andrey Kondrakhin. – This is the fastest analysis, as they say, here and now. It is made using a device that has a cartridge that detects only one infection, for example, a coronavirus. Apart from it, the device does not see anything, as with conventional PCR, but in this case the result can be obtained much faster."

What infections can be detected

Polymerase chain reaction makes it possible to detect infectious diseases in humans, for example:

– hepatitis;

– ureaplasmosis of the genitals;

– candidiasis (thrush);

– chlamydia;

– papillomavirus infection;

– herpes;

– the presence of cancer cells;

– mycoplasmosis;

– bacterial vaginosis;

– infectious mononucleosis;

– trichomoniasis;

– AIDS (HIV).

Testing also helps to identify a person's hereditary predisposition to certain diseases and assess the hormonal background.

PCR test for COVID-19

For a PCR test for coronavirus, a smear from the nose and oropharynx is taken from a person. During a pandemic, it is recommended to hand it over to everyone who is ill with SARS, influenza, pneumonia, etc. They take the material for analysis on an outpatient basis, and a specialist can also go home. According to the requirements, the patient is discharged after two negative tests for the presence of infection. PCR tests can give a false positive or false negative result, in which case a repeat study is necessary.

How to prepare for the analysis

"Special training is not required at all, except not to eat for three hours (at least) before taking the material, if they take a swab from the oral cavity. Food particles can distort the result. It is enough to rinse your mouth with water and go for analysis," the specialist explained.

Before the smear, it is also not recommended to drink coffee, tea, other beverages, brush your teeth, rinse your mouth with refreshing mixtures, chew gum, smoke. The nose should not be washed, drops should not be instilled there or smeared with medicines. If blood is taken, then it must be taken on an empty stomach so that the result is as accurate as possible. Urine should be collected in the morning on an empty stomach or 2-3 hours after eating. It is advisable to do this immediately after sleep.

Decoding of PCR analysis

After the test, the results are evaluated by both the equipment and medical specialists.

"At the end of the test, the machine reports what it found in the smear, and the laboratory technician describes the result," the expert explained. – If the program of the machine contains information about the quantities at which the test is considered positive, then the machine will do it. The analysis will be either yes or no. The person himself does not need to decipher anything, the only thing that a specialist can look at the machine is the number of pieces of RNA, because there are false positive or false negative results if the device has failed. All the same, there is control from the doctor."

How accurate is PCR diagnosis of infections

"PCR is absolutely accurate, almost 99.9%, because the device will build exactly the same RNA as a key cast, one in one. Nothing new can be done there. The set of particles increases to the amount that the machine can recognize. This can be done even if there are very few particles," Andrei Kondrakhin noted.

At the same time, due to the fact that errors often occur when taking biological material and there is a possibility of foreign particles entering, false positive or false negative test results for coronavirus may occur.

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