The polymerase chain reaction (PCR) method, invented by a scientist named Kary Mullis in 1984, is considered one of the revolutionary developments in the scientific world. Thanks to PCR and similar developments, a lot of information has been obtained about gene diversity and the interaction between genes. Microbiology laboratories are one of the areas where the PCR technique is used the most. In these laboratories, thanks to the PCR method, many microbes that cause various diseases in humans can be detected.
PCR (Polymerase Chain Reaction – Polymerase Chain Reaction) test is a test that is frequently brought up today due to the coronavirus outbreak. This test, which is used for coronavirus diagnosis, has many uses, especially in the field of health. In this article, the titles such as exactly what the PCR test is, for what purposes it is used, the way the PCR test is applied and what should be considered when it is applied, how it is used in the coronavirus have been examined in detail.
What is the PCR test?
PCR (Polymerase Chain Reaction – Polymerase Chain Reaction) is a general name given to reactions applied to enzymatically increase a specific region in DNA. PCR method can be briefly expressed as replicating or repeating DNA replication a certain number of times.
This method can also be described as “in vitro” ie in vitro nucleic acid sequence cloning. PCR test is a molecular recognition technique. This test, which has the ability to distinguish even a small number of microbes, is an advanced and highly sensitive technique among molecular diagnostic tests.
How is the PCR test done?
The PCR (Polymerase Chain Reaction) technique is based on the separation of the two strands of DNA by exposing them to high temperatures, in other words, to become a single strand, then the synthetic oligonucleotides bind to the target DNA to ensure the elongation of the chain and the repetition of this cycle for a certain number of times. Thanks to this feature, it becomes possible to detect even a small number of microbes by reproducing them with the PCR test.
What are the usage areas of the PCR test?
Finding nucleic acid replication techniques for diagnostic purposes is considered an important development in medical science. With the advancement in this technology, the PCR test, which is on the way to become a standard in clinical use, can also provide protection against important drug side effects that may occur during treatment by detecting the patient’s genes associated with this drug in personalized drug treatment planning.
Disrupting the normal life cycle of the cell and acquiring a malignant character with a tendency to divide continuously may result in cancer. Differences in genes related to cell cycle, DNA repair, and growth signal in continuously dividing cancer cells can be detected by the PCR method. PCR test, which is currently a limited area of application in the diagnostic approach for all cancer types, may contribute to the diagnosis of some types of cancer such as bladder cancer, breast cancer, colorectal cancer, and endometrial cancer.
The PCR method is also an effective test for the detection of viruses in samples belonging to the human body. In diseases caused by bacteria, it may contribute to the diagnosis of these diseases in the early stages, especially in diseases that are thought to be caused by bacteria that are late in culture.
The PCR test, which can provide diagnostic benefits in animal diseases as well as in infectious diseases in humans, is also used in veterinary diagnosis and treatment planning.
The PCR test is used in many types of research, studies, and determination of unknown features, the basis of which is diagnosis. Many uses of PCR tests in the field of health can be summarized as follows.
- Carrier and patient diagnosis in hereditary diseases
By increasing the gene sequences in the PCR test, defects in these genes can be detected. Being able to diagnose early even in carriers is a great advantage of this test.
- Prenatal diagnosis
Prenatal abnormalities in the fetus can be detected this way. These abnormalities are usually caused by genetic disease.
- Detection of pathogenic organisms in clinical samples
It has a great contribution in distinguishing pathogenic organisms by increasing even a small number of microbes. It is a fast and sensitive technique in disease diagnosis. In coronavirus detection, the pathogenic organism can be recognized and diagnosed in this way.
- Forensic Medicine
In this method, DNA analysis of criminals can be performed by replicating the gene sequences performed in the PCR test. Therefore, it has great importance in forensic medicine.
- Cancer-related research
Mutations in tumor-forming genes can be detected by PCR testing.
- Gene expression studies in the creation of probes/cloning
- Generation of a large number of DNA samples in DNA sequence analysis
- Determination of unknown gene sequences
- Studying past DNA and elucidating biological evolution
- Development of vaccines in the field of biotechnology
- Ensuring normal birth of the baby by performing pre-implantation genetic tests and then performing implantation in a single cell within Vitro fertilization
- Development of techniques used in the diagnosis of genetic inbreeding in many plants and animals such as PCR-derived RAPD, AFLP, SSR, and ISRR.
- Investigation of DNA protein interaction (footprinting)
PCR test is an examination that has many uses in sectors such as food and agriculture, as well as its uses in the health field mentioned above.
Food safety is one of the factors that significantly affect health, and serious illness can occur as a result of consuming contaminated or virus-infected foods. The PCR method can also be used to detect these factors that may be food health-related disease factors.
In which diseases can the PCR test be used?
PCR test is an examination that has an important use in the diagnosis of microbial diseases. PCR testing can be useful in the diagnosis of disorders caused by various bacterial, viral, parasitic, or fungal (fungal) conditions:
Use of PCR Test in Diagnosis of Bacterial Diseases
In addition to the detection of bacterial pathogens, PCR tests may also be useful in detecting resistance genes against antibiotics used in these living things. For example, in throat infections caused by streptococcus bacteria, by taking a swab sample from the throat or after antibiotic use, clostridium difficile, which can cause diarrhea, and e.coli bacteria, which can cause intestinal infections, can be obtained in a shorter time than the culture.
Apart from these bacteria, PCR tests can also be used in the diagnosis of lung infections caused by bacteria such as mycoplasma, chlamydia, and legionella, which are agents of atypical (different from normal) pneumonia. Culture results of bacteria causing disease in infections caused by atypical pneumonia factors are quite time-consuming. These bacteria can be detected in a shorter time by performing a PCR test on throat swab samples taken from these patients.
Use of PCR Test in Diagnosis of Viral Diseases
One of the first uses of PCR testing is to diagnose viral diseases. Herpes simplex virus (HSV), which causes the development of lesions known as cold sores, causes clinical symptoms involving the genital area, skin, and central nervous system. If this disease affects the brain structures and the patient does not receive appropriate treatment, the probability of the disease being fatal can increase up to 70%. Thanks to the PCR test of some genes in the DNA of this virus, the presence of this virus can be detected in the person 1 day after the onset of clinical symptoms.
With the PCR test other than this virus, varicella-zoster virus, which is the agent of chickenpox, Epstein-Barr virus, which can trigger the development of lymphoma, JC and BK viruses that can cause significant damage to the kidney and brain, parvovirus b19 and influenza, which causes the disease known as 5th disease in children, Respiratory discomfort factors such as adenovirus and coronavirus can also be detected.
What is the role of PCR testing in the diagnosis of Covid-19?
The PCR test is a very important test for the control and prevention of the epidemic in COVID-19 disease caused by the new type of coronavirus (SARS-CoV-2), which has affected the whole world in a short time. Real-time RT-PCR test is very helpful, especially in the diagnosis of early disease. There is a risk of false-positive or false-negative results based on the suspicion about this test.
No problems occur in the results of the majority of PCR tests performed for the diagnosis of Covid-19. Since the viral load that can be detected in the patient is not yet detected in the tests performed very early, this examination may result in a false negative. For this reason, samples must be taken correctly and at the right time for the PCR test to give correct results.
Coronavirus diagnostic tests are of two types as molecular and serological tests. The tests used in the diagnosis of Covid-19 can be done with the help of the genetic material of the virus (can be DNA or RNA) or the detection of antibodies created by defense cells against the virus by activating the immune system of the antigenic material. The tests used to detect the genetic material of the virus are called molecular tests, while the tests that try to detect the antigen-antibody of the virus are called serological tests.
Antibody-antigen tests, that is, serological tests, are tests that are used to detect the stages of the viral infection that has been or is currently undergoing. A real-time PCR test can be used to confirm serological tests in Coronavirus. The tests to be used may vary depending on the stage of the disease.
PCR test, which is an important part of the prevention and control studies regarding the coronavirus epidemic, can provide an early diagnosis of this disease thanks to its convenience and specificity. Detecting false negativity or false positivity is one of the main problems in the use of the PCR test in the diagnosis of Covid-19. Therefore, the compatibility of the symptoms and findings of the patient with the PCR test can be considered in the clinical diagnosis of COVID-19.
The main thing that the PCR test uses in the diagnosis of coronavirus is to detect the RNA polymerase enzyme gene, that is, its genetic material, which can be found depending on RNA in the virus. This test should be applied by a knowledgeable microbiologist and laboratory. Otherwise, it can lead to very wrong results. As a result of the wrong results, it may be that the disease cannot be diagnosed. This may allow the virus to spread further.
If the results are negative, the possibility of the test being performed incorrectly should be considered. The time that the sample was taken and whether the sample was taken correctly is also very important for the test to give an accurate result. The performance of the PCR test depends on the abundance of RNA in the sample, as it makes the diagnosis from RNA detection. Therefore, the quality of the sample is also very important for the efficiency of this test. In addition, improper transfer of the sample taken may cause inaccurate results.
Technical errors that are not caused by the person performing the test, such as virus mutation or PCR test inhibition, are among other errors that may occur during the application of the test.
There are two types of applications for PCR at this time. These types are PCR tests that give real-time results and give fast results. While it takes 3-4 hours to get results in real-time PCR tests, this time can be reduced to 1.5 hours in PCR tests that give rapid results. Real-time PCR test of these types is used in Turkey.
In the PCR test applied for the diagnosis of coronavirus, it is necessary to take a sample from an organ belonging to the respiratory tract, such as a nasopharyngeal swab (taking a swab from the nose and throat) or sputum sample from the lower respiratory tract. Different methods can be used to take this sample. Taking samples with throat swabs among these methods is a method that can give correct results for the first week.
In the continuation of the disease, that is, when the patient becomes pneumonia, the virus spreads rapidly in the lungs, while the number in the throat decreases and disappears, and therefore the sample taken from the throat may not be meaningful for diagnosis. Therefore, since the disease progresses after the first week of the disease, a diagnosis can be made through the sputum, which is the cough material.
After the samples are taken at the right time and in the right way, the samples are placed in a liquid viral transport medium that can allow the virus RNA to pass into the solution. The RNAs passing from the sample to the solution are then separated from this solution, and this RNA is then amplified to facilitate identification. From the RNA that is replicated, the type of virus that RNA belongs to is determined by several tests in the laboratory and thus the diagnosis is made.
PCR; Denaturation performed in the range of 94 ° C-98 ° C, separation of the two strands of DNA exposed to high temperature, annealing between 37 ° C-65 ° C, namely the binding of synthetic oligonucleotides to the target DNA (hybridization) and It consists of 3 stages, namely elongation stages, and these stages are performed by repeating a certain number of stages in a certain cycle.
In addition to the coronavirus tests that give direct results, the disadvantage of the PCR test is that it gives results at a later time, but the PCR method detects even a small number of pathogens, which indicates that this method is more sensitive than other methods. The fact that this test is more sensitive can be counted among the advantages of this test compared to other tests. The PCR technique is also a very advantageous method because it reduces the exposure to radioactivity resulting from some imaging tests.
Radioactive diagnostic methods are generally known for their damages to the body. Because of this feature, the PCR technique has become more prominent and more usable among diagnostic methods.
What are the symptoms that indicate that medical intervention is required in Covid-19 disease?
Covid-19 disease, which progresses with mild to moderate respiratory tract infection symptoms in some people, may cause shortness of breath that gradually worsens in some people. In another patient group, a decrease occurs in the oxygen values of the people, although they can breathe normally. Care should be taken, as this condition called silent hypoxia, and symptoms such as shortness of breath can progress to a health problem known as acute respiratory distress syndrome (ARDS) that requires immediate attention.
In addition to sudden onset of shortness of breath, other symptoms such as unexpected dizziness, rapid heartbeat, and increased sweating may accompany those who develop ARDS. There are various signs and symptoms in Covid-19 patients that indicate that the condition may progress to ARDS:
- Difficulty breathing or shortness of breath
- Compressive pain or discomfort felt in the chest or upper body that does not tend to regress
- Sudden confusion
- Blue-purple discoloration, especially in the nail bed, gums, eye area, and lips
- Fever that does not decrease despite various interventions
- Cold hands and feet
- Weakening of the pulse
Coronavirus diagnostic tests, which are applied in a professional laboratory at the right time and with the right methods, are an important part of the process of controlling the epidemic. Therefore, patients who think they have similar symptoms are recommended to apply to the nearest health center so that they can access appropriate treatment.
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