- PCR (test) - what is the PCR technique?
- PCR (test) - application of the PCR method
- PCR (test) and Lyme disease
- PCR - results. Can they be false positives?
- PCR - results. Can they be false-negative
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PCR is a test that has been used in the diagnosis of many diseases. The PCR method allows to show the direct presence of bacteria in the organism, and specifically its DNA. PCR is used, inter alia, in in the diagnosis of Lyme disease. What is the PCR technique? What is real time PCR?
Contents:
- PCR (test) - what is the PCR technique?
- PCR (test) - application of the PCR method
- PCR (test) - Lyme disease
- PCR (test) - results. Can they be false positives?
- PCR (test) - results. Can they be false-negative
PCR(polymerase chain reaction) is the primary method used in genetic and molecular laboratories. It was developed in 1983 by Kara Mullis, who was awarded the Nobel Prize in 1993 for it.
PCR (test) - what is the PCR technique?
PCR testconsists in multiple amplification (usually in 30-40 cycles) of a specific fragment of DNA, e.g. genes encoding Borrelia proteins, with the use of the polymerase enzyme.
Then, in order to visualize the amplified DNA fragment, special molecular dyes are used. PCR, thanks to the amplification of specific DNA fragments, is an ultra-sensitive method.
In the PCR method, DNA fragments are amplified using specific primer pairs for a given region of the bacterial DNA. For example, PCR target regions used in the diagnosis of Lyme disease are genes encoding proteins, e.g. 16S rDNA, recA, fla, p66, OspA.
The biological material from which the test can be performed is:
- blood
- skin section from migrating erythema
- cerebrospinal fluid
- articular or cartilage fluid
- urine
PCR (test) - application of the PCR method
In addition to the diagnosis of Lyme borreliosis, the PCR method can detect specific DNA fragments of other microorganisms or humans. Therefore, the PCR method is also used in:
- diagnosis and monitoring of viral infections: herpes viruses (herpes virus, herpes zoster, Epstein-Barr, cytomegalovirus), HIV, hepatitis viruses (HBV and HCV)
- diagnosis and monitoring of bacterial infections, especially those bacteria,which cannot be identified through cultures, e.g. tuberculosis
- diagnosis and monitoring of neoplastic diseases: leukemia, breast cancer, colorectal cancer, lung cancer
- diagnosis of cancer predisposition: breast and ovarian cancer (BRCA1 and BRCA2 genes), colorectal cancer (APC and CHEK2 genes)
- diagnosis of genetic diseases: hemochromatosis (HFE gene), thalassemia (HBA1 and HBA2 genes), celiac disease (HLA genes)
- paternity tests, which detect the DNA compatibility of the child and father
Currently, there are many modifications to the PCR method. For example, in the diagnosis of Lyme disease, the methods of nested PCR (nested PCR) and real-time PCR ( ) are mainly used. The latter is a quantitative method and allows to determine the exact number of Borrelia DNA copies in the tested biological material.
PCR (test) and Lyme disease
The basis for the diagnosis of Lyme disease is serological testing. However, there are clinical situations that may cause these tests to be false-negative:
- serological window, i.e. the initial period of infection in which the body does not yet produce specific IgM and IgG antibodies against Borrelia
- in immunocompromised people who do not produce enough antibodies
- presence of immune complexes that consist of Borrelia antigens and specific antibodies for them and prevent the detection of antibodies by serological methods
If the patient is suspected of having Lyme disease symptoms, and the serological test results are negative, a molecular test based on the PCR method can be performed.
DNA of Borrelia spirochetes can be detected in the early stage of infection, as the number of spirochetes in body fluids is then the highest.
In the diagnosis of Lyme borreliosis, serological methods such as ELISA are primarily used, but in some clinical situations genetic testing using the PCR method is helpful.
The longer the time from infection, the lower the probability of detecting bacterial DNA. Therefore, in some cases, before the PCR test is performed, bacteria are grown on special media for 1-2 weeks and then the test is performed.
This approach is used in the case of suspicion of Lyme Lyme disease and harvested cerebrospinal fluid. In the late phase of infection, it is more appropriate to perform an ELISA test.
The PCR method should not be used in the routine diagnosis of Lyme disease due to the lack of standardizationconcerning the occurrence of Borrelia burgdorferi varieties in Poland.
However, in the case of difficulties in diagnosis (especially in the early stage of the disease), the result of the PCR test is helpful in the diagnostic process.
A positive test result means that the presence of Borrelia burgdorferi DNA and its variants was detected in the tested material, but it does not explain whether the organism is actively infected. On the other hand, a negative result does not exclude the presence of bacteria in the body.
Some laboratories offer testing for the presence of Borrelia DNA in the tick. However, the result of such a test is not the basis for the diagnosis of Lyme disease in a bitten person.
PCR - results. Can they be false positives?
PCR is an ultra-sensitive method and at the same time very sensitive to contamination. Therefore, it is very important in PCR-based testing to select an appropriate internal control to rule out false-positive results.
Correct collection of material for tests as well as the experience and reliability of the diagnostician performing the test are also of key importance.
PCR - results. Can they be false-negative
As in any laboratory test, also in the PCR method, false negative results may appear. Here it is crucial to collect blood for an appropriate anticoagulant, as some of them, such as heparin, can inhibit the PCR reaction.
Collecting material after starting antibiotic therapy may decrease the efficiency of the PCR reaction and increase the likelihood of false-negative results.
It should also be remembered that Borrelia bacteria live for a very short time in the blood and body fluids, because thanks to special receptors they penetrate very quickly into "hard-to-reach" places in the body.
Therefore, in contrast to the diagnostics of other microorganisms, the number of Borrelia spirochetes in biological material is small, sometimes even at the limit of detection of the PCR method. This can also cause a false negative.
References
- Dunaj J. et al. The importance of the PCR method in the diagnosis of Lyme borreliosis. Browse Epidemiol. 2013, 67, 119 - 123.
- Laboratory diagnostics of tick-borne diseases Recommendations of the Working Group: National Chamber of Laboratory Diagnosticians, National Institute of Public He alth-National Institute of Hygiene, National Consultant in the field of infectious diseases, Department of Infectious Diseases and Neuroinfections, Medical University of Białystok, Polish Society of Virology, Warsaw 2014 .
- Ružić-Sabljić E. and Cerar T. Progress in the molecular diagnosis of Lyme disease. Expert Rev Mol Diagn. 2022Jan; 17 (1): 19-30.
- Valones M.A.A. et al. Principles and applications of polymerase chain reaction in medical diagnostic fields: a review. Braz J Microbiol. 2009 Jan-Mar; 40 (1): 1-11.
Karolina Karabin, MD, PhD, molecular biologist, laboratory diagnostician, Cambridge Diagnostics Polska A biologist by profession, specializing in microbiology, and a laboratory diagnostician with over 10 years of experience in laboratory work. A graduate of the College of Molecular Medicine and a member of the Polish Society of Human Genetics. Head of research grants at the Laboratory of Molecular Diagnostics at the Department of Hematology, Oncology and Internal Diseases of the Medical University of Warsaw. She defended the title of doctor of medical sciences in the field of medical biology at the 1st Faculty of Medicine of the Medical University of Warsaw. Author of many scientific and popular science works in the field of laboratory diagnostics, molecular biology and nutrition. On a daily basis, as a specialist in the field of laboratory diagnostics, he runs the content department at Cambridge Diagnostics Polska and cooperates with a team of nutritionists at the CD Dietary Clinic. He shares his practical knowledge on diagnostics and diet therapy of diseases with specialists at conferences, training sessions, and in magazines and websites. She is particularly interested in the influence of modern lifestyle on molecular processes in the body.