RU2696075C2 - Method of extracting viral nucleic acids from different fractions of extracellular vesicles - Google Patents

Abstract

FIELD: biotechnology; medicine.SUBSTANCE: invention refers to biotechnology, molecular biology and medicine. Disclosed is a method of extracting viral nucleic acids from various fractions of extracellular vesicles. Technical result consists in expansion of viral nucleic acids extraction from most fractions of human extracellular vesicles.EFFECT: invention can be applied in medicine.1 cl, 1 ex

Description

The invention relates to medicine, in particular to cellular and molecular technology, and can be used in medical research in virology to study the composition of viral nucleic acids in extracellular vesicles in order to identify the role of vesicular transport in the spread of viral infections in the body.

In recent years, the fundamental role of the immune system in the progression of atherosclerosis, as well as the destabilization and rupture of atherosclerotic plaques [Libby P. Inflammation in atherosclerosis. Arterioscler Thromb Vase Biol. - 2012. - No. 32. - P. 2045-2051; Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis.- 1986. - No. 6. - P. 131-138]. Despite intensive research in this area, the trigger for activation of the immune system remains unknown. One of the candidates for the role of a trigger in atherogenesis may be bacteria and viruses [Bruggeman C. Cytomegalovirus is involved in vascular pathology. Am Heart J. - 1999. - No. 138. - P. S473-475]. So, there is a whole series of data that the high content of antibodies to influenza viruses, hepatitis, chlamydia, enterobacteria, cytomegalovirus (CMV), human herpes viruses type 1 and 2, etc. correlates with the development and progression of atherosclerosis [Jha HC, Srivastava P , Sarkar R, Prasad J, Mittal A. Chlamydia pneumoniae IgA and elevated level of IL-6 may synergize to accelerate coronary artery disease. J Cardiol. - 2008. - No. 52. - P. 140-145; Crumpacker CS. Invited commentary: human cytomegalovirus, inflammation, cardiovascular disease, and mortality. Am J Epidemiol. - 2010. - No. 172. - P. 372-374; Alyan O, Kacmaz F, Ozdemir O, et al. Hepatitis C infection is associated with increased coronary artery atherosclerosis defined by modified Reardon severity score system. Circ J. - 2008. - No. 72. - P. 1960-1965; Siscovick DS, Schwartz SM, Corey L, et al. Chlamydia pneumoniae, herpes simplex virus type 1, and cytomegalovirus and incident myocardial infarction and coronary heart disease death in older adults: the Cardiovascular Health Study. Circulation. - 2000. - No. 102. - P. 2335-2340; Cheng J, Ke Q, Jin Z, et al. Cytomegalovirus infection causes an increase of arterial blood pressure. Plos pathog. - 2009. - No. 5. - P. e1000427]. In addition, deoxyribonucleic acids (DNA) of many of these agents were also found within the atherosclerotic plaques themselves [Keller TT, van der Meer JJ, Teeling P, et al. Selective expansion of influenza A virus-specific T cells in symptomatic human carotid artery atherosclerotic plaques. Stroke. - 2008. - No. 39. - P. 174-179; Reszka E, Jegier B, Wasowicz W, Lelonek M, Banach M, Jaszewski R. Detection of infectious agents by polymerase chain reaction in human aortic wall. Cardiovasc Pathol. - 2008. - No. 17. - P. 297-302]. Among the studied infectious agents - atherosclerosis triggers, a special place is occupied by the family of human herpes viruses, which are widespread in the population of pathogens that can cause chronic immune activation [Horvath R, Cerny J, Benedfk J, Hokl J, Jelmkova I, Benedfk J. The possible role of human cytomegalovirus (HCMV) in the origin of atherosclerosis. J Clin Virol. - 2000. - No. 16. - P. 17-24; Aguilar D, Fisher MR, O'Connor CM, et al. Metabolic syndrome, C-reactive protein, and prognosis in patients with established coronary artery disease. Am Heart J. - 2006. - No. 152. - P. 298-304]. However, the results of the described studies on the relationship between chronic herpesvirus infection and atherosclerosis are extremely contradictory, which is largely due to the complexity of determining not only the presence but also the signs of infection activation [Heybar H, Alavi SM, Farashahi Nejad M, Latifi M. Cytomegalovirus infection and atherosclerosisis in candidate of coronary artery bypass graft. Jundishapur J Microbiol. - 2015. - No. 8. -P. el5476; Nieto FJ, Adam E, Sorlie P, et al. Cohort study of cytomegalovirus infection as a risk factor for carotid intimal-medial thickening, a measure of subclinical atherosclerosis. Circulation. - 1996. - No. 94. - P. 922-927; Barbarash OL, Zykov MV, Pecherina TV, Kashtalap VV, Barbarash LS, Kutikhin AG. The prognostic value of peripheral artery diseases in patients with ST-segment elevation myocardial infarction. Dis Markers. - 2013. - No. 35. - P. 877-882]. In our preliminary work, we found that the presence and distribution of DNA of various herpes viruses in the tissue of atherosclerotic plaques in patients with coronary heart disease (IHD) and in the intact arteries of patients without IHD is not significantly different, which may be associated with the detection of latent forms of infection, not causing immune activation and atherogenesis [Nikitskaya EA, Grivel ZhSH, Ivanova OI, and others. The study of herpesvirus DNA in the coronary arteries of patients who died in the acute stage of myocardial infarction. Creative cardiology. - 2014. - No. 4. - S. 52-64]. However, in further studies, we found that cytomegalovirus is often reactivated in patients with acute coronary syndrome. Moreover, cytomegalovirus DNA is determined not only in blood cells, but also in plasma, where its amount is 2 times higher in patients with acute forms of coronary heart disease than in healthy volunteers [Nikitskaya EA, Grivel JC, Maryukhnich EV, et al. Cytomegalovirus in plasma of acute coronary syndrome patients. Acta Naturae. - 2016. - No. 8. - P. 102-107].

It is known that one of the mechanisms of transmission of viral material from cell to cell in a multicellular organism can be transport through extracellular vesicles (EV) [Schorey JS, Cheng Y, Singh PP, Smith VL. Exosomes and other extracellular vesicles in host-pathogen interactions. EMBO Rep.- 2015. - No. 16. - P. 24-43]. Viral proteins and nucleic acids (NKs) produced by infected cells and transmitted by microvesicles and exosomes can both enhance the immune response [Walker JD, Maier CL, Pober JS. Cytomegalovirus-infected human endothelial cells can stimulate allogeneic CD4 + memory T cells by releasing antigenic exosomes. J Immunol. - 2009. - No. 182. - P. 1548-1559], and inhibit it [Dukers DF, Meij P, Vervoort MB, et al. Direct immunosuppressive effects of EBV-encoded latent membrane protein 1. J Immunol. - 2000. - No. 165. -P. 663-670].

A separate class of extracellular vesicles, virus-like vesicles, inside which viral DNA, ribonucleic acid (RNA) and proteins were detected, was isolated. It was shown that these vesicles can participate in the transmission and spread of infection within the body, including cells of the immune system [Jorfi S, Inal JM. The role of microvesicles in cancer progression and drug resistance. Biochem Soc Trans. 2013. - No. 41. - P. 293-298]. This mechanism has been especially studied in detail for the human immunodeficiency virus [Wiley RD, Gummuluru S. Immature dendritic cell-derived exosomes can mediate HIV-1 trans infection. Proc Natl Acad Sci USA. - 2006. - No. 103. - P. 738-743]. A similar transmission of viral infection through extracellular vesicles for influenza virus, hepatitis B and C viruses, as well as for herpes virus type 1 and 2 [Meckes DG, Raab-Traub N. Microvesicles and viral infection. J Virol. - 2011. - No. 85. - P. 12844-12854]. We showed that, along with the activation of cytomegalovirus infection in patients with acute forms of coronary heart disease, there is a significant increase in the amount of EV, especially vesicles of platelet origin [Vagida M, Arakelyan A, Lebedeva A, et al. Flow analysis of individual blood extracellular vesicles in acute coronary syndrome. Platelets. - 2017. - No. 28. - P. 165-173].

The determination of the virus and the reservoir of infection in patients with atherosclerosis is complicated by the fact that viruses, potential triggers, in particular, cytomegalovirus and / or human immunodeficiency virus (HIV), are in the latent phase, and transmission of viral particles during reactivation can occur through intercellular transport through extracellular vesicles . Under these conditions, the number of copies of the detected virus can be extremely small, in addition, such packaging can make it difficult to isolate NK.

To solve these problems, it is necessary to create a methodological approach that allows you to combine the method of selective EV extraction, NK purification from various subpopulations of EV and high-precision detection of small amounts of various viral NK into a common workflow.

Methods based on the differential centrifugation of biological fluids and media with and without precipitating reagents and methods based on affinity isolation from cell-free media are most often used to isolate EV. Both approaches have their drawbacks and advantages, but for the task of isolating the EV of NK-bearing viruses, specific separation of particles produced by infected cells is needed. In addition, it was shown that vesicles are able to merge during centrifugation, which leads to cross-contamination of different populations [Momen-Heravi F, Balaj L, Alian S, et al. Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles. Front Physiol. - 2012. - No. 3. - P. 162]. Isolation of vesicles using antibodies specific for membrane molecules answers the questions of the origin of EV and allows distinguishing various subpopulations of EV. All the necessary requirements are satisfied by the principle of isolation and analysis of EV using magnetic nanoparticles conjugated with antibodies specific for various vesicular and viral markers. The use of digital polymerase reaction (PCR) and real-time quantitative PCR [Taylor SC, Laperriere G, Germain H. Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Sci rep. - 2017. - No. 7. - P. 2409].

For a more complete description of the composition of extracellular vesicles and their role in the activation and spread of infection in the body, it is necessary to analyze the composition of nucleic acids in various populations of extracellular vesicles in human plasma. One of the main obstacles to the use of modern polymerase chain reaction technology for a comprehensive study of the composition of nucleic acids in extracellular vesicles is the difficulty in isolating individual fractions of extracellular vesicles, from which it will be possible to extract nucleic acids in the future without contamination or loss during the isolation process.

There is a method of isolating enterovirus RNA from extracellular vesicles obtained from human neuroblastoma cell culture. The method uses the method of centrifugation and ultrafiltration of the culture medium to obtain exosome fractions (fractions of extracellular vesicles of small sizes) with further joint isolation of cellular DNA and viral RNA from the general exosome population. The viral RNA is then amplified by the real-time polymerase chain reaction technique to analyze its quantitative composition [Mao L, Wu J, Shen L, Yang J, Chen J, Xu H. Enterovirus 71 transmission by exosomes establishes a productive infection in human neuroblastoma cells . Virus Genes. - 2016. - No. 52 (2). - P. 189-194]. In this method, only small-size EVs isolated from the culture medium of human neuroblastoma cells were analyzed, which did not allow us to evaluate the composition of viral nucleic acids inside other types of extracellular vesicles. In addition, the method carried out the selection of nucleic acids from a common solution of exosomes, which did not allow to determine the phenotype of cells that secrete these vesicles with viral nucleic acids. Therefore, this method does not make it possible to evaluate the complete nucleic acid composition of enterovirus depending on the phenotype of various fractions of extracellular vesicles.

In addition, methods are known for isolating hepatitis C virus nucleic acids from exosomes obtained from human hepatoma cell lines [Ramakrishnaiah V, Thumann C, Fofana I, Habersetzer F, Pan Q, de Ruiter P, Willemsen R, Demmers J, Raj V, Jenster G, Kwekkeboom J, Tilanus H, Haagmans B, Baumert T, der Laan L. Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh 7.5 cells. Proc Natl Acad Sci USA.-2013.-No. 110 (32) .- P. 13109-13113; Saha B, Kodys K, Adejumo A, Szabo G. Circulating and Exosome-Packaged Hepatitis C Single-Stranded RNA Induce Monocyte Differentiation via TLR7 / 8 to Polarized Macrophages and Fibrocytes. J Immunol. - 2017. - No. 198 (5). - P. 1974-1984], where exosomes were isolated from the culture medium of human hepatocytes infected with hepatitis C virus using the sucrose gradient ultracentrifugation technique. Then, viral RNA was isolated from the general population of isolated exosomes. However, in these methods, nucleic acids were also isolated only from the exosome fraction, which did not allow us to evaluate the composition of viral nucleic acids inside other types of extracellular vesicles. In addition, in both methods, nucleic acids were isolated from the general exosome population, which did not allow us to determine the phenotype of cells that secrete these vesicles with viral nucleic acids.

Methods for determining the nucleic acids of hepatitis E and hepatitis B viruses as part of exosomes isolated from a culture of infected human hepatocytes are also described. In the methods, the exosome fraction was isolated by ultracentrifugation. After that, certain exosome fractions were separated using magnetic beads specific to surface markers typical of various exosome fractions. Subsequently, nucleic acids were extracted from the selected exosome fractions [Sanada T, Hirata Y, Naito Y, Yamamoto N, Kikkawa Y, Ishida Y, Yamasaki C, Tateno C, Ochiya T, Kohara M. Transmission of HBV DNA Mediated by Ceramide-Triggered Extracellular Vesicles. Cell Mol Gastroenterol Hepatol. - 2016. - No. 3 (2). - P. 272-283; Nagashima S, Takahashi M, Kobayashi T, Nishizawa T, Nishiyama T, Primadharsini PP, Okamoto H. Characterization of the Quasi-Enveloped Hepatitis E Virus Particles Released by the Cellular Exosomal Pathway. J Virol. - 2017. - No. 91 (22). - P. e00822-17]. As in the previous methods, the nucleic acids were isolated in these studies from the exosome fraction secreted by a single cell line, which did not allow us to evaluate the composition of NK in the remaining subpopulations of extracellular vesicles.

Thus, according to the literature, the existing methods for determining the composition of viral nucleic acids allow one to analyze them only within selected minor subpopulations of extracellular vesicles without a detailed determination of the EV phenotype and with the loss of a large number of vesicles due to the necessity of separating the vesicles from the cell fraction by gradient centrifugation or ultracentrifugation methods . However, it is important to note that a large number of nucleic acids of viruses can also be transferred by other fractions of EV, the separation of which from cellular structures is difficult due to the need for additional purification of EV from impurities of freely circulating NAs. In this regard, the selection of ND from all subpopulations of EVs requires the development of the stages of sorting and filtering samples for the selection of EVs without contamination with freely circulating nucleic acids and viral particles.

A known method for the isolation of nucleic acids of human immunodeficiency virus from subpopulations of leukocyte extracellular vesicles [Arakelyan A, Fitzgerald W, Zicari S, Vanpouille C, Margolis L. Extracellular Vesicles Carry HIV Env and Facilitate Hiv Infection of Human Lymphoid Tissue. Scientific Reports. - 2017. - No. 7. - P. 1695], which consists in the fact that as a material for determining viral nucleic acids using samples of the culture medium from cells and tissues infected with HIV. To obtain a fraction of extracellular vesicles, the culture medium is coupled with magnetic nanoparticles (MNPs) of size 15 nm, consisting of iron oxide and coated with carboxyl groups. At a preliminary stage, these particles are conjugated with antibodies specific for the selected surface marker of EV, which is practically not contained in the membrane of viral particles (CD45). For this, 1 mg of MNP is incubated for 10 minutes at room temperature in 200 μl of activation buffer containing 1.7 mM of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 0.76 mM of N-hydroxysulfosuccinimide. After activation, 500 μl of binding buffer is added to the magnetic nanoparticles, 1 mg of purified antibodies is immediately added to the resulting suspension. After two-hour incubation in a thermomixer at room temperature with stirring, the reaction is stopped by adding 10 μl of quenching solution and 2 washes with washing buffer using a magnetic separator at 4 ° C. MNP conjugated with antibodies are dissolved in 2 ml of storage buffer and stored at 4 ° C at a concentration of 0.5 mg / ml of iron oxide.

The complexes of MNPs conjugated with antibodies prepared in this way are incubated with the culture medium for 40 minutes at a temperature of 37 ° C. Then, the formed MNP-EV complexes are separated using magnetic columns in a strong magnetic field to clear them of viral particles and free nucleic acids . After immobilization of MNP-EV complexes in a magnetic field, the magnetic columns are washed three times with a washing solution containing phosphate buffer, 0.5% BSA, 2 mM EDTA. In the next step, the contents of the column are washed out of the magnetic field and dissolved in 400 μl of phosphate buffer. The obtained MNP-EV complexes are used for the extraction of nucleic acids on magnetic silicon particles under high salt conditions. To do this, the material is lysed by adding 200 μl of the obtained MNP-EV sample to a standard lysis buffer, then a high-salt buffer is added to the lysate to absorb the released NC on magnetic particles, during which the magnetic particles used to separate the EV are also absorbed, which allows you to clean the resulting solution NK. Next, the NC is separated from magnetic silicon particles by reducing the salt concentration of the solution using standard low-salt eluting buffers. Upon completion of the extraction, a solution of viral and cellular nucleic acids is obtained, isolated from fractions of extracellular vesicles, which is subjected to analysis by polymerase chain reaction. This method is selected for the prototype. The disadvantage of this method is that when isolating nucleic acids from extracellular vesicles, a medium from cell or tissue cultures is used, which contains a limited set of EV mainly of leukocyte origin, which does not correspond to the distribution of the population of EV in the human body that can participate in the transfer of viral NK . Moreover, for the separation of EV from free viral particles, only the surface marker CD45 is used, which allows one to analyze the composition of EV only of leukocyte origin. In this regard, the analysis of viral nucleic acids inside the EV of a different cellular origin using this method becomes impossible.

The objective of the invention is to increase the efficiency of assessing the composition of nucleic acids of viruses in various subpopulations of extracellular vesicles.

The technical result consists in the isolation of viral nucleic acids from various fractions of extracellular human vesicles, necessary to determine the mechanisms of the spread of viral infection in the human body using vesicular transport.

This is achieved due to the fact that the analysis uses the full composition of extracellular vesicles from platelet-depleted human blood plasma, when isolating vesicles from platelet-depleted plasma, magnetic sorting of viral particles by specific glycoprotein is used, and after that fractions of extracellular vesicles are isolated by using magnetic nanoparticles with antibodies specific for various surface markers of vesicles.

In the proposed method for the isolation of nucleic acids from subpopulations of extracellular vesicles, instead of the culture medium from infected cells and tissues, the platelet-depleted plasma of patients infected with the virus is used as a source of the sample of viral nucleic acids instead of the culture medium, due to which fractions of EV of any cellular origin that circulate in the blood become available for analysis .

In addition, instead of conducting magnetic sorting onto surface markers that are most rare for viral particles, the stage of conjugation of a solution of viral particles and extracellular vesicles with magnetic beads associated with antibodies to the virus glycoprotein necessary for its infection has been introduced. It was shown in experiments that glycoproteins of the large virus envelope are contained in the membrane in less than 15% of the total population of extracellular vesicles, which confirms the effectiveness of the proposed method. In this regard, for the analysis of viral nucleic acids inside the EV, it is possible to use a solution of vesicles that did not bind to the viral glycoprotein and pass through a magnetic column. Due to this, the separation of free viral particles from extracellular vesicles was carried out without loss of the main subpopulations of EV. Moreover, the next stage of conjugation of the solution of extracellular vesicles with magnetic nanoparticles bound to antibodies to various cell markers was also added. Due to this, nucleic acids were isolated from EV fractions of various cellular origin (platelet, leukocyte, etc.).

The method is as follows:

A sample of the blood of a patient infected with a virus taken from a peripheral vein into a vacuum tube with a 3.2% solution of sodium citrate is used as a material for the isolation of nucleic acids; the blood sample is stored at room temperature for no more than 15 minutes; blood tubes are centrifuged twice for 15 min at an acceleration of 3000 g at room temperature, the upper fraction is taken, obtaining platelet-poor plasma; after which the plasma solution is frozen, stored at a temperature not exceeding -80 ° C for at least 6 hours until the study is continued; then the plasma is thawed at room temperature; after which they carry out the binding of free viral particles from the prepared blood plasma with 15 nm nanoparticles, previously conjugated with antibodies specific for the envelope of the virus; to bind viral particles to MNPs and separate them from EV, the plasma solution is incubated for 40 minutes at 37 ° C with constant stirring; then, using magnetic columns in a magnetic field, the resulting MNP-virus particle complexes are separated by washing the magnetic columns three times with a washing solution containing phosphate buffer, 0.5% BSA, 2 mM EDTA; then a plasma solution is collected with EV, which is re-coupled with 15 nm nanoparticles, pre-conjugated with antibodies specific for different surface vesicle markers (CD45, CD31, CD41, etc.); after the immobilization of antibody-labeled MNP-EV complexes in a magnetic field, the magnetic columns are washed three times with a washing solution; then the contents of the column are washed out of the magnetic field and dissolved in 400 μl of phosphate buffer; then, the isolated fractions of the EV are lysed by adding 200 μl of the MNP-EV sample to a standard lysing buffer with lysate absorption in a high salt buffer on magnetic silicon particles; nucleic acids are separated from magnetic silicon particles by lowering the salt concentration of the solution and are subjected to polymerase chain reaction analysis.

Example No. 1.

The blood plasma of a 36-year-old male patient infected with cytomegalovirus was taken as the source of the test material. Blood was taken from a peripheral vein into a vacuum tube with a 3.2% solution of sodium citrate. A blood sample was stored at room temperature for 5 minutes. Then, blood samples were centrifuged twice for 15 minutes with an acceleration of 3000g. The upper fraction was taken from the tubes - blood plasma free from platelets. The plasma solution was frozen and stored until subsequent analysis for 1 month at a temperature of -83 ° C. Next, plasma samples were thawed at room temperature. Then, plasma samples were associated with MNPs, which at the preparatory stage were conjugated to antibodies specific for the cypomegalovirus gpB membrane glycoprotein. To bind viral particles to MNPs conjugated with antibodies, plasma samples were incubated with MNP complexes for 40 minutes at 37 ° C with constant stirring. Next, the magnetic columns were washed three times with a washing solution containing phosphate buffer, 0.5% BSA, 2 mM EDTA.

In the next step, the solution of EV that passed through the magnetic column was divided into 3 parts and associated with MNPs, which at the preparatory stage were conjugated with antibodies specific for surface markers of EV (CD31, CD41, CD45, CD63, CD81). Then, the resulting MNP-EV complexes were immobilized using magnetic columns in a strong magnetic field and the magnetic columns were washed three times with a washing solution. Then, the contents of the columns were washed out of the magnetic field, dissolved in 400 μl of phosphate buffer, and the isolated fractions of EV were lysed by adding 200 μl of the MNP-EV sample to a standard lysis buffer with lysate absorption in a high salt buffer on magnetic silicon particles. The obtained nucleic acids were separated from magnetic silicon particles by lowering the salt concentration of the solution and subjected to analysis of their concentration by polymerase chain reaction.

When analyzing a polymerase chain reaction of cytomegalovirus DNA isolated from various fractions of extracellular vesicles, the following results were obtained:

The amount of cytomegalovirus DNA in platelet-poor plasma = 6893 copies per 1 ml of blood plasma.

The amount of cytomegalovirus DNA in the total fraction of EV that do not carry viral glycoprotein on the surface = 4554 copies per 1 ml of blood plasma.

The amount of cytomegalovirus DNA in the EV fraction containing on its surface a marker CD31 = 924 copies per 1 ml of blood plasma.

The amount of cytomegalovirus DNA in the EV fraction containing on its surface a marker CD41 = 1320 copies per 1 ml of blood plasma.

The amount of cytomegalovirus DNA in the EV fraction carrying on its surface a marker CD45 = 858 copies per 1 ml of blood plasma.

The amount of cytomegalovirus DNA in the EV fraction containing on its surface a marker CD63 = 858 copies per 1 ml of blood plasma.

The amount of DNA of cytomegalovirus in the EV fraction, carrying on its surface a marker CD81 = 396 copies per 1 ml of blood plasma.

Thus, we have successfully studied the distribution of cytomegalovirus nucleic acids in various fractions of extracellular vesicles.

Claims (1)

A method for isolating viral nucleic acids from various fractions of extracellular vesicles, which consists in collecting blood plasma from patients infected with a virus by centrifuging for 15 minutes in a cooling centrifuge with a rotary-bucket rotor at an acceleration of 3000 g, separating the extracellular vesicles from the viral particles using magnetic sorting by the glycoprotein of the virus envelope after conjugation in a thermomixer with an orbit of 3 mm and an acceleration of 350 rpm, the fractions of extracellular vesicles are separated using sorting by cell surface markers CD45, CD31, CD41, lyses the extracted fractions of extracellular vesicles and extract nucleic acids using magnetic silicon particles 1-5 μm in size under standard high salt conditions followed by polymerase chain reaction, characterized in that the analysis uses a complete the composition of extracellular vesicles from platelet-depleted human blood plasma; when isolating vesicles from platelet-depleted plasma, magnetic sorting of viral parts is used it with a specific glycoprotein, and after this, fractions of extracellular vesicles are isolated by using magnetic nanoparticles with antibodies specific to various surface markers of the vesicles.