RU2806427C1 - Set of oligonucleotide primers and probes for species differentiation of human herpes virus 6a and human herpes virus 6b and method of its use - Google Patents

Abstract

FIELD: molecular biology.

SUBSTANCE: set of oligonucleotide primers and probes for species differentiation of human herpes virus 6A and human herpes virus 6B is described. A method of using the specified set of oligonucleotide primers and probes is also described. The method involves extracting DNA from biological material and performing RT-PCR using the above-mentioned set of oligonucleotide primers and probes, amplification, analysis and interpretation of the results.

EFFECT: development of a means for detecting the DNA of human herpes viruses, namely HHV-6A and HHV-6B, with a high degree of specificity.

5 cl, 1 tbl, 4 ex

Description

The invention relates to the field of medicine and molecular biology and can be used in clinical practice for species identification of DNA of human herpes viruses, in particular human herpes virus 6A (HHV-6A) and human herpes virus 6B (HHV-6B).

HHV-6A (Human betaherpesvirus 6A) and HHV-6B (Human betaherpesvirus 6B) - DNA-containing viruses of the Herpesviridae family of the Betaherpesvirinae subfamily of the genus Roseolavirus - are the closest genetic relatives of human cytomegalovirus (Human betaherpesvirus 5) and human herpes virus 7 (Human betaherpesvirus 7) . Currently, the common name for HHV-6B and HHV-6A is human herpes virus 6 (HHV-6, HHV-6A/B).

HHV-6 was first isolated in 1986 from peripheral blood B lymphocytes of HIV-infected patients with non-Hodgkin's lymphoma. In 1992, it was proposed to divide HHV-6 into two variants (hereinafter referred to as types) A and B, which in 2012 were classified as separate types: HHV-6A and HHV-6B. It has been established that HHV-6A and HHV-6B are able to effectively integrate into the telomeres of host cell chromosomes both in vivo and in vitro, due to the peculiarities of genome organization.

The virus exhibits tropism for a wide range of host cells: T cells, monocytes-macrophages, hematopoietic cells of the bone marrow, epithelial cells of the kidneys and salivary glands, endothelial cells, cells of the central nervous system (microglial cells, oligodendrocytes and astrocytes). HHV-6A/B, like other herpes viruses, is characterized by the ability to persist and latency in the body of an infected person. HHV-6A/B infection - anthroponosis. The source and reservoir of infection is a person with HHV-6A/B infection, as well as a virus carrier.

The high pathogenetic significance of HHV-6A/B has been shown: it can cause acute skin lesions in young children (sudden exanthema of newborns), fever in newborns with convulsive syndrome, mononucleosis-like syndrome; participate in the development of myalgic encephalomyelitis; in immunocompromised individuals, cause fever, pneumonia, hepatitis, and damage to the central nervous system. It has been proven that HHV-6A/B can also act as a cofactor for HIV. Along with the occurrence of a primary infection, reactivation of the virus is possible. Verification of the diagnosis of HHV-6A/B infection is carried out only with positive results of laboratory tests.

HHV-6A is more often detected in adults with chronic diseases, as well as in persons with established inherited chromosomally integrated HHV-6A/B status. HHV-6B is significantly more likely than HHV-6A to cause acute infections in children (sudden exanthema, fever without rash), and is also associated with febrile seizures and temporal lobe epilepsy. In addition, HHV-6B, unlike HHV-6A, is resistant to the antiviral effects of alpha and beta interferons.

HHV-6A and HHV-6B differ in nucleotide sequence, cultivation characteristics, and epidemiological data. The nucleotide sequences of HHV-6A and HHV-6B coincide in 75-95%, depending on the gene being compared. Viruses use different cellular receptors for penetration: HHV-6A - CD46, HHV-6B - CD134. These differences may explain the different tissue tropism of the viruses and the different spectrum of pathology.

Every year, information about the differences between HHV-6A and HHV-6B is updated; more often, molecular biological methods are used to differentiate HHV-6A and HHV-6B: PCR and sequencing.

There is a known method for differentiating HHV-6A and HHV-6B [Patent JP2003135100, filing date July 26, 2002] based on the difference in the lengths of amplified fragments for different subtypes, followed by electrophoretic detection. The U89/90 gene, characterized by the presence of insertions of 108 and 228 bp in size, was selected for analysis. for HHV-6B relative to HHV-6A. The disadvantage of this method is the use of electrophoretic detection for analysis and the variability of the size of insertions.

To detect HHV-6A, various methods are used to discriminate allelic variants: based on ligation [Landegren U., et al. A Ligase-Mediated Gene Detection Technique // Science. - 1988. - Vol. 241. - P. 1077-1080], mass spectroscopy [Griffin T. J., et al. Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry // Proc. Natl. Acad. Sci. - 1999. - Vol. 25. - P. 6301-6306]; DNA microarray [Wang D.G, et al. Large-Scale Identification, Mapping, and Genotyping of Single-Nucleotide Polymorphisms in the Human Genome // Science. - 1998 - Vol. 280. - P. 1077-1082]; restriction fragment length analysis [Saiki R.K., et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia // Science. - 1985. - Vol. 230. - No. 4732. - P. 1350-1354]; TaqMan type probes [Livak K.J., et al. 12 Oligonucleotides with Fluorescent Dyes at Ends Provide a Quenched Probe System Useful for Detecting PCR Product and Nucleic Acid Hybridization // PCR Wethods Appl. - 1995. - Vol. 4. - P. 357-362]; melting curve analysis [Ririe K. M., et al. Product Differentiation by Analysis of DNA Melting Curves during the Polymerase Chain Reaction//Anal. Biochem. 1997. Vol. 245 - P. 154-160].

A probe for detecting human herpes virus type 6A and/or 6B is known from the prior art (patent JP2018085961, filing date November 29, 2016), which is used in a kit for real-time PCR, and the type of fluorescent dye contained in the detection probe HHV6 type B is different from the type of fluorescent dye found in the HHV type 6A detection probe.

During the practical implementation of the mentioned solution, a fairly large number of false positive results were observed, which is its drawback.

Also known from the prior art is a method for identifying variants A and B of human herpes virus type 6 (patent RU 2627607, priority date September 28, 2016), which makes it possible to determine single nucleotide polymorphisms specific to HHV-6A and HHV-6B in the U67 gene of HHV-6 by real-time PCR using primers HHV6F 5'-CGGATACAGTAAGACGGGATAT-3' and HHV6R 5'-ACGTAAGCTTGCACAATGC-3' and two fluorescently labeled probes HHV6AZ F1-GCAATAGATTTGGAAACGCGCAT-Q1 and HHV6BZ F2-GCAATAGATTCGGAAATGCGGCAT-Q2, where o presence of HHV- 6A or HHV-6B is judged in the case of recording the exponential accumulation of the fluorescence signal in the channels corresponding to the probes. This method is based on the determination of single nucleotide polymorphisms in the U67 gene and does not contain data on its sensitivity.

Although the demand for fast, high-throughput, low-cost tests to determine the DNA of various infectious agents has led to the development of various laboratory techniques, there is a need to expand an accurate diagnostic solution that allows species differentiation of human herpes virus species in a short time and at the lowest cost. in particular, HHV-6A and HHV-6B, which would allow for quick, cost-effective and unambiguous interpretation of the results for diagnostic purposes and in population studies of the pathogen.

In this regard, there is a need to develop a set of oligonucleotides for the simultaneous detection and differentiation of HHV-6A and HHV-6B: primers and fluorescent probes for RT-PCR.

The technical result of the claimed invention is aimed at identifying the DNA of human herpes viruses, namely HHV-6A or HHV-6B, with a high degree of specificity in samples of biological material through a set of oligonucleotide primers and fluorescent probes that allow effectively differentiating types of HHV-6A/B from using widely used techniques and available materials.

The technical result is achieved through the use in RT-PCR of a set of chemically synthesized oligonucleotides that make it possible to determine HHV-6A DNA and HHV-6B DNA in a biological material sample:

forward primer HHV-6A/B-for - SEQ ID NO: 1;

reverse primer HHV-6A/B-rev - SEQ ID NO: 2;

fluorescently labeled probe HHV-6A-probe - SEQ ID NO: 3;

fluorescently labeled probe HHV-6B-probe - SEQ ID NO: 4.

Species differentiation of HHV-6A and HHV-6B is carried out by assessing the accumulation curves of fluorescent signals through the corresponding channels for fluorophores.

Primers are oligonucleotide sequences for amplification of a region of the U31 gene, fluorescent probes are oligonucleotides containing a fluorophore and a fluorescence quencher, allowing the detection of amplified fragments.

A set of oligonucleotide primers and probes for implementing the proposed method was developed on the basis of known sequences: the U31 Human betaherpesvirus 6A gene (reference genome of the reference exogenous strain Human betaherpesvirus 6A U1102, NC 001664.4), presented in the National Center for Biotechnology Information (NCBI) database [https ://www.ncbi.nlm.nih.gov/nuccore/NC_001664.4]; gene U31 Human betaherpesvirus 6B (reference genome of the reference exogenous strain Human betaherpesvirus 6B HST, AB021506.1), presented in the National Center for Biotechnology Information (NCBI) database [https://www.ncbi.nlm.nih.gov/nuccore /AB021506.1].

Primers and probes were selected for the selected fragments to determine a fragment of 200 bp in size: forward primer HHV-6A/B-for - SEQ ID NO: 1; reverse primer HHV-6A/B-rev SEQ ID NO: 2, fluorescent probe HHV-6A-probe SEQ ID NO: 3 and fluorescent probe HHV-6B-probe SEQ ID NO: 4.

The claimed invention is the result of a study conducted at the Central Research Institute of Epidemiology of Rospotrebnadzor (Moscow, Russia) as part of work to improve the diagnosis of human herpes viral diseases.

To select target sequences - sites for landing oligonucleotides, we used the sequences of the U31 gene fragment of HHV-6A Human betaherpesvirus 6A strains/isolates and the U31 gene fragment of Human betaherpesvirus 6B HHV-6B strains/isolates, presented in the National Center for Biotechnology Information (NCBI) database. [http://www.ncbi.nlm.nih.gov]. To search for conserved sequences, we used modern algorithms for in silico analysis of nucleotide sequences and publicly available programs, including Mega and Oligo Calculators from Thermo Fisher Scientific (USA). A list of unique sequences characteristic of HHV-6A and HHV-6B was compiled. Then the oligonucleotide sequences of the forward and reverse primers, as well as fluorescently labeled probes, were selected. The mentioned sequences of the set of oligonucleotide primers and probes are given in Table 1.

Analysis of these sequences using the Primer BLAST resource showed that when using the forward primer HHV-6A/B-for (SEQ ID NO: 1), reverse primer HHV-6A/B-rev (SEQ ID NO: 2), as well as fluorescent probes HHV-6A-probe - SEQ ID NO: 3 and HHV-6B-probe - SEQ ID NO: 4, the HHV-6A and HHV-6B regions are detected with 100% specificity.

As biological material, it is preferable to use whole blood, smears from the mucous membrane of the oropharynx, urine, and tissue material.

PCR research is carried out in accordance with MU 1.3.2569-09 “Organization of work in laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I-IV.” For DNA extraction, the RIBO-prep reagent kit (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) or any similar kit/reagent kit for DNA extraction can be used.

RT-PCR is carried out in a multiplex format using the proposed set of oligonucleotide primers and probe presented in Table 1, under the following conditions:

The total volume of the reaction mixture is 25 µl. PCR components are mixed as follows:

(a) 10 µl of a mixture containing:

- oligonucleotide primers SEQ ID NO: 1, 2 - 0.6 pmol/μl;

- fluorescent probe SEQ ID NO: 3, 4 - 0.15 pmol/μl;

- dNTPs - 0.44 mM.

(b) PCR buffer containing MgCl ₂ and recombinant enzyme Taq DNA polymerase, for example, 5 μl of PCR buffer-N (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) or any similar commercial reagent in accordance with the manufacturer’s instructions.

(c) DNA extracted from 10 μL biological samples. The staging and analysis of amplification results is carried out on a device with a fluorescent signal detection system in “real time”, for example, Rotor-Gene Q (Qiagen, Germany) in accordance with the manufacturer’s instructions or similar.

Amplification is carried out according to the following program: 1 cycle at 95°C for 15 minutes, then 45 cycles at a temperature of 95°C - 10 seconds / 60°C - 20 seconds. Fluorescence detection is carried out at the 60°C stage using specified channels for fluorophores F ₁ and F ₂ .

Analysis of the results obtained is carried out using the software of the device used for RT-PCR.

To analyze the results, set a threshold line corresponding to a value of 0.03. The results are interpreted based on the presence (or absence) of the intersection of the S-shaped (sigma-shaped) fluorescence curve with the threshold line set at the appropriate level. This determines whether or not the DNA sample being analyzed has a threshold cycle value. During the DNA amplification process, probes compete for binding to a DNA template containing two primers SEQ ID NO: 1 and SEQ ID NO: 2. In this case, the advantage is given to the probe corresponding to the type of human herpes virus 6A/B represented in the sample. If both HHV-6A/B species are present, both probes hybridize. Thus, samples for which the fluorescence curves cross the threshold line, and, at the same time, the kinetics of accumulation of the fluorescent signal is exponential, are positive: for HHV-6A - through the F ₁ channel, for HHV-6B - through the F ₂ channel, for HHV-6 6A and VHCh-6B - channels F ₁ and F ₂ simultaneously.

The implementation of the claimed invention is illustrated by the following examples: Example 1. Preparation of a set of oligonucleotide primers and probes for species differentiation of human herpes virus 6A (HHV-6A) and human herpes virus 6B (HHV-6B).

To select target sequences - oligonucleotide landing sites, we used known sequences: the U31 Human betaherpesvirus 6A gene (reference genome of the reference exogenous strain Human betaherpesvirus 6A U1102, NC_001664.4), presented in the National Center for Biotechnology Information (NCBI) database [https ://www.ncbi.nlm.nih.gov/nuccore/NC_001664.4]; gene U31 Human betaherpesvirus 6B (reference genome of the reference exogenous strain Human betaherpesvirus 6B HST, AB021506.1), presented in the National Center for Biotechnology Information (NCBI) database [https://www.ncbi.nlm.nih.gov/nuccore / AB021506.1]. We compiled a list of unique sequences characteristic of HHV-6A and HHV-6B, to which we selected unique oligonucleotide sequences of forward HHV-6A/B-for and reverse HHV-6A/B-rev primers, as well as fluorescent probes HHV-6A-probe and HHV-6B-probe.

A set of oligonucleotide primers and probes for species differentiation of HHV-6A and HHV-6B forward primer HHV-6A/B-for, reverse primer HHV-6A/B-rev, fluorescent probes HHV-6A-probe and HHV-6B-probe, presented unique sequences SEQ ID NO: 1-4, respectively.

Analysis of these sequences using the Primer BLAST resource showed that when using unique sequences SEQ ID NO: 1-3, the HHV-6A region is detected with 100% specificity, and when using unique sequences SEQ ID NO: 1, 2, 4 with 100% specificity the HHV-6B region is detected.

Example 2. Method of using a set of oligonucleotide primers and probes for species differentiation of HHV-6A and HHV-6B.

For species differentiation of HHV-6A and HHV-6B in samples of biological material, 24 samples were selected with the confirmed presence of HHV-6A/B DNA.

The following samples of biological material were used for the study: whole venous and umbilical cord blood (11/24), tissue material (4/24), and a smear from the mucous membrane of the oropharynx (9/24). The presence of HHV-6A/B DNA was confirmed laboratory using a set of reagents “AmpliSens® HHV6-screen-titer-FL” (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia).

The PCR study was carried out in accordance with MU 1.3.2569-09 “Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I-IV.” For DNA extraction, a set of reagents “RIBO-prep” (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) was used in accordance with the manufacturer’s instructions.

RT-PCR was carried out in a multiplex format using the proposed set of oligonucleotide primers and probes presented in Table 1, under the following conditions:

The total volume of the reaction mixture is 25 µl. PCR components were mixed as follows:

(a) 10 µl of a mixture containing:

- oligonucleotide primers SEQ ID NO: 1, 2 - 0.6 pmol/μl;

- fluorescent probe SEQ ID NO: 3, 4 - 0.15 pmol/μl;

- dNTPs - 0.44 mM.

(b) 5 μl of PCR buffer-N (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) containing MgCl ₂ , recombinant enzyme Taq DNA polymerase.

(c) DNA extracted from biological material samples - 10 µl. The amplification results were performed and analyzed on a device with a Rotor-Gene Q real-time fluorescent signal detection system (Qiagen, Germany) in accordance with the manufacturer’s instructions.

Amplification was carried out according to the following program: 1 cycle at 95°C for 15 minutes, 45 cycles at a temperature of 95°C - 10 seconds / 60°C - 20 seconds. Fluorescence detection was carried out at the 60°C stage using channels for fluorophores: R6G - for HHV-6A, ROX - for HHV-6B. To analyze the results, a threshold line corresponding to a value of 0.03 was set. For 9 samples, the fluorescence curve crossed the threshold line along the channel for the R6G fluorophore; for 15 samples, the fluorescence curve crossed the threshold line through the channel for the ROX fluorophore, while the kinetics of accumulation of fluorescent signals was exponential.

The presence of HHV-6A DNA was determined using the channel for the R6G fluorophore; the presence of HHV-6B DNA was determined using the channel for the ROX fluorophore.

The results of the study were confirmed using the method of direct sequencing of nucleotide sequences (Sanger), which was performed on an ABI 3500xL analyzer (Applied Biosystems, USA) in accordance with the manufacturer’s instructions. Alignment and analysis of the obtained sequences was carried out using the AlignX program (Thermo Fisher Scientific, USA). No discordant results were found.

Example 3. Method of using a set of oligonucleotide primers and probes for species differentiation of HHV-6A and HHV-6B.

For species differentiation of HHV-6A and HHV-6B in samples of biological material, 65 samples were selected with the confirmed presence of DNA of various types of human herpes virus.

The following samples of biological material were used for the study: whole venous blood (17/65), smear from the mucous membrane of the oropharynx (19/65), tissue material (13/65), urine (16/65). The presence of human herpes virus DNA was confirmed laboratory using reagent kits “AmpliSens® HHV6-screen-titer-FL”, “AmpliSens® HHV8-screen/monitor-FL”, “AmpliSens® HHV7-screen/monitor-FL”, “AmpliSens® HSV I, II-FL" (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia).

The PCR study was carried out in accordance with MU 1.3.2569-09 “Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I-IV.” For DNA extraction, a set of reagents “RIBO-prep” (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) was used in accordance with the manufacturer’s instructions.

RT-PCR was carried out in a multiplex format using the proposed set of oligonucleotide primers and probes presented in Table 1, under the following conditions:

The total volume of the reaction mixture is 25 µl.

PCR components were mixed as follows:

(a) 10 µl of a mixture containing:

- oligonucleotide primers SEQ ID NO: 1, 2 - 0.6 pmol/μl;

- fluorescent probe SEQ ID NO: 3, 4 - 0.15 pmol/μl;

- dNTPs - 0.44 mM.

(b) 5 μl of PCR buffer-N (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) containing MgCl ₂ , recombinant enzyme Taq DNA polymerase.

(c) DNA extracted from 10 μL biological samples.

The amplification results were performed and analyzed on a device with a Rotor-Gene Q real-time fluorescent signal detection system (Qiagen, Germany) in accordance with the manufacturer’s instructions.

Amplification was carried out according to the following program: 1 cycle at 95°C for 15 minutes, 45 cycles at a temperature of 95°C - 10 seconds / 60°C - 20 seconds. Fluorescence detection was carried out at 60°C using channels for fluorophores: FAM for HHV-6A, Cy5 for HHV-6B. To analyze the results, a threshold line corresponding to a value of 0.03 was set.

For 32/65 samples, the fluorescence curve crossed the threshold line along the channel for the FAM fluorophore; for 25/65 samples, the fluorescence curve crossed the threshold line through the channel for the Cy5 fluorophore, while the kinetics of accumulation of fluorescent signals was exponential.

The presence of HHV-6A DNA was determined using the channel for the FAM fluorophore; the presence of HHV-6B DNA was determined using the channel for the Cy5 fluorophore.

The results of the study were confirmed using the method of direct sequencing of nucleotide sequences (Sanger), which was performed on an ABI 3500xL analyzer (Applied Biosystems, USA) in accordance with the manufacturer’s instructions. Alignment and analysis of the obtained sequences was carried out using the AlignX program (Thermo Fisher Scientific, USA).

The belonging of 8/65 samples to other types of human herpes virus was confirmed using the method of direct sequencing of nucleotide sequences (Sanger), which was performed on an ABI 3500xL analyzer (Applied Biosystems, USA) in accordance with the manufacturer’s instructions. Alignment and analysis of the obtained sequences was carried out using the AlignX program (Thermo Fisher Scientific, USA). No discordant results were found.

Example 4. Method of using a set of oligonucleotide primers and probes for species differentiation of HHV-6A and HHV-6B.

For species differentiation of HHV-6A and HHV-6B in samples of biological material, 93 samples were selected, with the confirmed presence of DNA of various types of human herpes virus.

The following samples of biological material were used for the study: whole venous blood (31/93), smear from the mucous membrane of the oropharynx (39/93), tissue material (15/93), urine (8/93). The presence of human herpes virus DNA was confirmed laboratory using reagent kits “AmpliSens® HHV6-screen-titer-FL”, “AmpliSens® HHV8-screen/monitor-FL”, “AmpliSens® HHV7-screen/monitor-FL”, “AmpliSens® HSV I, II-FL" (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia).

The PCR study was carried out in accordance with MU 1.3.2569-09 “Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I-IV.” For DNA extraction, a set of reagents “RIBO-prep” (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) was used in accordance with the manufacturer’s instructions.

RT-PCR was carried out in a multiplex format using the proposed set of oligonucleotide primers and probe presented in Table 1, under the following conditions:

The total volume of the reaction mixture is 25 µl.

PCR components were mixed as follows:

(a) 10 µl of a mixture containing:

- oligonucleotide primers SEQ ID NO: 1, 2 - 0.6 pmol/μl;

- fluorescent probe SEQ ID NO: 3, 4 - 0.15 pmol/μl;

- dNTPs - 0.44 mM.

(b) 5 μl of PCR buffer-N (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) containing MgCl ₂ , recombinant enzyme Taq DNA polymerase.

(c) DNA extracted from 10 μL biological samples. The amplification results were performed and analyzed on a device with a Rotor-Gene Q real-time fluorescent signal detection system (Qiagen, Germany) in accordance with the manufacturer’s instructions.

Amplification was carried out according to the following program: 1 cycle at 95°C for 15 minutes, 45 cycles at a temperature of 95°C - 10 seconds / 60°C - 20 seconds. Fluorescence detection was carried out at the 60°C stage through the channels for fluorophores: ROX - for HHV-6A, FAM - for HHV-6B. To analyze the results, a threshold line corresponding to a value of 0.03 was set.

For 31/93 samples, the fluorescence curve crossed the threshold line along the channel for the ROX fluorophore; for 46/93 samples, the fluorescence curve crossed the threshold line through the channel for the FAM fluorophore; for 5 samples, the fluorescence curve crossed the threshold line through two channels for the ROX and FAM fluorophores simultaneously. In this case, the kinetics of accumulation of fluorescent signals was exponential.

The presence of HHV-6A DNA was determined using the channel for ROX fluorophores; the presence of HHV-6B DNA was determined using the FAM fluorophore channel; the presence of signals through two channels for the fluorophores ROX and FAM simultaneously confirmed the presence of DNA of two types of human herpes virus - HHV-6A and HHV- 6B. The results of the study were confirmed using the method of direct sequencing of nucleotide sequences (Sanger), which was performed on an ABI 3500xL analyzer (Applied Biosystems, USA) in accordance with the manufacturer’s instructions. Alignment and analysis of the obtained sequences was carried out using the AlignX program (Thermo Fisher Scientific, USA).

For 11/93 samples, the fluorescence curve did not cross the threshold line, which indicated that the sample did not contain samples containing HHV-6A DNA or HHV-6B DNA. The belonging of these 11/93 samples to other types of human herpes virus was confirmed using the method of direct sequencing of nucleotide sequences (Sanger), which was performed on an ABI 3500xL analyzer (Applied Biosystems, USA) in accordance with the manufacturer’s instructions. Alignment and analysis of the obtained sequences was carried out using the AlignX program (Thermo Fisher Scientific, USA). No discordant results were found.

Thus, the claimed invention allows, with 100% specificity, to carry out species differentiation of human herpes virus 6A and human herpes virus 6B) in samples of biological material using RT-PCR; the use of a set of synthesized unique oligonucleotide primers and probes SEQ ID NO: 1-4 eliminates the possibility of cross-linking reactions with other types of human herpes virus allow an unambiguous interpretation of the results obtained.

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<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..27</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier>

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>HHV-6B</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tgtctactcgcccatacaaccttagct</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (9)

1. A set of oligonucleotide primers and probes for species differentiation of human herpes virus 6A and human herpes virus 6B, having the following nucleotide composition: forward primer HHV-6A/B-for – SEQ ID NO: 1; reverse primer HHV-6A/B-rev – SEQ ID NO: 2; fluorescent probe HHV-6A-probe – SEQ ID NO: 3, fluorescent probe HHV-6B-probe – SEQ ID NO: 4. 2. A set of oligonucleotide primers and probes according to claim 1, where a fragment of the U31 Human betaherpesvirus 6A gene and a fragment of the U31 Human betaherpesvirus 6B gene are used to create forward and reverse primers and fluorescent probes . 3. Method of using a set of oligonucleotide primers and probes according to claim 1, including extracting DNA from biological material and performing RT-PCR using a set of oligonucleotide primers and probes according to claim 1, amplification, analysis and interpretation of results, where the presence of HHV-6A or HHV-6B is judged by evaluating the accumulation curves of fluorescent signals through the corresponding channels for the fluorophores. 4. The method of using a set of oligonucleotide primers and probes according to claim 3, in which the presence of HHV-6A DNA and HHV-6B DNA in the biological material sample is determined by assessing the accumulation curves of fluorescent signals in two channels simultaneously. 5. A method of using a set of oligonucleotide primers and probes according to claim 3 or 4, where a threshold line corresponding to a value of 0.03 is set to analyze the results.