RU2782428C1 - Multiparametric diagnostic test system for quantitative determination of mrna level of human rig-1, ifit-1, ifih-1 genes - Google Patents

Abstract

FIELD: medicine and biochemistry.

SUBSTANCE: invention relates to medicine and biochemistry, in particular to molecular diagnostics, namely to a multi-parameter diagnostic test system, and can be used to quantify mRNA levels of human RIG-1, IFIT-1, IFIH-1 genes in a biological sample. A multiparametric diagnostic test system for the quantitative determination of the mRNA level of the RIG-1, IFIT-1, and IFIH-1 genes by reverse transcription polymerase chain reaction has been proposed. The test system includes a set of oligonucleotide primers and fluorescent probes.

EFFECT: test system provides the ability to quickly and accurately determine the mRNA level of human RIG-1, IFIT-1, IFIH-1 genes in a biological sample.

2 cl, 2 tbl, 9 dwg

Description

The invention relates to medicine and biochemistry, in particular to molecular diagnostics, namely to a multi-parameter diagnostic test system, and can be used to quantify mRNA levels of human RIG-1, IFIT-1, IFIH-1 genes in a biological sample.

The first protective barrier against a viral infection is the skin and mucous membranes of the host organism. At the first stages, in response to the penetration of a viral infection, an emergency non-specific immune response is activated. There are various ways for a cell to recognize viral particles. Toll-like receptors (TLRs) are considered to be of the greatest importance. When a virus enters the cytoplasm, RLR, NLR, and cytosolic nucleic acid sensors come into play [1]. Signal transmission through TLR requires the involvement of protein adapters: MyD88 (Myeloid differentiation primary response gene 88), Mal (MyD88 adapter-like), TRIF (TIR-domain-containing adapter inducing IFNβ), and TRAM (Trif-related adapter molecule) [2] . Activation of molecular adapters is aimed at changing the activity of transcription factors (Nuclear factor κB (NFκB), IFN-regulatory factor 3 (IRF3), IFN-regulatory factor 7 (IRF7)) and activation of MAPK-dependent signaling pathways [1, 3].

The cell is capable of a TLR-independent response to pathogen entry mediated through cytosolic sensors. The most important links are RNA helicases belonging to the RLR family: Retinoic acid-inducible gene 1 (RIG-I), Melanoma differentiation-associated gene 5 (MDA-5 or IFIH-1), and Laboratory of genetics and physiology-2 (LGP- 2). The RIG-I and MDA-5 RNA sensors consist of the N-terminal CARD (Сaspase activation and recruitment domain) domain, which has the ATPase activity of the DExD/H box domain, and the C-terminal repressor domain. Under normal conditions, RIG-I is inactive due to the inhibitory activity of its regulatory domain. On the contrary, when a virus enters a cell, RIG-I undergoes conformational changes that induce multimerization of this RNA sensor [1]. The activated multimeric form of RIG-I or MDA-5 interacts with the Mitochondrial antiviral signaling protein (MAVS) adapter located on the mitochondrial membrane envelope or in peroxisomes [1]. Activation of MAVS-dependent signaling induces the activation of transcription factors IRF-1, IRF-3, IRF-7. It is known that these transcription factors are necessary for the production of interferons of the 1st and 3rd types, and, consequently, for the induction of the expression of hundreds of antiviral ISGs (Interferon-stimulated genes) [3].

RIG-I or MDA-5 RNA sensors play a key role in the antiviral protection of various cell types, including fibroblasts, epithelial cells, and dendritic cells [1]. Viral double-stranded RNA activates both RIG-I and MDA-5, while single-stranded RNA molecules with a 5'-triphosphate end without a cap structure can also act as activators of the first RNA helicase (RNA of this type is present in the genomes of many viruses ). It is important to note that RIG-I is unable to recognize single-stranded capped RNA that is produced by the cell. The MDA-5 RNA sensor distinguishes viral RNA from cellular RNA by the length of the molecule. Usually, long double-stranded RNAs are not characteristic of a healthy cell and are MDA-5 ligands [1]. It has been shown that these RNA sensors are necessary for cell recognition of such dangerous pathogens as rabies virus, measles virus, influenza A and B viruses, respiratory syncytial virus, hepatitis C virus, Ebola hemorrhagic fever, mengovirus. All these viruses are capable of producing long double-stranded RNA, which is the ligand for these RNA sensors.

There is a relationship between the RIG-1, IFIT-1 and IFIH-1 genes. Interferons, performing an autocrine or paracrine effect on cells, activate JAK/STAT-dependent signaling cascades and induce the expression of hundreds of ISGs. One of the most important ISGs is IFIT-1 (ISG-56). IFIT-1 is a gene with pronounced antiviral activity. It has been shown that IFIT-1 inhibits the replication of RNA and DNA viruses. Moreover, IFIT-1 silencing leads to increased HCV replication. On the other hand, IFIT-1 is involved in the induction of cell apoptosis. Thus, it was shown that IFIT-1 gene silencing was associated with a lower level of apoptosis in A549 cells treated with 5'-PPP-ssRNA, a synthetic RIG-1 agonist. Therefore, cell apoptosis associated with the action of type 1 interferons may be associated with overexpression of the IFIT-1 gene. Therefore, measurement of the IFIT-1 gene expression level can be considered not only as a marker of innate immune response activation, but also as a marker of type 1 interferon-associated apoptosis [4].

The joint determination of the level of expression of the RIG-1, IFIT-1, and IFIH-1 genes makes it possible to determine the level of activation of the innate immune response of the body and evaluate the activation of proapoptotic signaling cascades. This assessment is of particular relevance in the analysis of the pathogenesis of acute respiratory viral infections caused by influenza viruses, pneumoviruses, adenoviruses, and coronaviruses. Thus, a low level of expression of the RNA sensors MDA-5 and RIG-1 may indicate a weak activation of the innate interferon-mediated immune response to a respiratory infection, while overexpression of IFIT-1 may not only be a signal of the activation of the interferon defense system, but and a marker of possible cell death. The most sensitive and specific method for determining the level of gene expression is real-time RT-PCR. This technique assumes the presence of two primers and a fluorescently labeled oligonucleotide probe specific for the mRNA of interest.

There is a method for identifying genes, expression regulators, receptors, receptors for protein products and proteins that can regulate rhinovirus infections [5], including: a) contacting at least one compound with a target selected from certain genes (the group includes RIG-1, IFIT -1 and IFIH-1), proteins encoded by these genes, gene expression regulators, encoded protein receptors, gene encoded protein products, gene protein product receptors, and combinations thereof; b) determining if said compound binds to a target; and c) identifying those compounds that bind the target as compounds for regulating rhinovirus infection. However, this invention also relates to methods for identifying therapeutic compounds that can treat various disorders by regulating the expression and activity of genes, expression regulators, receptors, protein product receptors and identified proteins.

However, there are currently no known test systems that can be used to simultaneously determine the expression of the RIG-1, IFIT-1, and IFIH-1 genes by real-time RT-PCR. Thus, the relevance of the proposed invention is due to the need to determine the level of expression of RNA sensors and the IFIT-1 gene for a deeper understanding of the pathogenesis of acute respiratory viral infections and to determine further strategies for their treatment, to determine the level of activation of the interferon response and the risk of cell death.

The technical problem lies in the need to develop a test system and method for quantitative determination of the mRNA level of human RIG-1, IFIT-1, IFIH-1 genes, as well as the need to expand the range of respiratory disease diagnostic tools at early stages.

The technical result consists in providing the ability to quickly and accurately determine the level of mRNA of the human RIG-1, IFIT-1, IFIH-1 genes.

Human RIG-1, IFIT-1, and IFIH-1 sequences deposited in the GenBank database (NCBI) were used to select primers and oligonucleotide probes. The analyzed genes are localized on different chromosomes; the lengths of mature transcripts range from 600 to 4700 nucleotides. mRNA of the RIG-1 gene (DDX58) is represented by seven, of the IFIT-1 gene by five; and the IFIH-1 (MDA-5) gene by one transcription variant.

The original primer and TaqMan probe sequences were matched to conserved protein coding regions and should potentially reveal all transcriptional variants of the claimed genes. The selection of the required sequences was carried out based on the desired melting temperatures of the primers of 60°C and above, and of the oligonucleotide probe above 63°C, calculated at the selected salt concentration of 75 mM. Guided by rational design criteria, primers were selected so that they were separated by at least one intron per gene sequence and were predominantly located at the junction of exons. This approach makes it possible to distinguish the genomic PCR product from the PCR product obtained from cDNA and avoid additional DNase processing.

Sequences designed to detect human RIG-1, IFIT-1, and IFIH-1 mRNAs were analyzed by BLAST for their possible spectrum of non-specific hybridization. No sequences highly homologous to any other genes of this species have been identified.

The technical result is achieved by the fact that the multi-parameter diagnostic test system for quantitative determination of the mRNA level of the RIG-1, IFIT-1, IFIH-1 genes by reverse transcription polymerase chain reaction includes a set of oligonucleotide primers and fluorescent probes with the following structure:

forward primer of RIG-1 gene GAGCACTTGTGGACGCTTTA

reverse primer of RIG-1 gene ATACACTTCTGTGCCGGGAG

fluorescent probe of the RIG-1 gene Rox-CCTGGCATATTGACTGGACGTGGC-BHQ2

forward primer of the IFIT-1 gene AAACTTCGGAGAAAGGCATTAGAT

reverse primer of the IFIT-1 gene TGAAATGAAATGTGAAAGTGGCTG

IFIT-1 gene fluorescent probe Hex-CCTGAGACTGGCTGCTGACTTTGAGAAC-BHQ1

forward primer of the IFIH-1 gene AAACCCATGACACAGAATGAACA

reverse primer of the IFIH-1 gene TGTGAGCAACCAGGACGTAG

IFIH-1 gene fluorescent probe Cy5.5-CACAGTGGCAGAAGAAGGTCTGGA-BHQ3

The forward primer of the RIG-1 gene is shown in SEQ NO:1; the reverse primer of the RIG-1 gene is shown in SEQ NO:2; a fluorescent probe for the RIG-1 gene is shown in SEQ NO:3; the forward primer of the IFIT-1 gene is shown in SEQ NO:4; the reverse primer of the IFIT-1 gene is shown in SEQ NO:5; a fluorescent probe for the IFIT-1 gene is shown in SEQ NO:6; the forward primer of the IFIH-1 gene is shown in SEQ NO:7; the reverse primer of the IFIH-1 gene is shown in SEQ NO:8; a fluorescent probe for the IFIH-1 gene is shown in SEQ NO:9.

With the help of this test system, the level of mRNA of the human RIG-1, IFIT-1, IFIH-1 genes is quantified, during which a reverse transcription polymerase chain reaction is carried out, and the primary denaturation is carried out for 5 minutes at a temperature of 95 ° C, then 40 cycles are carried out, consisting of two stages: denaturation for 10 s at a temperature of 95°C, then primer annealing and chain elongation for 30 s at a temperature of 61.3°C.

Description of figures

Figure 1 shows the analysis by electrophoretic separation in agarose gel of PCR products obtained at the corresponding annealing temperatures (indicated above in °C): A - PCR products for quantitative detection of mRNA of the RIG-1 gene; B – PCR products for quantitative detection of mRNA of the IFIT-1 gene; C – PCR products for quantitative detection of mRNA of the IFIH-1 gene; (d) PCR products for quantitative detection of mRNA of the HPRT-1 gene.

In FIG. Figure 2 shows the calculation of the efficiency of the PCR reaction for the quantitative detection of RIG-1 gene mRNA, carried out in the monoplex mode: A – fluorescence growth curves obtained for a range of sample dilutions (from –10 to –2) in real-time fluorescence analysis on the ROX channel; B – curve constructed from the obtained values of Cq, used to calculate the reaction efficiency.

In FIG. Figure 3 shows the Calculation of the efficiency of the PCR reaction for the quantitative detection of the IFIT-1 gene mRNA, carried out in the monoplex mode: A - fluorescence growth curves obtained for a line of sample dilutions (from –10 to –2) in the analysis of fluorescence on the ROX channel in real time; B – curve constructed from the obtained values of Cq, used to calculate the reaction efficiency.

In FIG. Figure 4 shows the calculation of the efficiency of the PCR reaction for the quantitative detection of IFIH-1 mRNA, carried out in monoplex mode: A - fluorescence growth curves obtained for a line of sample dilutions (from –10 to –2) when analyzing fluorescence on the Cy5.5 channel in real mode time; B – curve constructed from the obtained values of Cq, used to calculate the reaction efficiency.

In FIG. Figure 5 shows the analysis by capillary electrophoresis using the Bioanalyzer 2100 system (Agilent) of PCR products obtained under optimal conditions in multiplex and monoplex modes: A - electrophoregram of PCR products: Sample 1–3 contain monoplex PCR products for RIG-1, IFIT-1 genes and IFIH-1, respectively, Sample 4 – multiplex PCR products for these genes with their simultaneous amplification in one tube, obtained from a sample isolated from A549 cells, Sample 5 – a mixture of monoplex PCR products obtained by mixing Samples 1–3, Sample 6 – products of multiplex PCR for RIG-1, IFIT-1 and IFIH-1 genes with their simultaneous amplification in one tube, obtained from a sample isolated from peripheral blood mononuclear cells; B — spectrophoregram reflecting the overlay of PCR results in multiplex mode (Sample 4 containing all three specific products RIG-1, IFIT-1 and IFIH-1) and monoplex modes, Sample 1 contains a specific product of the RIG-1 gene, Sample 2 and Sample-3 are products of the IFIT-1 and IFIH-1 genes, respectively.

In FIG. Figure 6 shows a comparison of the efficiencies of PCR reactions in monoplex and multiplex formats: A - shows standard calibration curves used to calculate the amplification efficiencies of RIG-1, IFIT-1 and IFIH-1 in multiplex format (simultaneous detection in one tube); B – standard calibration curve obtained in the multiplex PCR format (blue dots) compared to the calibration curve in monoplex setting (orange dots) for the RIG-1 gene; C – standard calibration curve obtained in multiplex PCR format (blue dots) compared to the calibration curve in monoplex setting (orange dots) for the IFIH-1 gene; D – standard calibration curve obtained in multiplex PCR format (blue dots) compared to the calibration curve in monoplex setting (orange dots) for the IFIT-1 gene.

In FIG. Figure 7 shows the amplification specificity of the required products using various PCR matrices: cDNA - complementary DNA obtained from the mRNA preparation by reverse transcription; RNA - preparation of total RNA isolated from stimulated A549 cells, water - used as a negative PCR control. The signatures of the RIG-1, IFIT-1 and IFIH-1 genes mean that the reaction was performed in a monoplex format for the corresponding genes, MIX is a multiplex PCR format.

In FIG. Figure 8 shows the determination of the expression level of the RIG-1, IFIT-1, IFIH-1 genes using the developed multiplex PCR in A549 cell culture stimulated with low molecular weight (LMW) and high molecular weight (HMW) poly(I:C) dsRNA. Delivery of LMW and HMW into cells was carried out using the Lipofectamine MessengerMAX reagent, the designation KK_LP is the control of cells stimulated only with the Lipofectamine MessengerMAX reagent (without dsRNA), KK is the intact control of cells. The significance of differences was determined using the ANOVA test (analysis of variance) with multiple comparisons. **** - P < 0.0001.

In FIG. Figure 9 shows the determination of the level of expression of the RIG-1, IFIT-1, IFIH-1 genes in peripheral blood mononuclear cells of healthy volunteers and patients with influenza and new coronavirus infection. Significance of differences was determined using the non-parametric Kruskal-Wallis test with multiple comparisons. * – P < 0.05; ** - P < 0.01.

Primers and oligonucleotide probes were selected to determine the expression level of the RIG-1, IFIT-1, and IFIH-1 genes (Table 1).

The human RIG-1 sensor RNA is represented in the database by 7 transcriptional variants. Therefore, multiple alignment of these variants was carried out, and a pair of primers and an oligonucleotide probe were matched to the identical site.

The IFIT-1 gene is represented by 5 transcriptional variants. Therefore, multiple alignment of these variants was carried out, and a pair of primers and an oligonucleotide probe were matched to the identical site.

The MDA-5 (IFIH-1) sensor RNA is represented by only one transcription variant.

The HPRT-1 gene was chosen as a reference gene suitable for real-time RT-PCR when cells were infected with respiratory infections.

Table 1 : Matched primers and TaqMan probes for RIG-1, IFIT-1, IFIH-1 expression

GENE mRNA SEQUENCE NAME SELECTED PRIMERS (5'-3') PCR product length A RIG-1 NM_014314.4 hRIG-1_F GAGCACTTGTGGACGCTTTA 175 hRIG-1_R ATACACTTCTGTGCCGGGAG hRIG-1_O ROX-CCTGGCATATTGACTGGACGTGGC-BHQ2 IFIT-1 NM_001548.5 hIFIT-1_F AAACTTCGGAGAAAGGCATTAGAT 187 hIFIT-1_R TGAAATGAAATGTGAAAGTGGCTG hIFIT-1_O HEX-CCTGAGACTGGCTGCTGACTTTGAGAAC-BHQ1 IFIH-1 NM_022168.4 hIFIH-1_F AAACCCATGACACAGAATGAACA 216 hIFIH-1_R TGTGAGCAACCAGGACGTAG hIFIH-1_O Cy5.5-CACAGTGGCAGAAGAAGGTCTGGA-BHQ3 B HPRT-1 NM_000194.3 hHPRT_F CCTGGCGTCGTGATTAGTG 228 hHPRT_R TGTAATCCAGCAGGTCAGCA hHPRT_O FAM-AGGCCATCACATTGTAGCCCTCTGTGT-BHQ1

Selection and optimization of PCR conditions

At this stage of the work, we experimentally evaluated the quality of the selected pairs of primers and probes, as well as the possibility of their formation of hetero- and homodimers and nonspecific products. The material for this study was cDNA samples obtained from total RNA preparations isolated from A549 cells infected with influenza A virus. The work was carried out using the PCR kit HS 2× PCR mix (Biolabmix, Russia). 5 pmol of primers and probes (0.5 µl from the stock of primers and probes with concentrations of 10 pmol/µl) were added to the reaction with a volume of 25 µl.

To determine the optimal thermal profile, the following PCR was performed with an annealing gradient:

1) primary denaturation 95°C - 5 min,

followed by 40 three-stage cycles:

2) denaturation 95°С – 10 s;

3) primer annealing at temperatures ranging from 57.5°C to 63.5°C - 30 s (the exact temperatures were 63.5°, 63.2°, 62.5°, 61.3°, 59.9 °, 58.7°, 57.9°, 57.5°C);

4) elongation 72°С – 30 s;

The detection of the results was carried out by the growth of fluorescence, the presence of nonspecific products was assessed by electrophoretic separation of the products in an agarose gel. On FIG. 1 shows the results of electrophoretic separation of PCR products for RIG-1 (Fig. 1 A), IFIT-1 (Fig. 1 B), IFIH-1 (Fig. 1 C) and for the HPRT1 gene (Fig. 1 D), proposed in as an endogenous control, when carrying out the reaction at appropriate annealing temperatures. Electrophoresis was performed in 1% agarose in 1x TBE buffer and stained with ethidium bromide.

Taking into account the results obtained, it was proposed to use the following as the optimal temperature profile for PCR:

1) primary denaturation 95°C - 5 min,

further 40 two-stage cycles:

2) denaturation 95°С – 10 s;

3) primer annealing and chain elongation 61.3°С – 30 s.

Calculation of PCR efficiencies

For a correct relative quantitative analysis of the expression of RIG-1 genes of similar receptors in the multiplex format, the values of the amplification efficiencies of the corresponding genes should be identical or as close as possible to each other. Therefore, further, under the selected optimal conditions, we calculated the efficiency of PCR.

The efficiency of amplification of PCR products was calculated from the slope of the curves obtained by a series of successive tenfold dilutions of cDNA samples.

On FIG. Figure 2 shows the results of calculating the efficiency of PCR when amplifying the RIG-1 gene in a monoplex format. As seen in FIG. 2A, the expression sections of the accumulation curves obtained for the corresponding dilutions of the sample are parallel, the distance between successive dilutions is constant, the slopes of the curves are the same. The calculated PCR efficiency on RIG-1 (FIG. 2B) was 99.81%, which is an almost ideal amplification parameter. Similarly, PCR amplification efficiencies were obtained for IFIT-1 (FIG. 3) and for IFIH-1 (FIG. 4). Thus, it was demonstrated that in the monoplex format, the efficiency of amplification of the products of the three genes RIG-1, IFIT-1 and IFIH-1 was: 99.81%, 99.81%, 99.87%, respectively. Graphically, this is expressed in the fact that the standard calibration curves (shown in Fig. 2–4 B) have similar slope angles, i.e. are parallel.

For multiplex PCR, amplification of all RIG-1, IFIT-1, and IFIH-1 genes was performed simultaneously in one tube. For this, the PCR sample, in addition to the DNA-dependent DNA polymerase enzyme and the buffer attached to it, simultaneously contained all the selected pairs of primers and oligonucleotide probes that specifically detect the claimed genes. The final concentrations of primers and probes contained in the sample during multiplex PCR are presented in Table 2.

Table 2 : Composition of the multiplex reaction

TITLE CONCENTRATION, nM h RIG-1_F 200 h RIG-1_R 200 h RIG-1_O 200 h IFIT-1_F 200 h IFIT-1_R 200 h IFIT-1_O 200 h IFIH_F 750 h IFIH_R 750 h IFIH_O 250

Due to the fact that primers can give nonspecific chimeric products when in the same tube, the analysis of PCR products in multiplex and monoplex modes was carried out by capillary electrophoresis (Fig. 5). As shown in FIG. 5 A, when performing monoplex PCR in test tubes, only products corresponding to the declared genes RIG-1, IFIT-1, IFIH-1 were amplified, with lengths of 175 bp, 187 bp. and 216 b.p. for everybody. When conducting multiplex PCR in a test tube, all three genes with similar lengths were simultaneously detected. The spectrophorogram of the products obtained in the multiplex format, superimposed on the spectrophorograms of the PCR products formed in the monoplex formats, had identical peaks (Fig. 5B). Thus, the multiplexing did not lead to the formation of any unwanted random products.

Further, the efficiency of amplification of each of the declared genes in the multiplex PCR format was calculated. As shown in FIG. 6 A, the amplification efficiencies of all specific products during PCR in one tube are almost identical and exceed 98% (for RIG-1, IFIT-1 and IFIH-1 were: 99.75%, 99.09%, 98.80%, respectively). In addition, primer multiplexing resulted in a decrease in amplification of no more than 1–2% compared to monoplex amplification for each of the specific products (Fig. 6C–D). These results indicate that during multiplex PCR, doubling of specific products occurs in each round.

To assess the appearance of possible nonspecific products due to genomic contamination of complementary cDNA probes, the PCR developed in multiplex and monoplex formats was tested using various matrices (RNA or cDNA). As can be seen from the electrophoretic separation of the respective PCR products shown in FIG. 7, PCR for IFIT-1 gives positive specific products obtained from genomic DNA (RNA probe in FIG. 7). Such results are expected and were predicted at the stage of selection of primer sequences and a probe specifically detecting IFIT-1. However, these results indicate a strong need for pretreatment of the RNA sample tested in PCR with DNases to remove genomic DNA.

Verification of the test system in vitro and on clinical specimens

The developed quantitative multiplex PCR system was used to analyze the expression of the RIG-1, IFIT-1, and IFIH-1 genes in stimulated A549 cell cultures and in peripheral blood mononuclear cells of healthy volunteers and patients with influenza and novel coronavirus infection.

It is known that double-stranded RNA molecules (dsRNA) are a ligand that activates sensory components of the innate immune system. First of all, TLR-3 and cytosolic sensors IFIH-1 (MDA-5) are activated in response to dsRNA penetration into the cell. Long dsRNAs are not characteristic of healthy cells. Usually their presence is associated with the penetration of viral pathogens [1]. The synthetic analogue of viral dsRNA is poly(I:C) dsRNA. This molecule consists of two chains: the first is a polymer of inosinic acid, the second is cytidinic acid. This agonist is used in research practice to induce interferons and ISGs. The introduction of poly(I:C) dsRNA into the cell medium simulates the situation of a viral infection. Two types of synthetic agonists are used: high molecular weight poly(I:C) dsRNA (High molecular weight poly(I:C) dsRNA, HMW) and low molecular weight analog (Low molecular weight poly(I:C) dsRNA, LMW). HMW is 1500 to 8000 base pairs long, while LMW is 200 to 1000 base pairs long.

For the first stage of verification, in our work, we assessed changes in the expression of the RIG-1, IFIT-1, and IFIH-1 genes upon stimulation of A549 HMW and LMW poly(I:C) dsRNA cells. A549 cells are a cell line that is a model of type II alveolar pneumocytes. We have shown that HMW and LMW significantly increase the level of mRNA of the RIG-1, IFIT-1, IFIH-1 genes (Fig. 8), which is reflected in the scientific literature as a well-known fact. Therefore, the test system we are developing is suitable for assessing the activation of interferon-mediated mechanisms of the innate immune system.

At the second stage of verification, the developed multiplex PCR was used to simultaneously measure the expression of the RIG-1, IFIT-1, IFIH-1 genes in peripheral blood mononuclear cells of healthy donors and patients diagnosed with COVID-19 or influenza. Diagnosis of these infections was carried out with PCR kits certified for such studies. As a result of the measurement, it was shown that in patients suffering from respiratory infections (influenza, SARS-CoV-2), there is an increase in the expression level of the cytosolic RNA sensor RIG-1 (Fig. 9). Thus, using a multiplex PCR system, it was shown that RIG-1 is a marker of a viral infection that is nonspecifically activated in response to infection of the body with an influenza or new coronavirus infection. On the contrary, the expression of IFIT-1 and IFIH-1 was increased only in patients infected with the influenza virus. In the case of COVID-19 infection, the level of these markers seems to depend on the severity of the disease.

In general, the use of the developed multiplex PCR test system allows high-efficiency quantitative analysis of the RIG-1, IFIT-1, IFIH-1 genes both in clinical samples and in cell culture.

Bibliography

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3 Randall RE, Goodbourn S. Interferons and viruses: an interplay between induction, signaling, antiviral responses and virus countermeasures// Journal of General Virology . - 2008. - T. 89. - No. 1. - S. 1-47.

4 Yap, GL et al., Annexin-A1 promotes RIG-I-dependent signaling and apoptosis via regulation of the IRF3–IFNAR–STAT1–IFIT1 pathway in A549 lung epithelial cells// Cell death & disease , – 2020. – T. 11. - no. 6. - S. 1-12.

5 Methods and targets for identifying compounds for regulating rhinovirus infection: patent appl. CA2679412A1, Canada, appl. CA2679412A, 02/28/2008, publ. 09/04/2009.

Claims (14)

1. Multiparametric diagnostic test system for quantitative determination of mRNA levels of RIG-1, IFIT-1, IFIH-1 genes by reverse transcription polymerase chain reaction, including a set of oligonucleotide primers and fluorescent probes with the following structure: forward primer of the RIG-1 gene GAGCACTTGTGGACGCTTTA, reverse primer of RIG-1 gene ATACACTTCTGTGCCGGGAG, fluorescent probe for the RIG-1 gene Rox-CCTGGCATATTGACTGGACGTGGC-BHQ2, direct primer of the IFIT-1 gene AAACTTCGGAGAAAGGCATTAGAT, reverse primer of the IFIT-1 gene TGAAATGAAATGTGAAAGTGGCTG, fluorescent probe for the IFIT-1 gene Hex-CCTGAGACTGGCTGCTGACTTTGAGAAC-BHQ1, direct primer of the IFIH-1 gene AAACCCATGACACAGAATGAACA, reverse primer of the IFIH-1 gene TGTGAGCAACCAGGACGTAG, IFIH-1 gene fluorescent probe Cy5.5-CACAGTGGCAGAAGAAGGTCTGGA-BHQ3. 2. The test system according to claim 1, characterized in that the optimal concentration of primers and probes is 200 nM for primers and a probe for the RIG-1 gene, 200 nM for primers and a probe for the IFIT-1 gene, 750 nM for primers for the gene IFIH-1, 250 nM for IFIH-1 gene probe.

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