RU2460803C2 - Differential diagnostic technique for respiratory viral infections by real-time multiplex pcr and sequence list for implementation thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is described is a differential diagnostic technique for respiratory viral infections by multiplex reverse transcription and real-time PCR. The technique provides analysis of each test sample for the presence of nucleic acids of 11 respiratory viruses in 3 reaction mixtures. The technique enables differential detection of nucleic acids of primary ARVI agents - influenza A and B viruses, coronaviruses, type 1, 2, 3, 4 parainfluenza viruses, adenoviruses, respiratory syncytial virus, rhinoviruses and enteroviruses in biological samples. The presence of nucleic acid of any respiratory virus in the test sample is determined by an increasing fluorescent signal of specific dye in one of the reaction mixture.

EFFECT: invention may be used in preventative disinfection departments for the purpose of epidemiologic situation monitoring and fast interpretation of ARVI breakouts.

6 cl, 4 tbl, 6 ex

Description

The invention relates to the field of laboratory diagnostics, medical virology, molecular biology and epidemiology. The invention is intended to identify and identify in clinical samples 11 groups of respiratory viruses (influenza A and B viruses (HAV and HBV), coronaviruses (HF), parainfluenza viruses 1, 2, 3, 4 types (HSV 1, 2, 3, 4) , adenoviruses (ADV), respiratory syncytial virus (RSV), rhinoviruses (RV) and enteroviruses (EV) in the presence of internal positive control (MIC) using real-time multiplex PCR, which allows to improve the PCR analysis technique for for the diagnosis of acute respiratory viral infections facies (ARVI) and can be used in epidemiological services to monitor the epidemiological situation and quickly decipher ARVI outbreaks.

State of the art

SARS are a ubiquitous disease that over the years has outnumbered all other infectious diseases combined. SARS is characterized by a very wide variety of pathogens that can cause diseases with similar symptoms. The main contribution to the structure of acute respiratory viral infections is made by influenza viruses, parainfluenza, respiratory syncytial virus, coronaviruses, adenoviruses, rhinoviruses and some enteroviruses. Respiratory viral infections can be accurately identified only with laboratory diagnostic methods. A wide variety of respiratory viruses makes etiological diagnosis extremely difficult. Traditional methods for identifying respiratory viruses have disadvantages, primarily associated with insufficient sensitivity (immunofluorescence methods), long duration, laboriousness, non-universal procedure (culture method). The detection of antibodies to viruses in the serum of patients using solid-phase ELISA is a retrospective method (it does not detect the pathogen itself, but records the body's response to infection). ELISA detection of viral antigens in clinical samples is limited by insufficient sensitivity. The use of immunochemical and serological methods also complicates the high antigenic diversity of some groups of respiratory viruses - adenoviruses, rhinoviruses, enteroviruses, etc. Traditional methods, due to the limitations listed above, do not allow the simultaneous detection of the main groups of respiratory viruses in clinical samples with high sensitivity.

The solution to this problem in recent years has been associated with the development of molecular methods for the amplification of nucleic acids (MANK), the specificity of which is based on the uniqueness of the nucleotide sequences of the viral genomes. IASCs include various modifications of the polymerase chain reaction (PCR), including real-time detection PCR (PCR-RV). The introduction of the PCR method in laboratory practice has become one of the most important events in clinical laboratory diagnostics. The advantages of the PCR method are high specificity, sensitivity, universality of the procedure, simplicity and convenience of analysis, process automation, the ability to identify several pathogens in one tube at once, provided that several pairs of corresponding primers are present in the reaction mixture (multiplex PCR). The multiplex PCR-RV format reduces the complexity, cost and duration of the analysis.

All MANK include three stages:

- sample preparation, usually consisting in the isolation and concentration of nucleic acids (NK), as well as in the release of inhibitors present in the sample;

- enzymatic amplification of NK;

- identification of amplification products.

Scientific publications are known that describe the use of PCR for the detection and identification of respiratory viruses in biological samples (J Med Virol. - 2004. - No. 72. - P. 484-495; J Clin Microbiol. - 2007. - No. 45. - P .2965-2670; J Clin Microbiol. - 2004. - No. 42. - P.1564-1569, J Clin Virol. - 2008, No. 41, P.53-56), as well as a number of patents (US No. 6881835 B2, No. 0229211 A1, No. 0014140 A1, No. 0172835 A1, No. 0233314 A1, WO 2007/058499 A1, WO 2008/042450 A2). The listed publications and patents describe various PCR formats that are used for the diagnosis of acute respiratory viral infections, including multiplex PCR with agarose gel detection, hybrid enzyme analysis and PCR-RV. The closest analogs are PCR test systems presented in Templeton K. et al. (J Clin Microbiol. - 2004. - No. 42. - P.1564-1569) and WO 2008/042450 A2. The features distinguishing the present invention from the listed analogues are:

- nucleotide sequences of primers and probes listed in the sequence listing;

- the possibility of differential diagnosis of rhinovirus and enterovirus infection;

- the use of internal positive control, represented by an RNA-containing virus, which is added directly to the clinical sample before isolation of nucleic acids and allows you to control the efficiency of NK isolation and the presence of inhibitors.

An object of the invention is to provide a method for identifying respiratory viruses using multiplex PCR analysis with the detection of amplification products in real time. This approach allows the simultaneous detection of nucleic acids of 11 groups of respiratory viruses in biological samples without resorting to the use of additional methods for detecting amplicons (DNA electrophoresis in agarose gel, DNA microarrays), thereby reducing the risk of contamination with amplification products, the percentage of technological errors. The multiplex PCR-RV format reduces the complexity, cost and duration of the analysis.

1. This problem is achieved due to the fact that the method of differential diagnosis of respiratory viral infections, characterized by the detection in clinical samples of nucleic acids of the main pathogens of respiratory viral infections of a person - influenza viruses A and B, coronaviruses, parainfluenza viruses of types 1, 2, 3, 4, adenoviruses, respiratory syncytial virus, rhinoviruses and enteroviruses in the presence of internal positive control, providing for the implementation of a multiplex reverse transcription reaction (RT) and P Real-time R amplifications with real-time detection, and clinical samples of choice are: nasal swabs, throats, nasopharynx, nasopharyngeal aspirates, nasal swabs, sputum, bronchoalveolar lavage, sectional material, culture fluid samples, characterized in that for its implementation using oligonucleotides - SEQ ID NO 1-45, on the basis of which prepare a mixture of primers, probes for PCR-PB; then, using selected primers and probes, reaction mixtures for multiplex RT and PCR are formed, in which fluorescently labeled probes are distributed among the reaction mixtures so that probes labeled with different dyes are in the same mixture; then carry out a multiplex reverse transcription reaction and multiplex PCR-RV (each sample is analyzed in three reaction mixtures); the presence of a particular respiratory virus in the studied sample of nucleic acids is determined by the growth of the fluorescence signal of a specific dye in one of the reaction mixtures; moreover, during the analysis, a number of controls are carried out to exclude false positive and false negative results, for which form pools of positive control samples (FFP), which, along with the internal positive control (MIC), are analyzed in the analysis of each series of samples, and laboratory strains are included in the pool of positive controls respiratory viruses, diluted and mixed in a certain concentration so that in multiplex PCR-PB they are determined on the 31-33 cycle; in the case when during the PCR-RV formulation there is a delay in the values of the threshold cycles of the MIC, this indicates the possible presence of inhibitors in the sample that reduced the sensitivity of the analysis, or errors in the isolation of RNA or formulation of the reaction; such a sample is re-analyzed. In addition, to exclude false positive results, along with all samples, a negative control sample (OKO) is analyzed, using distilled water as the one. In addition, at the PCR stage with real-time detection, the following oligonucleotides (distributed over three reaction mixtures) are used: mixture No. 1: SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17; mixture No. 2: SEQ ID NO 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33; mixture No. 3: SEQ ID NO 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45. In addition, each virus-specific set of primers and probes can be used to identify the corresponding nucleic acid samples viruses in a monospecific format, including for the detection of adenoviruses - SEQ ID NO 1, 2, 3; coronaviruses SEQ ID NO 4, 5, 6, 7, 8, 9, 10, 11; enteroviruses - SEQ ID NO 12, 13, 14; Influenza B virus - SEQ ID NO 18, 19, 20; respiratory syncytial virus - SEQ ID NO 21, 22, 23; influenza A viruses - SEQ ID NO 24, 25, 26; rhinoviruses - SEQ ID NO 27, 28, 29, 30, 31, 32, 33; Parainfluenza virus type 1 - SEQ ID NO 34, 35, 36; Parainfluenza virus type 2 - SEQ ID NO 37, 38, 39; parainfluenza virus type 3 - SEQ ID NO 40, 41, 42; parainfluenza virus type 4 - SEQ ID NO 43, 44, 45. In addition, the analysis provides for the identification of the MIC, represented by an RNA-containing virus in order to control the efficiency and quality of nucleic acid excretion, including the presence of reverse transcription and PCR inhibitors in the sample. In addition, when analyzing samples in reaction mixtures No. 1 and No. 2, differential identification of rhinoviruses and enteroviruses is carried out.

The method allows to differentially detect nucleic acids in biological samples of the main pathogens of ARVI - influenza A and B viruses, coronaviruses, parainfluenza viruses 1, 2, 3, 4 types, adenoviruses, respiratory syncytial virus, rhinoviruses and enteroviruses. The presence of a particular respiratory virus in the studied sample of nucleic acids is determined by the growth of the fluorescence signal of a certain dye in one of the reaction mixtures.

The method can be implemented based on the following sequence listing.

Nucleotide sequences of primers and probes for multiplex PCR-PB.

The problem was solved by selecting 12 sets of virus-specific primers (specific to 11 respiratory viruses and MIC) for the stages of analysis of RT and PCR-RV, as well as the formation of optimal reaction mixtures for analysis. The choice of primers for PCR-RV was limited by several criteria, such as specificity for the genomic sequences of the viruses of the corresponding group, high PCR efficiency, melting point 55-60 ° C, G / C ratio in the range from 40 to 65%, size of PCR products up to 250 bp

Sequences of 12 hybridization probes were also targeted to the region between the primers. The 5'-terminal nucleotide of each probe is modified with one of 4 fluorescent dyes (FAM, ROX, R6G, Cy5 or similar), and the 3'-end with one of the fluorescence quenchers (BHQ1, BHQ2, BHQ3 or similar). The "flare-up" of a fluorescent signal during the accumulation of a specific amplicon during PCR-RV occurs according to the principle of cleavage of the 5'-end probe label. Primers and probes were sent to the following sections of the viral genomes: HAV and HBV to the M gene; KB - to the NSP13 gene; RSV - to the polymerase L gene; EV and PB - to the 5'-untranslated region of the genome (NTR); ADV - to the hexon gene; HSV 1-3 - to the HN hemagglutinin ruminidase gene; HSV4 - to the phosphoprotein gene. Primers and probes are synthesized by Syntol (Russia) or another company specializing in the synthesis of oligonucleotides and fluorescently-labeled probes for PCR-PB.

The sequences of primers and probes for PCR-PB, the corresponding calculated sizes of amplification products, viruses and target genes detected with their help are presented in the sequence listing and in Table 1.

Table 1

Characterization of oligonucleotides for multiplex PCR-PB.

Title The size of the PCR product, bp Detectable virus
target gene SEQ ID NO 1 159 Adenoviruses,
hexon gene SEQ ID NO 2 SEQ ID NO 3 SEQ ID NO 4 94-103 Coronaviruses SEQ ID NO 5 NSP13 gene SEQ ID NO 6 SEQ ID NO 7 SEQ ID NO 8 SEQ ID NO 9 SEQ ID NO 10 SEQ ID NO 11 SEQ ID NO 12 162 Enteroviruses
5'NTR SEQ ID NO 13 SEQ ID NO 14 SEQ ID NO 15 119 Defense industry SEQ ID NO 16 SEQ ID NO 17 SEQ ID NO 18 146 Flu virus b
M-gene (7 segment) SEQ ID NO 19 SEQ ID NO 20 SEQ ID NO 21 120 Respiratory syncytial virus,
L gene SEQ ID NO 22 SEQ ID NO 23 SEQ ID NO 24 227 Influenza A virus
M-gene (7 segment) SEQ ID NO 25 SEQ ID NO 26 SEQ ID NO 27 204 Rhinoviruses
5'NTR SEQ ID NO 28 SEQ ID NO 29 SEQ ID NO 30 SEQ ID NO 31 SEQ ID NO 32 SEQ ID NO 33 SEQ ID NO 34 162 Parainfluenza virus type 1,
HN gene SEQ ID NO 35 SEQ ID NO 36 SEQ ID NO 37 241 Parainfluenza virus type 2,
HN gene SEQ ID NO 38 SEQ ID NO 39 SEQ ID NO 40 143 Parainfluenza virus type 3,
HN gene SEQ ID NO 41 SEQ ID NO 42 SEQ ID NO 43 199 Parainfluenza virus type 4,
phosphoprotein gene SEQ ID NO 44 SEQ ID NO 45

Using selected primers and probes, 3 reaction mixtures for multiplex RT and PCR were formed, which include the optimal combinations of primers and probes: mixture No. 1 - for the detection of ADV, KV, EV, VPK; mixture No. 2 - HAV, HBV, RV, RSV; mixture No. 3 - HSV 1, 2, 3, 4 (table 2). Fluorescently labeled probes are distributed over the reaction mixtures so that the probes labeled with different dyes are in the same mixture.

table 2

Distribution of virus-specific primers and probes for reaction mixtures in multiplex PCR-RV.

Dye Reaction mixture No. 1 Number 2 Number 3 Fam ADV HBV HSV1 Rox HF RSV HSV2 R6g EV CAA HSV3 Cy5 Defense industry RV HSV4

Since all of the listed viruses (except adenoviruses) are RNA-containing, the stage of reverse transcription (RT) is necessary to detect them. As primers for conducting a multiplex reverse transcription reaction, oligonucleotides complementary to a fragment of the viral genome, which are either part of the corresponding primer for PCR-PB, or oligonucleotides complementary to the region of the viral genome outside the amplification region, are used. RT primers are characterized by a melting point below 50 ° C and primary structure features that reduce the likelihood of nonspecific annealing of RT primers on the RNA matrix and PCR-PB primers on the cDNA matrix, thereby increasing the amplification efficiency in samples with a high content of non-specific NC .

The use of the developed multiplex PCR-RV implies that for the analysis of one sample for the presence of 11 respiratory viruses, it must be analyzed in 3 reaction mixtures (Table 2). The presence of a particular respiratory virus in the studied sample of nucleic acids is determined by the growth of the fluorescence signal of a certain dye in one of the reaction mixtures.

During the analysis, a number of controls are provided to exclude false positive and false negative results. To do this, pools of positive control samples (PKO-1, PKO-2, PKO-3) were formed, which, along with the MIC, are analyzed during the analysis of each series of samples and control all stages of the analysis - nucleic acid extraction, RT and PCR. The pools of positive controls include laboratory strains of respiratory viruses, diluted and mixed in a certain concentration so that in multiplex PCR-PB they are determined on the 31-33 cycle. PKO-1 contains inactivated drugs ADV, KV, EV, VPK; PKO-2 - CAA, HBV, RSV, RV; FFP-3 - HSV-1, 2, 3, 4. Positive control samples are bred in order to control the possibility of detecting viruses in samples with a low virus content. In each test sample include MIC, which in the absence of inhibitors should be determined at 30-33, but no later than 34 cycles. In the case when during the PCR-RV formulation there is a delay in the values of the threshold cycles of the MIC, this indicates the possible presence of inhibitors in the sample that reduced the sensitivity of the analysis, or errors in the formulation of the reaction (for example, the MIC instead of the 30-33 cycle is determined by 36- 37 or not defined at all). Such a sample must be re-analyzed. Thus, the setting of a number of controls eliminates false negative samples. To exclude false positive results, along with all samples, a negative control sample (OKO) is analyzed, which can be used as distilled water.

Thus, the essence of the invention lies in the fact that an approach has been developed for the simultaneous detection in clinical samples of nucleic acids of the main pathogens of human respiratory viral infections - influenza A and B viruses, coronaviruses, parainfluenza viruses 1, 2, 3, 4 types, adenoviruses, respiratory syncytial virus, rhinoviruses and enteroviruses in the presence of internal positive control (MIC). Samples from the nose, throat, nasopharynx, nasopharyngeal aspirates, nasal swabs, sputum, bronchoalveolar lavage, sectional material, and samples of culture fluid can be used as samples for research.

The method for differential diagnosis of respiratory viral infections by multiplex PCR with real-time detection includes the following steps:

1) preparation of mixtures of primers, probes for RT and PCR-RV and control samples (MIC, PKO1, PKO2, PKO3 and OKO);

2) the selection of nucleic acids of respiratory viruses from the studied and control samples;

3) conducting a multiplex reverse transcription reaction and multiplex PCR-RV (each sample is analyzed in three test tubes);

4) accounting and interpretation of analysis results.

Stage 2 (conducting a multiplex reverse transcription reaction and multiplex PCR-RV) is carried out in one of three options.

The 1st option is “Two-step RT and PCR-RV”, which provides for sequential RT and PCR reactions in separate tubes. After the RT reaction is completed, part of the RT products (cDNA) are transferred to test tubes, strips, or 96-well PCR plates for PCR-PB, to which the required amount of the reaction mixture for PCR-PB is then added.

2nd option - “Two-step RT and PCR-RV in one test tube”, providing for consecutive RT and PCR reactions in one test tube. The RT reaction is carried out in test tubes, strips or 96-well PCR plates suitable for PCR-RV, in a small volume up to 25 μl. After completion of the RT reaction, the reaction mixture for PCR-RV is introduced into test tubes containing RT products (cDNA).

The third option is “One-step RT and PCR-RV”, which provides for RT and PCR reactions in one tube in a reaction mixture that initially contains all the necessary components for RT and PCR-RV reactions. “One-step RT and PCR” is the most convenient and least time-consuming version of RT and PCR.

The implementation of the invention

Example 1. Isolation of viral nucleic acids

Total nucleic acid (NK) is isolated using one of the commercial kits: QIAmp Viral RNA mini kit (Qiagen, Germany), ZR Viral RNA Kit ™ (Zymo Research, USA), RIBO-sorb (InterLabService, Russia) or other specialized commercial kits for the simultaneous isolation of viral RNA and DNA, following the instructions of the manufacturer. The listed commercial kits are based on modifications of the NK extraction method proposed by Boom R. et al. [Boom R. et al. Rapid and simple method for purification of nucleic acids // J Clin Microbiol. - 1990. - No. 28. - P.495-503]. Before isolation of NK, 10 μl of VPA is added to each test sample and OCO, while the VPA sample must be diluted so that it is detected in PCR-RR by 30-33, but no later than 34 cycles. Simultaneously with the selection of NK from the studied samples, NK is isolated from the control samples - PKO1, PKO2, PKO3 and OKO.

Example 2. Carrying out PCR analysis in the format "Two-phase RT and PCR-RV"

At the first stage of the analysis, in the multiplex RT reaction, cDNA is obtained, which at the PCR stage serves as a matrix for amplification. The composition of the reaction mixture for multiplex RT includes primers divided into 3 groups, which are introduced into test tubes, strips or PCR plates in the amount of 6 pmol of each primer (in a volume of 2 μl) per reaction mixture.

As primers for conducting a multiplex reverse transcription reaction, oligonucleotides complementary to a fragment of the viral genome, which are either part of the corresponding primer for PCR-PB, or oligonucleotides complementary to the region of the viral genome outside the amplification region, are used. RT primers are characterized by a melting point below 50 ° C and primary structure features that reduce the likelihood of nonspecific annealing of RT primers on the RNA matrix and PCR-PB primers on the cDNA matrix, thereby increasing the amplification efficiency in samples with a high content of non-specific NC .

In tubes with primers, 10 μl of RNA isolated from the studied samples PKO-1, PKO-2, PKO-3 and OKO are added, heated for 5 minutes at 65 ° C, then cooled to room temperature for 3 minutes. Sample tubes are centrifuged to precipitate condensate. Next, the volume of the reaction mixture was adjusted to 30 μl with a pre-prepared mixture containing M-MLV revertase buffer, dNTP 0.5 mM, 30 units. Moloni leukemia virus revertase, and cDNA was obtained for 30 minutes at 42 ° C. All reagents for the OT reaction manufactured by Syntol (Russia) or similar. To inactivate the enzyme, the mixture is heated at 95 ° C for 5 minutes and, after cooling, centrifuged to precipitate condensate. Next, PCR-PB is carried out with cDNA samples.

The reaction of multiplex PCR-RV is carried out in test tubes, strips or tablets for PCR in a volume of 50 μl. Each sample is analyzed in 3 tubes (reaction mixture No. 1, No. 2 and No. 3), each of which contains a PCR reaction mixture (“Set of reagents for PCR-PB with Taq DNA polymerase and antibodies inhibiting enzyme activity”, cat. No. M-428, Synthol, Russia or equivalent), a mixture of 4 sets of primers (6 pmol of each primer - Table 3), 4 probes (5 pmol of each probe - Table 3), 2.5 units . Taq DNA polymerase with inhibitory enzyme activity antibodies (SibEnzyme, cat. No. E351, Russia or equivalent) and 5 μl of cDNA. The reaction is carried out in one of the following thermal cyclers: DT-96 (DNA-Technology, Russia), ANK-32 (Research Institute of Analytical Instrumentation RAS, Russia), Rotor-Gene ™ 6000 (Corbett Research, Australia), ICycler IQ5, Bio-Rad (United States) or similar. PCR program: 95˚C - 120 sec - 1 cycle; 95˚C - 20 sec, 58˚C - 40 sec - 45 cycles. The determination of threshold cycle values is carried out in accordance with the instrument manual.

Table 3

Oligonucleotides used in the composition of the reaction mixtures in multiplex PCR-RV.

Reaction mixture No. 1 Number 2 Number 3 Name of primers and probes SEQ ID NO 1
SEQ ID NO 2
SEQ ID NO 3
SEQ ID NO 5
SEQ ID NO 6
SEQ ID NO 7
SEQ ID NO 9
SEQ ID NO 10
SEQ ID NO 11
SEQ ID NO 13
SEQ ID NO 14
SEQ ID NO 15
SEQ ID NO 17 SEQ ID NO 18
SEQ ID NO 19
SEQ ID NO 21
SEQ ID NO 22
SEQ ID NO 23
SEQ ID NO 24
SEQ ID NO 25
SEQ ID NO 26
SEQ ID NO 27
SEQ ID NO 28
SEQ ID NO 29
SEQ ID NO 30
SEQ ID NO 31
SEQ ID NO 32
SEQ ID NO 33 SEQ ID NO 34
SEQ ID NO 35
SEQ ID NO 36
SEQ ID NO 37
SEQ ID NO 38
SEQ ID NO 39
SEQ ID NO 40
SEQ ID NO 41
SEQ ID NO 42
SEQ ID NO 43
SEQ ID NO 44
SEQ ID NO 45

When planning the analysis, it should be borne in mind that each test sample is analyzed in three reaction mixtures for the presence of NK 11 respiratory viruses. The quality of each reaction mixture is controlled by the appropriate FFP and OCO. For example, in order to analyze 10 clinical samples, at the RT and PCR stages it is necessary to use 36 tubes - 12 for each reaction mixture (10 test samples corresponding to PKO and OCO) (Table 4).

Table - 4. Scheme of analysis of 10 clinical samples.

Samples after isolation of NK one 2 3 four 5 6 7 8 9 10 PKO-1 PKO-2 PKO-3 EYE Reak mixture No. 1 one 2 3 four 5 6 7 8 9 10 eleven 12 Reak mixture No. 2 13 fourteen fifteen 16 17 eighteen 19 twenty 21 22 23 24 Reak mixture No. 3 25 26 27 27 29th thirty 31 32 33 34 35 36

Example 3. PCR analysis in the format "Two-phase RT and PCR-RV in one test tube"

A significant difference between the PCR analysis in the format "Two-step RT and PCR-RV in one tube" from the PCR analysis in the format "Two-step RT and PCR-RV" is the lack of the need to transfer cDNA from tubes with RT products to separate tubes for PCR RV RT and PCR-RV reactions are carried out in a single test tube (in test tubes, strips, plates that meet the requirements of PCR-RV equipment). PCR analysis in the format "Two-phase RT and PCR-RV in one test tube" is carried out as follows. When setting the RT reaction, the primer mixtures are introduced into test tubes, strips, or PCR plates in an amount of 3 pmol of each primer (in a volume of 1 μl) per reaction mixture.

As primers for conducting a multiplex reverse transcription reaction, oligonucleotides complementary to a fragment of the viral genome, which are either part of the corresponding primer for PCR-PB, or oligonucleotides complementary to the region of the viral genome outside the amplification region, are used. RT primers are characterized by a melting point below 50 ° C and primary structure features that reduce the likelihood of nonspecific annealing of RT primers on the RNA matrix and PCR-PB primers on the cDNA matrix, thereby increasing the amplification efficiency in samples with a high content of non-specific NC .

5 μl of RNA isolated from the studied samples PKO-1, PKO-2, PKO-3 and OKO are added to the tubes with primers and heated for 5 minutes at 65 ° C, then cooled to room temperature for 3 minutes. Sample tubes are centrifuged to precipitate condensate. Next, bring the volume of the reaction mixture to 25 μl with a pre-prepared RT mixture containing the reaction mixture for PCR-RV (Cat. No. M-428, Synthol, Russia, or the like), 30 units. Moloni leukemia virus revertase, and cDNA was obtained for 30 minutes at 42 ° C. To inactivate the enzyme, the mixture is heated at 95 ° C for 5 minutes. After the RT stage, the samples are centrifuged to precipitate condensate, after which the reaction mixture for PCR-RV (cat. No. M-428, Synthol, Russia, or the like) is added to cDNA samples, a mixture of 4 sets of primers (6 pmol of each primer - table 3), 4 probes (5 pmol of each probe - table 3), 2.5 units. Taq DNA polymerase with inhibitory enzyme activity antibodies (SibEnzyme, cat. No. E351, Russia, or similar), bringing the volume of the mixture to 50 μl, and carry out the PCR-PB reaction. The reaction is carried out in one of the following thermal cyclers: DT-96 (DNA-Technology, Russia), ANK-32 (Research Institute of Analytical Instrumentation RAS, Russia), Rotor-Gene ™ 6000 (Corbett Research, Australia), ICycler IQ5, Bio-Rad (United States) or similar. When planning the analysis, it should be borne in mind that each test sample is analyzed in three reaction mixtures in the presence of control samples. For example, in order to analyze 10 clinical samples, 36 tubes should be used at the RT and PCR stages - 12 for each reaction mixture (10 test samples corresponding to PKO and OCO) (Table 4).

Example 4. Carrying out PCR analysis in the format "One-stage RT and PCR-RV"

When setting up a PCR analysis in the format of “One-step RT and PCR-RV”, the same initial reagents are used as in Example 3. A significant difference between PCR analysis in the format “One-step RT and PCR-RV” and PCR analysis in the formats “Two-step RT and PCR-RV ”and“ Two-step RT and PCR-RV in one test tube ”consists in the absence of the need to open tubes with RT products in order to introduce the reaction mixture for PCR-RV, as all necessary components for the RT and PCR reactions are initially present in the reaction mixture. The primer mixtures are added to test tubes, strips or tablets that meet the requirements of the PCR-RV equipment in an amount of 6 pmol of each primer per reaction mixture (in a volume of 2 μl).

As primers for conducting a multiplex reverse transcription reaction, oligonucleotides complementary to a fragment of the viral genome, which are either part of the corresponding primer for PCR-PB, or oligonucleotides complementary to the region of the viral genome outside the amplification region, are used. RT primers are characterized by a melting point below 50 ° C and primary structure features that reduce the likelihood of nonspecific annealing of RT primers on the RNA matrix and PCR-PB primers on the cDNA matrix, thereby increasing the amplification efficiency in samples with a high content of non-specific NC .

10 μl of the studied RNA and RNA isolated from PKO-1, PKO-2, PKO-3 and OKO are added to the primer tubes and heated for 5 minutes at 65 ° C, then cooled to room temperature for 3 minutes. Sample tubes are centrifuged to precipitate condensate. Next, bring the volume of the reaction mixture to 50 μl with a pre-prepared mixture containing the reaction mixture for PCR, a mixture of 4 sets of primers (6 pmol of each primer - table 3), 4 probes (5 pmol of each probe - table 3), 30 units Moloni leukemia virus revertase, 2.5 units Taq DNA polymerase with inhibitory enzyme activity antibodies. The reaction is carried out in one of the following thermal cyclers: DT-96 (DNA-Technology, Russia), ANK-32 (Research Institute of Analytical Instrumentation RAS, Russia), Rotor-Gene ™ 6000 (Corbett Research, Australia), ICycler IQ5, Bio-Rad (United States) or similar. The program for one-stage RT and PCR-RV: 42ºС - 5 min, 95˚C - 120 sec - 1 cycle; 95˚C - 20 sec, 58˚C - 40 sec - 45 cycles. The determination of threshold cycle values is carried out in accordance with the instrument manual. When planning the analysis, it should be borne in mind that each test sample is analyzed in three reaction mixtures in the presence of control samples. For example, in order to analyze 10 clinical samples, 36 tubes should be used at the RT and PCR stages - 12 for each reaction mixture (10 test samples corresponding to PKO and OCO) (Table 4).

Example 5. Accounting and interpretation of analysis results

The analysis results are taken into account, threshold cycles are calculated using the software for the PCR-RV device on which the analysis was carried out, guided by the manufacturer's instructions. A sample is considered to be positive, in the analysis of which an increase in the fluorescent signal is observed on one of the color channels of the amplifier. To interpret the results of the analysis, Table 2 should be used. For example, when analyzing the sample in mixture No. 3, the signal growth on the FAM channel indicates the presence of type 1 parainfluenza virus in the NC sample, type 2 parainfluenza virus on the ROX channel, and type 3 parainfluenza virus R6G, Cy5 - parainfluenza virus type 4, etc. When analyzing clinical samples, samples that are determined up to 33 cycles are considered positive. If the sample is determined on the 34th cycle or later, but the threshold cycles of the FFP and MIC are within the normal range, and negative control is not determined, it is considered controversial and re-analyzed. If the result is retained when re-setting, the sample is considered positive. Attention should be paid to the values of threshold cycles for FFP and MIC. The composition of PKO includes laboratory strains of respiratory viruses, diluted and mixed in a certain concentration so that in multiplex PCR-PB they are determined on the 30-33 cycle. Also, in each study include MIC, which in the absence of inhibitors should be determined at 30-33, but no later than 34 cycles. If during the PCR-RV formulation, there is a delay in the values of the threshold cycles of the MIC, for example, instead of the 30-33 cycle, it is determined to be 36-37 or not determined at all, this indicates the possible presence of inhibitors in the sample that reduced the sensitivity of the analysis, and errors in the formulation reactions or errors in the allocation of NK. Such a sample should be re-analyzed. Thus, the setting of controls with FFP and MIC allows eliminating false negative samples. To exclude false positive results, along with all samples, a negative control sample (OKO) is analyzed, which can be used as distilled water.

Example 6. Differential detection of rhinoviruses and enteroviruses.

According to modern classification, rhinoviruses and enteroviruses belong to the same genus (Enterovirus) of the Picornaviridae family and, therefore, are phylogenetically related viruses. Each of these groups includes more than 100 serological types. Primers and probes for the detection of rhinoviruses and enteroviruses in the RT and PCR-RV reactions are directed to the untranslated part of the genome that is highly conservative for the entire family, which leads to the risk of cross-reactions that make it difficult to interpret the analysis result. When interpreting the results of the analysis of samples in reaction mixtures No. 1 and No. 2, the following rules should be used.

1. If there is a simultaneous increase in the fluorescence signal on channel R6G in reaction mixture No. 1 and on channel Cy5 in mixture No. 2 or only on channel R6G in mixture No. 1, then consider such a sample to be positive for enterovirus.

2. If the growth of the fluorescent signal is observed only in the mixture for No. 2 on the channel Cy5, then such a sample is considered positive for rhinovirus.

Claims (6)

1. A method for the differential diagnosis of respiratory viral infections, characterized by the detection in clinical samples of nucleic acids of the main pathogens of human respiratory viral infections - influenza A and B viruses (HAV and HBV), coronaviruses (KB), parainfluenza viruses 1, 2, 3, 4 types ( HSV 1, 2, 3, 4), adenoviruses (ADV), respiratory syncytial virus (RSV), rhinoviruses (RV) and enteroviruses (EV) in the presence of internal positive control (MIC), which provides for a multiplex reverse transcription reaction and PCR am plififications with real-time detection, and as clinical samples are used optionally: nasal swabs, throats, nasopharynx, nasopharyngeal aspirates, nasal swabs, sputum, bronchoalveolar lavage, sectional material, culture fluid samples, characterized in that for its implementation use oligonucleotides - SEQ ID NO 1-45, on the basis of which a mixture of primers, probes for RT and PCR-PB is prepared; then, using selected primers and probes, reaction mixtures for multiplex PCR are formed in which the oligonucleotides SEQ ID NOS 1-45 are distributed in three reaction mixtures as follows: mixture No. 1: SEQ ID NOS 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17; mixture No. 2: SEQ ID NO 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33; mixture No. 3: SEQ ID NO 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45; moreover, fluorescence-labeled probes are distributed over the reaction mixtures so that the probes labeled with different dyes in accordance with Table 2 are in the same mixture, then a multiplex reverse transcription reaction and multiplex PCR-RV are performed (each sample is analyzed in three reaction mixtures); the presence in the sample of nucleic acids of a particular respiratory virus in the sample is determined by the growth of the fluorescence signal of a specific dye in one of the reaction mixtures in accordance with the table above; moreover, during the analysis, a series of controls are carried out to exclude false positive and false negative results, for which pools of positive control samples are formed, which, along with the MIC, are analyzed in the analysis of each series of samples, and the respiratory virus strains diluted and mixed are included in the pools of positive controls in a certain concentration so that in multiplex PCR-RV they are determined on the 31-33 cycle; in the case when during the PCR-RV formulation there is a delay in the values of the threshold cycles of the MIC, this indicates the possible presence of inhibitors in the sample that reduced the sensitivity of the analysis, or errors in the formulation of the reaction; such a sample is re-analyzed. 2. The method according to claim 1, characterized in that each virus-specific set of primers and probes can be used to detect in the nucleic acid samples of the corresponding viruses in a monospecific format, including the detection of adenoviruses - SEQ ID NO 1, 2, 3; coronaviruses SEQ ID NO 4, 5, 6, 7, 8, 9, 10, 11; enteroviruses - SEQ ID NO 12, 13, 14; Influenza B virus - SEQ ID NO 18, 19, 20; respiratory syncytial virus - SEQ ID NO 21, 22, 23; influenza A viruses - SEQ ID NO 24, 25, 26; rhinoviruses - SEQ ID NO 27, 28, 29, 30, 31, 32, 33; Parainfluenza virus type 1 - SEQ ID NO 34, 35, 36; Parainfluenza virus type 2 - SEQ ID NO 37, 38, 39; parainfluenza virus type 3 - SEQ ID NO 40, 41, 42; Parainfluenza virus type 4 - SEQ ID NO 43, 44, 45. 3. The method according to claim 1, characterized in that in order to exclude false positive results, along with all samples, a negative control sample (OKO) is analyzed, which is used as distilled water. 4. The method according to claim 1, characterized in that the analysis involves the identification of an internal positive control represented by an RNA-containing virus in order to control the efficiency and quality of nucleic acid isolation, including the presence of reverse transcription and PCR inhibitors in the sample. 5. The method according to claim 3 or 4, characterized in that when analyzing the samples in the reaction mixtures No. 1 and No. 2, differential identification of rhinoviruses and enteroviruses is carried out. 6. The kit for implementing the method of differential diagnosis of respiratory viral infections according to claim 1 includes primers and probes for multiplex PCR-RV with the following nucleotide sequences:
5'-3 '
SEQ ID NO 1 CTCCAGCAACTTCATGTCYATGGG
SEQ ID NO 2 CACTCTGACCACGTCGAARACTTC
SEQ ID NO 3 FAM-AGGGTGGGCTCRTCCATGGGRTCCA-BHQ1
SEQ ID NO 4 GTGGCTGGGATGATATGTT
SEQ ID NO 5 TTATGGTGGTTGGAATAATATGTT
SEQ ID NO 6 GCGGGTGGGATAATATGTT
SEQ ID NO 7 TGGCATAGCACGATCACA
SEQ ID NO 8 TGTTAGGCAAAGCTCTATCACA
SEQ ID NO 9 GAGGGCATAGCTCTATCACA
SEQ ID NO 10 ROX-ATGGGTTGGGATTATCCTAAGTGTGAT-BHQ2
SEQ ID NO 11 ROX-ATGGGATGGGACTATCCTAAGTGTGAT-BHQ2
SEQ ID NO 12 CTCCGGCCCCTGAAT
SEQ ID NO 13 RATTGTCACCATAAGCAGCC
SEQ ID NO 14 R6G-CGGAACCGACTACTTTGGGTGTCCG-BHQ1
SEQ ID NO 15 GTCATCACCCACCGTTGT
SEQ ID NO 16 CCTTCTGGAGGTCCTCCAT
SEQ ID NO 17 Cy-5-AGAGCCCCAGGGTGCCCGAAT-BHQ3
SEQ ID NO 18 GGAATGGGRACAACAGCAAC
SEQ ID NO 19 AGGTACATGACCATGAGACAR
SEQ ID NO 20 FAM-CATGAAGCATTTGAAATAGCAGAAGGC-BHQ-1
SEQ ID NO 21 CAGTCAGTAGTAGACCATGTGAATTC
SEQ ID NO 22 ATATCTTCATCWCCATACTTTTCTGT
SEQ ID NO 23 ROX-GTTCTATAAGCTGGTATTGATGCAGGGA-BHQ2
SEQ ID NO 24 TTCTAACCGAGGTCGAAACG
SEQ ID NO 25 CGTCTACGYTGCAGTCC
SEQ ID NO 26 R6G-TGGCTAAAGACAAGACCAATCCTGTCACC-BHQ-1
SEQ ID NO 27 TGCGTGGCTGCCTGC
SEQ ID NO 28 TGCGTGGCGGCCAAC
SEQ ID NO 29 TGCGTGGCGGCCAGC
SEQ ID NO 30 CTGCGTGGCTGCCT
SEQ ID NO 31 AGCCYGCGTGGTGC
SEQ ID NO 32 AACACGGACACCCAAAGTAGT
SEQ ID NO 33 Cy-5-TTAGCCRCATTCAGGGGGCCG-BHQ3
SEQ ID NO 34 CTATGACATCAACGACAACAGG
SEQ ID NO 35 TTTGGTCTTTCCCTTGAGATCTA
SEQ ID NO 36 FAM-TGCAGGAACAAGGGGGTTATCAGTTATG-BHQ1
SEQ ID NO 37 CAGGACTATGAAAACCATTTACCT
SEQ ID NO 38 TGCAGGATTGATTGTGGC
SEQ ID NO 39 ROX-CTGATCTAGCTGAACTGAGACTTGCTTTC-BHQ2
SEQ ID NO 40 ATGGYTCAATCTCAACAACAAG
SEQ ID NO 41 TTGGATGTTCAAGACCTCCAT
SEQ ID NO 42 R6G-TACCCATCTGTTGGACCAGGGATATACT-BHQ1
SEQ ID NO 43 CCTGGAGTCCCATCAAAAGTAAG
SEQ ID NO 44 GCATCTATACGAACRCCTGCTC
SEQ ID NO 45 Cy5-TTTGTTGATCAAGACAATACAATTACACTTGA-BHQ3;
where FAM, ROX, R6G, Cy5 are fluorescent dyes; BHQ1, BHQ2, BHQ3 - fluorescence quenchers; R is A or G, Y is C or T.