RU2619179C1 - Method for evaluating viral contamination of air - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method comprises sampling air by sampler "Flora-100" which is placed in a specific pattern, and identification of microorganisms, wherein the further adjusted membrane filter into a sampler, in a cup with a selected sample was placed 10 ml of a medium for the preservation and stabilization of RNA viruses and identification of viruses was carried out by determination of the nucleic acids of pathogens of acute respiratory viral infections in man by multiplex PCR with the detection in real time.

EFFECT: invention improves the estimation accuracy and can be used to assess the viral contamination of the air in rooms of enterprises and organizations in various fields.

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Description

The invention relates to medicine, in particular to microbiology, virology, and can be used to assess viral contamination of air in the premises of enterprises and organizations of various profiles with RNA-containing viruses, pathogens of acute respiratory viral infections with the aim of their prevention.

In the frequency and number of diseases, acute respiratory infections (ARI) occupy first place in the world, accounting for 95% of all infectious diseases. Mortality from acute respiratory infections today remains stably high. Thus, 2 million people die of influenza every year in the world. In the Russian Federation, according to the “Report on the state of population’s health and healthcare organization based on the results of the activities of the executive authorities of the constituent entities of the Russian Federation for 2014” annually per 100 thousand people account for 4,573.9 cases of infectious and parasitic diseases (growth rate of 0.2%) . In the structure of infectious diseases, an important place is given to infectious diseases of the respiratory system.

Acute respiratory diseases are a group of diseases most often caused by viruses (influenza, parainfluenza, representatives of the Paramixoviridae family, adenoviruses, coronaviruses, rhinoviruses, etc.) and characterized by extremely rapid spread in the form of epidemics and pandemics. Acute respiratory viral infections (SARS) - infectious diseases characterized by lesions of the upper and lower respiratory tract, etc. [Respiratory syncytial virus infection: clinic, diagnosis, treatment / Cheshik SG, Vartanyan R.V. Methodological recommendations of Rospotrebnadzor (project) "laboratory diagnosis of influenza and other acute respiratory viral infections by the polymerase chain reaction" / Reference book of the head of the Department 2014. No7. S. 50-69].

Viruses that cause ARI and SARS are transmitted by airborne droplets from sick people and healthy virus carriers [Rational therapy of acute respiratory infections and influenza / Ovsyannikova EM, Korovina NA, Morgunova SL, Stoyko T.Yu. , Bodarevskaya O.P. Medical advice. 2015. No1; The role of respiratory viruses in the development of allergies / Gervazieva VB, Sveranovskaya VV, Sternshis Yu.A., Semenov B.F. / Cytokines and inflammation. 2003. T. 2. No. 3. S. 1-8; Respiratory syncytial viral infection (PC infection)]. A person, as a source of transmission of respiratory viral infections, secretes viruses into the external environment with coughing, sneezing, saliva, mucus, etc. So, a person infected with the influenza virus is contagious from the first hours of the disease until the 5-7th day of the disease; infected with paramyxoviruses (including parainfluenza) and coronaviruses - contagious from the first two to three days (maximum risk of infection) to ten days of illness (the likelihood of illness is reduced); infected with adenoviruses is dangerous until 3-4 weeks of illness [Infectious immunology / journal medical immunology 2005. V. 7. No. 2-3. S. 287-335; Actual issues of symptomatic and pathogenetic treatment of acute respiratory viral infections / Selkova EP Handbook of outpatient physician. 2013. No1. S. 9-13; Acute respiratory viral infections / Lioznov D.A., Lapotnikov V.A., Petrov V.N. / Nurse. 2011. No4. S. 3-9; Modern laboratory support of epidemiological studies and preventive measures / Infection and immunity. 2012.Vol. 2. No. 1-2. S. 234-349].

Viruses that cause respiratory infections and are mainly RNA-containing (with the exception of DNA-containing adenovirus) are characterized by different structures, small sizes (up to 250 nm) [Mass spectrometric approaches to the study of enveloped viruses: new opportunities for structural biology and preventive medicine review / Kordyukova L.V., Serebryakova M.V. Journal of Biochemistry. No. 8. S. 1002-1016. 2012; Molecular and cellular basis of immunoregulation, immunodiagnostics and immunocorrection (experimental models) / Medical immunology. 2006. V. 8. No. 2-3. S. 113-194; Respiratory syncytial viral infection: a modern view of the problem / Bevza S.L., Kharlamova F.S. / Children's infections. 2008. v. 7. No. 1. S. 43-51; Identification of respiratory group adenoviruses by PCR / Shtyrov A.A., Orlova S.V. / Magazine Youth collection of scientific articles "Scientific aspirations" No. 2/2012 / - P. 63; Modern laboratory support of epidemiological studies and preventive measures / Infection and immunity. 2012.Vol. 2. No. 1-2. S. 234-349; Acute respiratory viral infections / Averyanov A.V. / Journal of Practical Pulmonology No. 4/2003 - S. 33; Modern aspects of therapy and prevention of influenza / Popov S.F., Levshin V.K. / Ivanova G.F. / Kuvshinova T.D. 2010. Journal of the medicinal bulletin. No. 6 (38). S. 17-22; Construction of Deoxyribozymes For Inhibition of Reproduction of the Influenza Virus A / Evdokimov A.A., Mazurkova N.A., Malygin E.G., Zarytova V.F., Levina A.S., Repkova M.N., Zagrebelny S.N. , Netesova; Molecular and cellular bases of immunoregulation, immunodiagnostics and immunocorrection (Experimental); SARS (Severe Acute Respiratory Syndrome), or SARS (severe acute respiratory syndrome) / N.V. Astafieva, E.G. Belova / Medical Scientific and Practical Journal. Therapist. No. 9. 2003; The method of differential diagnosis of respiratory viral infections by multiplex PCR with real-time detection and a list of sequences for its implementation / Fayzuloev EB, Nikonova AA, Oceania AS, Lobodanov SA, Malakho S.G. ., Zverev V.V. / Patent for invention RUS 2460803, 10.27.2010] and various degrees of stability in the external environment, and some of them can remain in a viable state for a long period of time. So, parainfluenza viruses at room temperature remain in the air for 4 hours; coronaviruses - on a plastic surface last up to 2 days, in sewage waters up to 4 days, respiratory syncytial viruses remain in a viable state on environmental objects from 20 minutes to 5-6 hours; adenoviruses - up to 14 days [Methodical Recommendations of Rospotrebnadzor (Project) “Laboratory Diagnosis of Influenza and Other Orvi by the Polymerase Chain Reaction Method” / Reference book of the head of the Laboratory 2014. No7. S. 50-69; Modern laboratory support of epidemiological studies and preventive measures / Infection and immunity. 2012. T. 2. No. 1-2. S. 234-349; Respiratory syncytial viral infection (PC infection); Infectious immunology / Journal of Medical Immunology 2005. V. 7. No. 2-3. S. 287-335].

A number of methods are known for sampling, conducting research, and taking into account the results described in various regulatory documents for assessing bacterial and mycological airborne contamination in the premises of organizations and enterprises of various profiles [Order of the Ministry of Health of the USSR of September 30, 1991 No. 254 “On the development of disinfection business "; MUK 4.2.1089-02 Use of the air disinfection unit UOV "POTOK 150-M-01" and control of microbial contamination of air during its operation; MU 28-6 / 15-86 "Methodological guidelines for the organization and conduct of a complex of sanitary and anti-epidemic measures in aseptic departments (blocks) and wards"; MUK 4.2.2942-11. "Control methods. Biological and microbiological factors. Methods of sanitary-bacteriological studies of environmental objects, air and sterility control in medical organizations. Guidelines "; Order of the Ministry of Health of the USSR No. 720 of July 31, 1978" On improving medical care for patients with purulent surgical diseases and strengthening measures to combat nosocomial infection "; MU 42-51-4-93" Control of microbial air contamination of industrial premises "; MU 3182-84" Guidelines for microbiological control in pharmacies "; Appendix Order of the Ministry of Health of the USSR dated December 28, 89 N 691" Instructions for the organization and conduct of a complex of sanitary and anti-epidemic measures in obstetric hospitals "; MU-44-116 (approved. dep by the Code of Sanitary Inspection of the Ministry of Health of the Russian Federation 05/19/1997) "Medical immunobiological preparations. Aseptic production of medical immunobiological preparations; MU 64-02-005-2002" Guidelines classification and organization of premises for the production of non-sterile medicines "; SanPiN 2.1.3.2630-10" Sanitary epidemiological requirements for organizations engaged in medical activities "; MU 2657-82. Guidelines for sanitary and bacteriological control in catering and food trade bubbled products. However, none of these documents reflects a method for assessing viral airborne contamination.

A known method for the differential diagnosis of respiratory viral infections by multiplex PCR with real-time detection and a list of sequences for its implementation [patent RU No. 2460803], providing for the analysis of each test sample for the presence of nucleic acids of 11 respiratory viruses in 3 reaction mixtures. 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 disadvantage of this method is that the method does not provide for sampling air with adsorption of viral nucleic acids, as well as its complexity and the long duration (up to two days) of determining the total microbial contamination of the air environment, which does not allow the study in real time (in the form express forecast).

A known method for express forecasting the total microbial contamination of the air environment [Patent RU 2491349, 2013], including determining the number of aerosol particles with a diameter of 0.3 μm, 0.5 μm and 1.0 μm per unit volume of air using an aerosol particle counter , the calculation of the predicted total microbial contamination of the air according to the formula: Y = 0,0003 (n _0,5 + n _1,0 ) -1,2, at least under one of the conditions n _0,3 ≤2,95n _{0, 5} and / or n _0.5 ≤ 3.99n _1.0 , where: Y is the predicted total microbial contamination of the air, CFU per unit volume of air; n _0,3 - the number of aerosol particles with a diameter of 0.3 microns per unit volume of air; n _0,5 - the number of aerosol particles with a diameter of 0.5 microns per unit volume of air; n _1,0 - the number of aerosol particles with a diameter of 1.0 μm per unit volume of air; 0,0003; 2.95 and 3.99 - coefficients; 1,2 - correcting dimensionless quantity. However, this method is calculated, in addition, it does not capture and does not identify viruses.

The prototype of the invention is a method for assessing bacterial air contamination [Methodical instructions MUK 4.2.734-99 "Microbiological monitoring of the production environment". Moscow 1999, Ministry of Health of Russia], including air sampling using a Flora-100 sampler on nutrient media, followed by incubation and identification of the isolated bacteria. The disadvantage of this method is that it captures and identifies only bacteria without isolation and identification of viruses, the method is applicable only to manufacturers of medical immunobiological preparations.

The objective of the invention is to develop a method that allows with high reliability to evaluate the viral contamination of air by RNA-containing viruses, pathogens of acute respiratory viral infections in the premises of enterprises and organizations of various profiles, with the aim of preventing acute respiratory viral infections.

The technical result when using the invention is to increase the accuracy of the assessment by determining the viral contamination of air with RNA-containing viruses, the causative agents of acute respiratory viral infections in rooms in 1 m ^{3 of} air.

The invention is illustrated by figures 1-2, which depict the points at which the sampling of air.

The proposed method for assessing viral air contamination is carried out as follows: take air samples using the Flora-100 microbiological impactor, fill in with RNA medium, determine air viral contamination by real-time multiplex PCR detection.

The sampler - microbiological air impactor Flora-100 - is placed at a height of at least 50 cm from the floor and at least 30 cm from the wall or any objects that impede the free passage of air. Sampling of air in a room with an area of up to 15 m ^{2 is} taken at one point in the center of the room; in rooms with an area of more than 15 m ² - samples are taken at five points according to the "envelope principle" (Fig. 1); in narrow long rooms (with a ratio of width to length> 1: 5), samples at points at a distance of not more than 5 m from each other

(Fig. 2). The specified method of air selection is used to fully cover the entire volume of air in the room.

An empty sterile Petri dish with a Vladipor membrane filter of the MFAS-OS-1 type (pore diameter 0.22 μm, disk diameter 47 mm) is installed in the sampler into the sampler holder, the microbiological Flora-100 air impactor.

A button is pressed that has a marking of the required volume of sampled air of 1000 l, while a red indicator light lights up above the button. An air sample is taken by the aspiration method by pressing the [NETWORK] button located on the left side of the sampler. After taking a predetermined volume of air, the indicator lamp goes out.

10 ml of RNA medium is stored in a sample cup to preserve and stabilize the RNA of viruses (production environment of the Central Research Institute of Epidemiology of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare, catalog number 981, supplied by Interlabservis LLC). The medium is intended for storage and transportation with simultaneous stabilization of intracellular mRNA for subsequent isolation of total RNA. The transport medium contains a component that stabilizes the expression profile [http://www.opt-union.ru/i_store/item_1000380764/sreda-rnk.html].

The contents of the cup are mixed.

Determine the content of nucleic acids of viruses by the method of multiplex PNR with real-time detection, the method includes the following steps:

1) preparation of mixtures of primers, probes for RT and PCR-RV and control samples (internal positive control, positive control 1, positive control 2, positive control 3, negative control);

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

3) real-time multiplex reverse transcription reaction and multiplex PCR (each sample is analyzed in three tubes);

4) accounting and interpretation of analysis results.

Stage 2 (conducting a multiplex reverse transcription reaction and multiplex PCR-RV) to identify and identify specific nucleic acid fragments of the causative agents of acute respiratory viral infections of humans (ARVI): RNA of the respiratory syncytial virus (human Respiratory Syncytial virus - hRSv), metapneumovirus (human Metapneumovirus hMpv), parainfluenza viruses of types 1, 2, 3 and 4 (human Parainfluenza virus-1-4 - hPiv), coronaviruses of the species OC43, E229, NL63, HKUI (human Coronavirus - hCov), rhinoviruses (human Rhino virus - hRv), DNA of adenoviruses of groups B, C and E (human Adenovirus - hAdv) and bocavirus (human Bocavirus - hBov) by PCR hybridization-fluorescence detection of the amplification products according to the manual of the kit of reagents for detection of pathogens of acute respiratory viral infections in man (ARI) (OOO "InterLabService", part number R-V57).

- To carry out a quantitative analysis, a calibration graph is constructed for the dependence of the threshold cycle Ct on the decimal logarithm of DNA concentration. By the value of the threshold cycle of three calibrators with a known concentration, using a computer program, the RNA concentration of the studied viruses is calculated, according to the manual for the device.

- Calculate the concentration of the virus in the air according to the formula:

K = M / 2 (copies / m ³ ),

where M is the concentration of the virus in the solution of the transport RNA medium, which was determined in the previous paragraph. The resulting number of copies is multiplied by 10 (in terms of 10 ml of medium).

The proposed method is illustrated by the following examples.

Example 1. The room area of 14.35 m ² . Air is taken in the center of the room at one point, the sampler, the microbiological air impactor Flora-100, is placed at a height of 50 cm from the floor on a stool, an empty sterile Petri dish with a Vladipor membrane filter of the MFAS-OS-1 type is installed in the sampler ( pore diameter 0.22 μm, disk diameter 47 mm) in the holder of the sampler - microbiological air impactor Flora-100. A button is pressed that has a marking of the required volume of sampled air of 1000 l, while a red indicator light lights up above the button. An air sample is taken by the aspiration method by pressing the [NETWORK] button located on the left side of the sampler. After taking a predetermined volume of air, the indicator lamp goes out. 10 ml of RNA medium is placed in a sample cup to preserve and stabilize RNA viruses. The contents of the cup are mixed.

The content of virus nucleic acids is determined by multiplex PCR with real-time detection.

1) prepare a mixture of primers, probes for RT and PCR-RV and control samples (internal positive control, positive control 1, positive control 2, positive control 3, negative control);

2) isolated nucleic acids of respiratory viruses from the studied samples;

3) carry out a multiplex reverse transcription reaction and multiplex real-time PCR (each sample is analyzed in three tubes);

4) take into account and interpret the results of the analysis.

Stage 2 (conducting multiplex reverse transcription reactions and multiplex PCR-RV) to identify and identify specific nucleic acid fragments of the causative agents of acute respiratory viral infections of humans (ARVI): RNA of the respiratory syncytial virus (human Respiratory Syncytial virus - hRSv), metapneumovirus (human Metapneumovirus hMpv), parainfluenza viruses of types 1, 2, 3 and 4 (human Parainfluenza virus-1-4 - hPiv), coronaviruses of the species OC43, E229, NL63, HKUI (human Coronavirus - hCov), rhinoviruses (human Rhino virus - hRv), DNA of adenoviruses of groups B, C and E (human Adenovirus - hAdv) and bocavirus (human Bocavirus - hBov) by PCR hybridization-fluorescence detection of the amplification products according to the manual of the kit of reagents for detection of pathogens of acute respiratory viral infections in man (ARI) (OOO "InterLabService", part number R-V57).

- To carry out a quantitative analysis, a calibration graph is constructed for the dependence of the threshold cycle Ct on the decimal logarithm of DNA concentration. By the value of the threshold cycle of three calibrators with a known concentration, using a computer program, the RNA concentration of the studied viruses is calculated, according to the manual for the device.

- Calculate the concentration of the virus in the air according to the formula:

K = M / 2 (copies / m ³ ),

where M is the concentration of the virus in the solution of the transport RNA medium, which was determined in the previous paragraph.

K = 2/2 = 1. Multiply by 10. 10 copies / m ^{3 of the} virus of the causative agent of respiratory diseases.

Example 2. A room with an area of 22.31 m ² (square room). Air is taken at five points according to the principle of the envelope, the sampler - the microbiological air impactor Flora-100 - is placed at a height of 50 cm from the floor on a stool, an empty sterile Petri dish with a Vladipor membrane filter of the MFAS-OS-1 type is installed in the sampler ( pore diameter 0.22 μm, disk diameter 47 mm) in the holder of the sampler - microbiological air impactor Flora-100. A button is pressed that has a marking of the required volume of sampled air of 1000 l, while a red indicator light lights up above the button. An air sample is taken by the aspiration method by pressing the [NETWORK] button located on the left side of the sampler. After taking a predetermined volume of air, the indicator lamp goes out. 10 ml of RNA medium is placed in all sampled dishes to preserve and stabilize the RNA of viruses. The contents of the cup are mixed.

The content of virus nucleic acids in all plates was determined by multiplex PCR with real-time detection.

1) prepare a mixture of primers, probes for RT and PCR-RV and control samples (internal positive control, positive control 1, positive control 2, positive control 3, negative control);

2) isolated nucleic acids of respiratory viruses from the studied samples;

3) carry out a multiplex reverse transcription reaction and multiplex real-time PCR (each sample is analyzed in three tubes);

4) take into account and interpret the results of the analysis.

Stage 2 (conducting a multiplex reverse transcription reaction and multiplex PCR-RV) to identify and identify specific nucleic acid fragments of the causative agents of acute respiratory viral infections of humans (ARVI): RNA of the respiratory syncytial virus (human Respiratory Syncytial virus - hRSv), metapneumovirus (human Metapneumovirus hMpv), parainfluenza viruses of types 1, 2, 3 and 4 (human Parainfluenza virus-1-4 - hPiv), coronaviruses of the species OC43, E229, NL63, HKUI (human Coronavirus - hCov), rhinoviruses (human Rhinovirus - hRv), DNA adenoviruses of groups B, C and E (human Adenovirus - hAdv) and bocavirus (human Bocavirus - hBov) by PCR with hybridization-fluorescence detection of amplification products according to the instructions for the reagent kit for identifying pathogens of acute respiratory viral infections of humans (ARVI) (LLC Interlabservis, catalog number R-V57).

- To carry out a quantitative analysis, a calibration graph is constructed for the dependence of the threshold cycle Ct on the decimal logarithm of DNA concentration. By the value of the threshold cycle of three calibrators with a known concentration, using a computer program, the RNA concentration of the studied viruses is calculated, according to the manual for the device.

- Calculate the concentration of virus in the air in each cup according to the formula:

K = M / 2 (copies / m ³ ),

where M is the concentration of the virus in the solution of the transport RNA medium, which was determined in the previous paragraph.

K ₁ = 0/2 = 0, K ₂ = 1/2 = 0.5, K ₃ = 2/2 = 1, K ₄ = 0/2 = 0, K ₅ = 0/2 = 0.

Calculate the amount of copies on all cups K = 1.5 copy / m ³ . Multiplied by 10. Get 15 copies / m ^{3 the} virus of the causative agent of respiratory diseases.

Example 3. A room with an area of 20.0 m ² (corridor). Air is taken at two points, at a distance of 5 meters from each other. The sampler - microbiological air impactor Flora-100 - is placed at a height of 50 cm from the floor on a stool, an empty sterile Petri dish with a Vladipor membrane filter of the MFAS-OS-1 type (pore diameter 0.22 μm, disk diameter is installed in the sampler) 47 mm) into the sampler holder - microbiological air impactor Flora-100. A button is pressed that has a marking of the required volume of sampled air of 1000 l, while a red indicator light lights up above the button. An air sample is taken by the aspiration method by pressing the [NETWORK] button located on the left side of the sampler. After taking a predetermined volume of air, the indicator lamp goes out. 10 ml of RNA medium is placed in all sampled dishes to preserve and stabilize the RNA of viruses. The contents of the cup are mixed.

The content of virus nucleic acids in all plates was determined by multiplex PCR with real-time detection.

1) prepare a mixture of primers, probes for RT and PCR-RV and control samples (internal positive control, positive control 1, positive control 2, positive control 3, negative control);

2) isolated nucleic acids of respiratory viruses from the studied samples;

3) carry out a multiplex reverse transcription reaction and multiplex real-time PCR (each sample is analyzed in three tubes);

4) take into account and interpret the results of the analysis.

Stage 2 (conducting a multiplex reverse transcription reaction and multiplex PCR-RV) to identify and identify specific nucleic acid fragments of the causative agents of acute respiratory viral infections of humans (ARVI): RNA of the respiratory syncytial virus (human Respiratory Syncytial virus - hRSv), metapneumovirus (human Metapneumovirus hMpv), parainfluenza viruses of types 1, 2, 3 and 4 (human Parainfluenza virus-1-4 - hPiv), coronaviruses of the species OC43, E229, NL63, HKUI (human Coronavirus - hCov), rhinoviruses (human Rhino virus - hRv), DNA of adenoviruses of groups B, C and E (human Adenovirus - hAdv) and bocavirus (human Bocavirus - hBov) by PCR hybridization-fluorescence detection of the amplification products according to the manual of the kit of reagents for detection of pathogens of acute respiratory viral infections in man (ARI) (OOO "InterLabService", part number R-V57).

- To carry out a quantitative analysis, a calibration graph is constructed for the dependence of the threshold cycle Ct on the decimal logarithm of DNA concentration. By the value of the threshold cycle of three calibrators with a known concentration, using a computer program, the RNA concentration of the studied viruses is calculated, according to the manual for the device.

- Calculate the concentration of virus in the air in each cup according to the formula:

K = M / 2 (copies / m ³ ),

where M is the concentration of the virus in the solution of the transport RNA medium, which was determined in the previous paragraph.

K, = 0/2 = 0, K ₂ = 1/2 / = 0.5

Calculate the amount of copies on all cups K = 0.5.

Multiply by 10. 5 copies / m ³ of the respiratory pathogen virus.

Example 4. A room with an area of 20.0 m ² (corridor). Air is taken at two points, at a distance of 5 m from each other. The sampler - microbiological air impactor Flora-100 - is placed on the floor, an empty sterile Petri dish with a Vladipor membrane filter of the MFAS-OS-1 type (pore diameter 0.22 μm, disk diameter 47 mm) is placed in the sampler into the sampler holder - microbiological air impactor Flora-100. A button is pressed that has a marking of the required volume of sampled air of 1000 l, while a red indicator light lights up above the button. An air sample is taken by the aspiration method by pressing the [NETWORK] button located on the left side of the sampler. After taking a predetermined volume of air, the indicator lamp goes out. 10 ml of RNA medium is placed in all sampled dishes to preserve and stabilize the RNA of viruses.

The content of virus nucleic acids in all plates was determined by multiplex PCR with real-time detection.

1) prepare a mixture of primers, probes for RT and PCR-RV and control samples (internal positive control, positive control 1, positive control 2, positive control 3, negative control);

2) isolated nucleic acids of respiratory viruses from the studied samples;

3) carry out a multiplex reverse transcription reaction and multiplex real-time PCR (each sample is analyzed in three tubes);

4) take into account and interpret the results of the analysis.

Stage 2 (conducting a multiplex reverse transcription reaction and multiplex PCR-RV) identification of specific

nucleic acid fragments of the causative agents of acute respiratory viral infections of humans (ARVI): RNA of the respiratory syncytial virus (human Respiratory Syncytial virus - hRSv), metapneumovirus (human Metapneumovirus - hMpv), type 1, 2, 3 and 4 parainfluenza viruses (human Parainfluenza virus 1-4 - hPiv), coronaviruses of the species OC43, E229, NL63, HKUI (human Coronaviras - hCov), rhinoviruses (human Rhinovirus - hRv), DNA of groups B, C and E adenoviruses (human Adenovirus - hAdv) and bocavirus (human Bocavirus - hBov) by PCR with hybridization-fluorescence detection of amplification products according to the instructions for a set of reagents to identify excitatory Lei acute respiratory viral infections in man (SARS) (000 "InterLabService" catalog number R-V57).

- To carry out a quantitative analysis, a calibration graph is constructed for the dependence of the threshold cycle Ct on the decimal logarithm of DNA concentration. By the value of the threshold cycle of three calibrators with a known concentration, using a computer program, the RNA concentration of the studied viruses is calculated, according to the manual for the device.

- Calculate the concentration of virus in the air in each cup according to the formula:

K = M / 2 (copies / m ³ ),

where M is the concentration of the virus in the solution of the transport RNA medium, which was determined in the previous paragraph.

K ₁ = 0/2 = 0, K ₂ = 1/2 = 0

Calculate the amount of copies on all cups K = 0

Multiply by 10.

0 copies / m ³ .

Claims (1)

A method for assessing viral air contamination with RNA-containing viruses, causative agents of acute respiratory viral infections in a room, including air sampling using a Flora-100 sampler, identification of microorganisms, characterized in that they additionally install a membrane filter in the sampler, which is placed at a height not less than 50 cm from the floor and at a distance of at least 30 cm from the wall or objects that impede the free passage of air, and a sample of air in a room with an area of up to 15 m ^{2 is} taken at one the center of the room; in a room with an area of more than 15 m ^{2, a} sample is taken at five points, namely at corner and central points; in rooms with a width to length ratio of more than 1: 5, a sample is taken at points at a distance of no more than 5 m from each other, 10 ml of medium is placed in a cup with the selected sample to preserve and stabilize the RNA of viruses, and virus identification is carried out by determining the content of nucleic acids causative agents of acute human respiratory viral infections by multiplex PCR with real-time detection.

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