RU2709460C9 - Method for biopathogens detection in air - Google Patents

Abstract

FIELD: ecology.

SUBSTANCE: invention relates to ecology, namely to detecting biopathogens in air. Detection is carried out step-by-step, so at the first stage after sampling of aerosol sample, it is transferred into liquid phase, then sample in liquid phase is treated with ultrasound. At the second stage, the sample is divided into three streams: 1 – sample preparation for immunological analysis, 2 – sample preparation for PCR analysis and archive. At the third stage, two sample preparations are performed in parallel for immunological analysis (IA) and PCR analysis. At the fourth stage, the suspension from prepared samples for analysis is sent to an immunological analyzer and a PCR unit for further analysis.

EFFECT: invention allows higher accuracy and faster determination of biopathogens in air up to 80 detected targets with maximum coverage of potential spectra of potential biopathogens in air – viruses, bacteria, toxins.

3 cl, 2 dwg

Description

The technical field to which the invention relates.

The invention relates to the field of research or analysis of air for the presence of biopathogens in it, to protect humans or animals from the harmful effects of bacteria, viruses, genetic vectors and nanotechnology objects.

The following terms are used in this description:

AKDB - Automatic biopathogen analyzer

NK - nucleic acids

IA - immunological analyzer

PCR - diagnostics (polymerase chain reaction) - a highly accurate method for the diagnosis of numerous infections, which is based on the study of human genetic material (DNA and RNA)

ASP is an automatic signaling device for special impurities.

The prior art.

The prior art method without sample air monitoring, including the sounding of space by pulsed coherent radiation in the UV region and recording the spectral course of the fluorescence intensity of protein-containing substances. In addition, a selective assessment of the normalized fluorescence intensities of protein-containing substances and interfering impurities at various excitation wavelengths within the spectral course of fluorescence relative to the intensity at a wavelength of 284 nm is carried out.

Also known from the prior art is an automatic signaling device for special impurities (ASP) designed for continuous monitoring of atmospheric air in order to detect aerosols of special impurities (bacteria, rickettsia, viruses) in it.

The composition of the TSA includes: a sensor; voltage converter 13 V to 26 V (power supply) or electric cable; sound signaling device; CIS winter and summer; Spare parts; documentation (http://www.mil.bv/print.php?ELEMENT_ID=7857&clear_cache=Y).

In addition, there is a known method for detecting the presence of an analyte in a liquid sample, a method for detecting the presence of a pathogen in a whole blood sample, a method for detecting the presence of a virus in a whole blood sample, a method for detecting the presence of a target nucleic acid in a whole blood sample, a method for detecting the presence of Candida species in a liquid sample, systems for detecting one or more nucleic acid analytes in a liquid sample and a replaceable cartridge for placing analysis reagents and consumables Ialov in the system. The presented inventions are characterized by the fact that in their implementation they use magnetic particles having an average diameter in the range from 700 nm to 950 nm, a relaxivity value of T ₂ per particle of 1 × 10 ⁹ to 1 × 10 ¹² mmol ^-1 s ^-1 , and have binding residues on their surface, and these binding residues act by altering the aggregation of magnetic particles in the presence of an analyte. The invention provides multiplex detection of several types of various molecules and can be used in clinical practice (RU 2653451 05/08/2018).

Along with the above, a method for detecting biological particles in an aerosol is known. For this, aerosol particles are deposited on the surface of the substrate. Irradiate a surface with a light source deposited by the sample. The emission of fluorescence and phosphorescence of a sample is detected at several wavelengths with the release of a phosphorescence signal with a time delay between the act of excitation and the act of receiving the emission signal. Biological particles are determined by the ratio of fluorescence and phosphorescence signals. In this case, during the deposition process, the concentration of aerosol particles on the substrate surface is controlled by the level of the scattering signal of the substrate surface with particles, which is compared with a predetermined limit value of the scattering level, determined taking into account the resolution of the optical signal detection system, which is assumed to be equivalent to the maximum size of the desired particles. Upon reaching a given level of scattering, the deposition of particles is stopped and the emission of fluorescence and phosphorescence of each particle is detected separately (RU 2495426 10.10.2013).

However, previously known methods for detecting pathogens in the air have the following disadvantages: a long analysis time: 4 or more hours (depending on the operating mode), the inability to detect cover the maximum possible spectrum of potential biopathogens in the air, as containing nucleic acids (viruses, bacteria), and not containing.

The implementation of the invention.

The claimed method includes the following methods: collecting aerosol particles, transferring them to a liquid state, subsequent analysis of the collected sample in parallel by two independent methods: using multiplex immunological analysis and real-time PCR on a single-use chip, while using two analysis methods allows you to cover the maximum possible spectrum of potential biopathogens in the air, both containing nucleic acids (viruses, bacteria), and not containing. Moreover, the number of simultaneously detected targets can be more than 80.

The objective of the method is to increase the accuracy of determination of biopathogens in the air, the rapid detection of biopathogens in the air, for 2 hours - more than 80 detected targets.

The technical result of the present invention is to increase the accuracy and accelerate the determination of biopathogens in the air to 80 detectable targets, as well as to maximally cover the possible spectrum of potential biopathogens in the air, both containing nucleic acids (viruses, bacteria), and not containing.

In addition, aerosol safety control is an extremely urgent task at the current stage of development of society. Life in modern megacities and the cramped economic ties between countries give a pathogen with an aerosol transmission mechanism colossal opportunities for initiating pandemics. A solution to the problem could be ways to detect biopathogens in the air in public places that can identify infectious agents before mass infection of people. Early information on the spread of pathogens in the environment, taking into account the latent period, can reduce the response time of the healthcare system by several days, and if measures are taken in a timely manner, drastically reduce the number of people exposed to infection.

Disclosure of the invention.

The specified technical result is realized due to the following techniques of the method for detecting biopathogens in the air. Air analysis is carried out in several stages. So, at the first stage, an aerosol sample is collected. Then transfer it to the liquid phase. After that, the sample in liquid form is treated with ultrasound in order to destroy the cell walls of biological objects and release nucleic acids (NK) and antigens suitable for further analysis into the solution. At the next stage - the second, the solution after sonication is divided into three streams: 1 - sample preparation for immunological analysis, 2 - sample preparation for PCR analysis and sample for archive. At the third stage, in parallel to each other, sample preparation of a divided sample for immunological analysis (IA) and PCR reaction is performed. In the fourth stage, after all sample preparation operations have been completed, suspensions from the prepared samples for analysis are sent to the immunological analyzer and the PCR unit for further analysis.

The specified method can be carried out by means of an automatic analyzer of biopathogens in the air, which can be presented in the following form and is a complex of equipment located in a single metal frame structure (Fig. 1). The layout of the device is made in the form of two compartments. In the upper compartment (2) are all the nodes and subsystems that are in direct contact with biological samples and reagents - from an aerosol collection device to PCR - and immunological analyzers. In this compartment, a controlled temperature and humidity regime is maintained. In the lower compartment (3) is located all the auxiliary equipment and control electronics: power sources and distributors, backup batteries, a refrigeration unit, a control computer and automation units. The refrigeration unit provides cooling of the circulating water to a temperature of + 4 ° C; this water, in particular, is used to cool reagents in order to ensure the autonomy of the device for 7 days. The sample preparation system of an automatic analyzer of biopathogens in the air is built according to a closed scheme.

The air analysis process begins with the collection of the aerosol sample and its transfer to the liquid phase. Air collection can be carried out by an impactor - a device that creates a flow of intake air up to 4 m ³ per minute and capable of concentrating aerosol particles in the range from 0.3 to 10 μm by about 10 times. Aerosol transfer can be carried out by means of an aerosol collector constructed on the principle of a water cyclone. A field of liquid transfer to the next stages of processing. The sample is processed in the liquid phase by ultrasound; an ultrasonic homogenizer can be used for sonication. After sonication, the sample solution is divided into three streams: 1 - sample preparation for immunological analysis, 2 - sample preparation for PCR analysis and archive.

At the same time, the sample preparation system can be built according to a closed scheme (see Fig. 2), which includes an impactor (1), an aerosol collector (5), an ultrasonic homogenizer (6), a first rotary valve (K1), and an NK separation unit (8 ), containing: a reservoir with reagents for the selection of NK (8a), a third rotary valve (K3), a reactor for the selection of NK (8b), a sample preparation unit IA (9), including: a tank with microspheres, antibodies and dyes (9a), a second rotational a valve (K2) and an IA reactor (9b); a reservoir containing washing liquids (7) such as: buffer, or 70% alcohol, or hypochlorite, or alkali; immunological analyzer (10); PCR reagent reservoir (12); PCR analyzer (13); sample archive (11); reservoir for cooled circulating water (14); reservoir of liquid intended for discharge (15). In this case, if necessary, I perform 16 - cooling or 17 - heating and / or carry out the action of 18 - magnet and / or 19 - mixing.

For the movement of liquids in this scheme, automatic syringe pumps are provided. The working fluid in all pumps is distilled water. To control the flow of liquids, rotary valves are used (in Fig. 2 - K1-K3), made of chemically inert materials that can be washed with an alkaline solution, sodium hypochlorite solution and ethyl alcohol. The hydraulic connections are made of tubes made of ETFE fluoropolymer (ethylene-tetrafluoroethylene copolymer), which also allow washing with the solutions listed above. The washing of the entire sample preparation system is carried out after each sampling and each analysis, according to the protocol of the device.

The implementation of the invention.

A method for identifying biopathogens in the air is as follows.

Stage 1. Perform an aerosol sampling, translate it into the liquid phase. Then, the sample in the liquid phase is treated with ultrasound in order to destroy the cell walls of biological objects and release nucleic acids and antigens suitable for further analysis into the solution. Moreover, to prevent excessive heating of the sample, the sample is cooled with circulating water.

Stage 2. After sonication, the sample solution is divided into three streams: 1 - sample preparation for immunological analysis, 2 - sample preparation for PCR analysis and archive. In this case, the archive can be represented by a set of cooled containers, in which 1 ml of the sample is placed in turn at each measurement. If the analysis does not subsequently detect pathogenic agents in the sample, the containers are washed and reused. If the analysis is positive, the sample from the archive can be additionally verified by independent methods.

Step 3. Perform sample preparation for immunological analysis (IA) and PCR analysis. Sample preparation for IA includes sample incubation with magnetic microspheres, biotinylated antibodies with fluorescent labels (streptovidin-phycoerythrin conjugates, SAPE), as well as appropriate washes. These operations can be performed in an IA reactor, which is a 1.5 ml polypropylene tank in which the required temperature is maintained (usually 37-38 ° C; this water, in particular, is used to cool the reagents to C). By means of an electromechanical drive, an annular permanent magnet can be supplied to this container to hold the magnetic particles during the washings. The reactor is mounted on the shaft of an orbital mixer capable of driving it at speeds of up to 1500 rpm. Reagents for sample preparation of IA (magnetic microspheres, antibodies, SAPE) are stored at 4 ° C; what can be used a cooled platform mounted on the shaft of the orbital mixer. Sample preparation for PCR analysis consists in the isolation and purification of nucleic acids (NK) from a sample solution. The principle of NC extraction is based on the use of magnetic particles. The selection of NK occurs in the reactor, the design of which is similar to the reactor sample preparation IA. It should be noted that the IA and PCR sample preparation operations are performed in parallel, which reduces the total analysis time of the sample by two methods.

Stage 4. after all sample preparation operations have been completed, suspensions from the reactors are sent to the immunological analyzer and PCR unit for further analysis.

Thus, due to a certain sequence and stages of analysis of air samples in order to identify biopathogens, take an aerosol sample, transfer it to the liquid phase, process the sample in the liquid phase with ultrasound, then divide the sample into three parts, conduct parallel sample preparation for the analysis of IA and PCR analysis, subsequent analysis of IA and PCR ensures the achievement of the technical result achieved - improving the accuracy and accelerating the determination of biopathogens in the air to 80 detectors Mykh targets, as well as the maximum coverage possible range of potential biopathogenic in the air as containing nucleic acids (viruses, bacteria) and containing no.

Industrial applicability.

A method for detecting biopathogens in the air can be carried out by a specialist in practice and, when implemented, ensures the implementation of the declared purpose. The possibility of implementation in practice follows from the fact that for each feature included in the claims on the basis of the description, the material equivalent is known, which allows us to conclude that the criterion of "industrial applicability" for the invention and the criterion of "completeness of disclosure" for the invention are met.

A method for detecting biopathogens in the air can be used in public places, and allows you to identify pathogens of infectious diseases until mass infection of people. Early information on the spread of pathogens in the environment, taking into account the latent period, can reduce the response time of the healthcare system by several days, and if measures are taken in a timely manner, drastically reduce the number of people exposed to infection.

Claims (3)

1. A method for detecting biopathogens in the air, including sampling an aerosol sample, characterized in that the method is carried out in stages, so at the first stage after sampling an aerosol sample, it is transferred to the liquid phase, then the sample in the liquid phase is treated with ultrasound; at the second stage, the sample is divided into three streams: 1 - sample preparation for immunological analysis, 2 - sample preparation for PCR analysis and archive; at the third stage two samples are prepared in parallel for immunological analysis (IA) and PCR analysis; at the fourth stage, suspensions from the prepared samples for analysis are sent to the immunological analyzer and the PCR unit for further analysis. 2. A method for detecting biopathogens in the air according to claim 1, characterized in that during sample preparation for IA, the sample is incubated with magnetic microspheres, biotinylated antibodies with fluorescent labels. 3. A method for detecting biopathogens in air according to claim 1, characterized in that the sample preparation of the PCR analysis consists in the isolation and purification of nucleic acids (NK) from the sample solution, while the principle of NK extraction is based on the use of magnetic particles.

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