Device for identifying mycobacterium tuberculosis

Abstract

The invention relates to a device for automatically extracting, purifying and amplifying (PCR) regions of Mycobacterium tuberculosis DNA and recording results in real time. The device consists of a single-use cartridge, an ultrasonic generator, an emitter, and an analyzer in the form of a device capable of interacting with the single-use cartridge and managing the processes of DNA extraction and subsequent PCR and detecting a fluorescent signal in real time inside the enclosed space of the cartridge. The device further comprises a filter element for keeping a pathogen in a ventilation well of a cell of the single-use cartridge into which a sample to be analyzed is placed, as well as at least one source of UV light arranged inside and providing for the disinfection of the inner cavities of the device. The technical result is that of protecting an inner cavity between a lid and the surrounding environment against contamination of the analyzer and of the surrounding space by a pathogenic agent.

Description

[0001] A device for automatic isolation, purification and amplification of Mycobacterium tuberculosis DNA sections with real-time recording of results

[0002] Field of technology

[0003] The invention relates to measuring equipment, namely to a device for identifying the tuberculosis pathogen Mycobacterium tuberculosis and non-tuberculous mycobacteria for use in medicine, molecular biology and biotechnology for the purpose of differential diagnosis of tuberculosis and determining the resistance of mycobacteria to antibacterial drugs.

[0004] State of the art

[0005] Improving the quality of life of patients with tuberculosis and reducing tuberculosis-related morbidity and mortality is an important goal for phthisiology and public health in general. Despite year-on-year improvements in the number of newly diagnosed tuberculosis cases, this serious infectious disease remains a major cause of disability and mortality worldwide. The emergence and spread of antibiotic-resistant mycobacteria strains significantly contributes to the spread of morbidity and high mortality. According to the WHO, the global priority in the fight against tuberculosis is the earliest possible diagnosis and the ability to rapidly determine the antibiotic resistance profile of identified M. tuberculosis strains or non-tuberculous mycobacteria. Traditional laboratory methods are too slow and require well-equipped microbiology or molecular biology laboratories, as well as highly qualified personnel performing the analysis.In this regard, new technologies for rapid detection are the focus of research and development work on tuberculosis diagnostics.

[0006] The prior art includes hardware systems and devices for the automated isolation, purification, and amplification of M. tuberculosis DNA regions with real-time results recording. These devices consist of disposable cartridges or tubes for mycobacterial DNA amplification and sample preparation systems (Jia Mei Hong et al., Point-of-care diagnostic tests for tuberculosis disease. Sci. Transl. Med. l4, eabj4124 (2022). DOI: 10.1126 / scitranslmed.abj4124). Currently, several different technologies for nucleic acid (NA) amplification in disposable cartridges exist. These devices are commonly used to diagnose infections, identify genetic mutations, and for other applications in biology and medicine.

[0007] The use of disposable cartridges ensures convenience and safety when working with infected biomaterial and minimizes the risk of sample contamination, which is especially important when working with genetic material. Each cartridge contains all the necessary reagents and templates for NA amplification, which makes the process fast and automated (Chakravorty S, Simmons AM, Rowneki M, Parmar H, Cao Y, Ryan J, Banada PP, Deshpande S, Shenai S, Gall A, Glass J, Krieswirth B, Schumacher SG, Nabeta P, Tukvadze N, Rodrigues C, Skrahina A, Tagliani E, Cirillo DM, Davidow A, Denkinger CM, Persing D, Kwiatkowski R, Jones M, Alland D. The New Xpert MTB / RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing. mBio. 2017 Aug 29;8(4):e00812-17.).

[0008] The operating principle of such devices may vary depending on the technology, but typically involves the following steps.

[0009] 1. Sample preparation. For analysis, a sample is collected and prepared in the cartridge. This sample may be tissue, blood, sputum, saliva, or other sources of genetic material.

[0010] 2. NA extraction. Extraction methods are used to isolate nucleic acids from the sample in the cartridge. This allows for the production of pure NA for further reactions.

[0011] 3. Amplification (PCR). The cartridge typically uses polymerase chain reaction (PCR), which increases the amount of specific DNA regions so they can be detected and analyzed.

[0012] 4. Detection. The cartridge or a separate amplifier unit can be equipped with sensors or special tags that detect the presence of amplified DNA fragments. This can be achieved using optical, electrical, or other detection technologies.

[0013] 5. Results analysis. The obtained data is processed by built-in software or transferred to a computer for further interpretation and analysis. A set of molecular biological test systems, reagents and analytical instruments for the detection, identification and genetic typing of pathogens of particularly dangerous infectious diseases is known, consisting of a complex for the extraction of nucleic acids (KVNK), an ANK-32 device for real-time polymerase chain reaction (RT-PCR) and a Nanofor 05 device for genetic typing of pathogens of bacterial and viral infectious diseases (Kurochkin V.E., Alekseev Ya.I., Petrov D.G., Evstrapov A.A. Domestic devices for molecular genetic analysis: developments of the IAN RAS and Sintol LLC / / Bulletin of the Russian Military Medical Academy. 2021. Vol. 40. No. 3. pp. 69-74. DOI: https: / / doi.org / 10.17816 / rmmar76918).The KVNK complex uses disposable cartridges to isolate and purify nucleic acid from various samples in a disposable polymer cartridge. However, the nucleic acid isolation complex cannot perform nucleic acid analysis in the same cartridge used for nucleic acid isolation and purification. This device requires transferring the sample and extracted nucleic acid to a test tube and performing nucleic acid analysis in a third-party device—a thermal cycler.

[0014] A well-known automated system from the American company Cepheid, called "GenXpert," consists of a disposable cartridge and analyzer. This system allows for nucleic acid extraction and purification, followed by DNA testing, with real-time results recording, all within the same cartridge (US6374684). However, the disposable cartridge of this device contains only one DNA testing cell, limiting the number of targets that can be simultaneously detected and the ability to use a separate physical detection channel.

[0015] In addition, a device from the South Korean company SD Biosensor Inc. is known, which allows for the isolation and purification of nucleic acid and PCR simultaneously in several cells (US20210283595A1).

[0016] A DNA amplifier with real-time results recording is also known. It comprises a thermal cycler, including a heat-conducting element with means for accommodating reaction mixtures, automatic temperature control, an optical system including a radiation source and receiver, and a microprocessor-based signal processing and control unit (RU209636). The "Indicator-Bio" amplifier enables high-speed nucleic acid amplification on a disposable flat polymer chip and uses a multispectral sensor to detect the fluorescent signal. However, the "Indicator-Bio" device does not include a nucleic acid extraction module. This precludes fully automated use, a significant drawback that limits the use of the "Indicator-Bio" in clinical practice.Furthermore, the single-cell nature of the disposable polymer chip used for the PCR reaction prevents the use of a negative control sample, making it difficult to use this amplifier for clinical laboratory diagnostics. The lack of a negative control sample makes it impossible to identify sample contamination.

[0017] As a rule, material potentially containing Mycobacterium tuberculosis cannot be used as a PCR matrix due to its high levels of inhibitory impurities, high viscosity, and low mycobacterial DNA concentration. Mycobacteria themselves possess an extremely resistant double membrane and mycolic acid layer, preventing the use of traditional DNA extraction methods.

[0018] In this regard, since the beginning of the use of PNR as a molecular genetic method for detecting mycobacteria, ultrasound irradiation of the sample has been used for sample preparation of the biospecimen and subsequent isolation of NC (Buck GE, O'Hara LC, Summersgill JT. Rapid, simple method for treating clinical specimens containing Mycobacterium tuberculosis to remove DNA for polymerase chain reaction. J Clin Microbiol. 1992 May;30(5):1331-4.).

[0019] Therefore, devices specialized for working with M. tuberculosis must have certain distinctive features. They must have a separate or integrated sample preparation module that allows for highly efficient nucleic acid extraction from mycobacteria, as well as design elements that protect the operator from contamination when handling potentially biohazardous biomaterial.

[0020] A device for DNA extraction is known, consisting of a plate with multiple wells, a plate holder, rods for mixing the sample and magnetic retention of microparticles, as well as an ultrasonic emitter, which only allows for the extraction of nucleic acid from a biological sample, but cannot perform DNA analysis using amplification and determine the presence of target sequences (KR102528711).

[0021] Similar devices are described in 1) patent CN109576151, which allows for the extraction of nucleic acid from various samples using magnetic microparticles, or 2) patent CN 114752466 using paraffin-embedded samples and ultrasound.

[0022] A device is known for DNA extraction and subsequent amplification of a target sequence with real-time recording of the result, consisting of a rotating sample container component, a shaft lifting unit located in the upper part of the sample cartridge rotation unit and having a piston capable of moving samples in the sample cartridge between different chambers; an ultrasonic unit located in the lower part of the cartridge rotation unit and having an ultrasonic amplitude transducer for ultrasonic processing of a sample in the cartridge for extraction of the sample nucleic acid; a temperature increasing and decreasing unit; a unit providing the ability to isolate nucleic acid using magnetic microparticles and ultrasound with the ability to perform GID* with detection of amplification results in real time (CN215162719). However, the reaction occurs in a single test tube and is not isolated from the environment.This requires special conditions when working with contagious samples, such as the tuberculosis pathogen.

[0023] The closest analogue of the claimed device is a system for the isolation of nucleic acids and their subsequent amplification, which consists of at least one analyzer, a control console or a computer on which software is installed and a disposable cartridge with the analyzed sample (RU2813921).

[0024] However, this system is not a specialized device adapted for use with mycobacteria. It also does not provide for limiting exposure of the disposable cartridge's contents to the environment or for disinfecting the receiving device after analysis.

[0025] Thus, the technical problem solved by the claimed invention is the need to overcome the shortcomings inherent in analogues and the prototype by creating a specialized device for the automatic isolation of M. tuberculosis DNA and conducting multiplex real-time DNA analysis for the rapid and safe diagnosis of tuberculosis.

[0026] Disclosure of invention

[0027] The technical result of the invention is the creation of a specialized device capable of detecting the presence of Mycobacterium tuberculosis in biological samples and determining the resistance of the tuberculosis pathogen to antimicrobial drugs through the simultaneous amplification of several specific DNA regions in real time and automatically within no more than three hours from the time the sample is loaded. Key advantages of the device include reduced risks of contamination of the analyzer and the sample chamber, portability, suitability for use in unequipped laboratories, ease of use, high operator safety, low operator qualification requirements, and high analytical multiplexity.

[0028] The technical result is achieved due to the fact that the device for automatic isolation, purification and amplification of Mycobacterium tuberculosis DNA sections with recording of results in real time consists of a disposable cartridge with a filter system for sample preparation of a biological sample containing mycobacteria, isolation and purification of mycobacteria nucleic acids and PCR in real time, an ultrasonic generator and emitter, as well as an analyzer with a disinfection system for the cartridge receiving tray.

[0029] A disposable cartridge (Fig. 1) consists of a base with reservoir containers (1), a lid with capillaries for liquid movement (2) and holes for air movement - ventilation wells (3), which when assembled form reservoir containers into which the analyzed sample is loaded or chemical agents are located for carrying out the processes of nucleic acid extraction and PCR.

[0030] The ventilation wells (3) are directly connected to the atmosphere and serve to equalize atmospheric pressure within the cell when it is filled or emptied. When the cell is empty, atmospheric air enters the cell through the ventilation well (3), and when it is filled, it is forced out of the cell into the atmosphere. In this case, if the cell contains non-inactivated Mycobacterium tuberculosis, there is a possibility of transferring the pathogen outside the cell, causing contamination of the instrument and the room. To prevent this, a filter element with a pore diameter of 0.05 - 0.5 μm (4) is placed in the ventilation well of the cell into which the sample is loaded. This filter element retains the pathogen without clogging the ventilation duct or impeding airflow through it.This means that when using a filter element (4), the disposable cartridge protects the internal cavity between the lid and the surrounding environment from pathogen contamination of the analyzer and surrounding space, while maintaining the overall functionality of the platform. Any commercially available filter element can be used, such as those from Biocomma Limited (Part Number BF025-40-50, China).

[0031] An ultrasonic homogenizer and emitter are connected to the wall of the container (reactor) - in the cartridge, where the biological sample potentially containing mycobacteria is prepared, while the ultrasonic emitter is closely adjacent to the wall of the reactor, made in the form of a convex lens.

[0032] The cells (1) of the disposable cartridge contain reagents for the extraction and purification of DNA from the analyzed sample, followed by PCR inside the closed volume of the cartridge in a special cell or cells (5), with the possibility of introducing the analyzed sample into the cartridge in a cell with a sealed lid (6).

[0033] The analyzer (Fig. 2) is a device capable of interacting with a disposable cartridge and controlling the processes of DNA extraction and subsequent DNA testing with detection in real time within the closed volume of the cartridge.

[0034] The operator manually loads the disposable cartridge (8) into the analyzer (9). The operator places the cartridge (8) into the tray (10) and then manually closes the lid (11) away from him / herself until it stops. The cartridge (8) in the tray (10) moves into the analyzer and is fixed in the closed volume of the analytical instrument. The Analyzer's operating protocol with the cartridge is then started, which includes the movement of liquids between the cartridge cells and the operation of the ultrasound module. Filter elements (4) are located in the ventilation wells of the cartridge (3). In the event that a pathogenic agent escapes the cartridge and contaminates the analyzer due to cartridge depressurization, the analyzer is equipped with at least one LED UV radiation source (12) 260-370 nm, with a power of at least 2 mW, for disinfection.

[0035] These sources will be launched automatically after each analysis, as well as before each analyzer is turned on, and will disinfect the internal cavities of the analyzer.

[0036] Brief description of the drawings

[0037] Fig. 1 shows a general view of the cartridge of the device for automatic isolation, purification and amplification of sections of Mycobacterium tuberculosis DNA with recording of results in real time, where the position

[0038] 1 - Base with reservoir tanks.

[0039] 2 - Lid with capillaries for liquid movement.

[0040] 3 - Ventilation wells

[0041] 4 - Filter element

[0042] 5 - cells for commissioning

[0043] 6 - lid and hole under it (port) for introducing the sample

[0044] 7 - rotary valve

[0045] Fig. 2 shows a part of the analyzer with installed UV radiation sources, where

[0046] 8 - Disposable cartridge

[0047] 9 - Analyzer

[0048] 10 - Lodgement

[0049] 11 - Lid

[0050] 12 - Sources of UV radiation

[0051] 13 - Drive shaft

[0052] 14 - Permanent magnet

[0053] Implementation of the invention

[0054] The device for automatic isolation, purification and amplification of Mycobacterium tuberculosis DNA sections with recording of results in real time consists of a disposable cartridge (8) and an analyzer (9), with the ability to connect to an external personal computer.

[0055] The cartridge consists of multifunctional cells (a cell for introducing a sample; cells containing reagents: lysis, washing solutions, elution solution, PCR reagents; a reactor cell) (1); filter elements (4) Biocomma Limited Article BF025-40-50 (China) with a pore diameter of 50 μm are placed in the ventilation wells of the cartridge (3). A PCR chip with three independent cells (containers) (5, Fig. 1) and a rotary valve (7, Fig. 1), which combines all these units.

[0056] Fluids are transferred using the cartridge's rotary valve, which engages the analyzer's drive shaft (13, Fig. 2) when the cartridge is inserted. The rotary valve also contains a cylinder with a movable piston, which engages the analyzer's movable rod when the cartridge is inserted and performs a reciprocating motion with it. Fluids are transferred between the cartridge's cells as follows: The analyzer rotates the rotary valve and positions it directly over the desired cartridge cell. The movable rod begins to move downward, pulling the piston along with it, creating a negative pressure in the rotary valve. The fluid in the cell then flows into the rotary valve's cylinder. The distance the rod travels determines the volume of fluid to be withdrawn, similar to a syringe.The discharge of liquid from the rotary valve occurs in the reverse order: the rotary valve is oriented towards the desired cell, the piston moves upward and displaces the liquid from the cylinder into the cell.

[0057] The ultrasonic emitter is connected to the outer wall of the cartridge where the biological sample is prepared (1, Fig. 1). The ultrasonic emitter is tightly pressed against the reactor wall. The ultrasonic pulse duration is 1 second, the pause between pulses is 1 second, and the total number of pulses ranges from 40 to 60.

[0058] DNA extraction is performed using magnetic particles functionalized with surface molecules that specifically bind to DNA and adsorb it onto their surface. The analyzer has a movable permanent magnet (14, Fig. 2) that, when brought close to the cartridge cell, initiates the collection of magnetic particles from the solution on the cell wall and their immobilization during fluid aspiration. This allows for the purification of adsorbed DNA molecules from impurities by sequentially washing the cell containing the immobilized magnetic particles, followed by elution and desorption of DNA into the eluate. The resulting eluate is transferred via a rotary valve to the cell containing the PCR reagents, after which the resulting reaction mixture is transferred via a rotary valve to the cell (container) of the cartridge's microfluidic chip, where the PCR takes place. The DNA amplifier then operates in a known manner, for example as described in Bartlett, J. M. S., & Stirling, D.(2003). PCR Protocols. Methods in Molecular Biology (Vol. 226). Humana Press. ISBN 978-1-59259-384-2.

[0059] The amplification results are collected and analyzed using the analyzer software.

[0060] After each analysis, and before each analyzer is turned on, the 2 UVC 275 nm LEDs (12), located in the cavity where the cartridge is installed, automatically activate and disinfect the analyzer's internal cavities. Please indicate which UV emitters were used.

[0061] Example 1. Isolation of M. tuberculosis Rif+ DNA from a biological sample with subsequent amplification of two genome regions simultaneously.

[0062] The sample was introduced into the sample introduction cell using standard equipment (a dispenser). The reagent cells contained all the reagents required for sample preparation and PCR amplification. A rotary valve ensured the movement of reagents within the cartridge during sample preparation for amplification. The PCR chip is designed for automated PCR within a confined space.

[0063] The Amplitube-RV PCR kit, a reagent kit for extraction using magnetic silica, was used as the reagent for the tests.

[0064] A clinical sputum sample containing M. tuberculosis Rif+ with confirmed resistance to rifampicin was used. The device cartridge contained 500 μl of lysis solution L, 10 μl of magnetic particle suspension, 100 μl of the test sample, 600 μl of wash solution W1, 400 μl of wash solution W2, 400 μl of TE buffer W3, 600 μl of wash solution, and 400 μl of elution solution (TE buffer).

[0065] DNA was extracted from a clinical sample manually using the same reagents and in the same volumes as in the Device to obtain reference values ​​of fluorescence intensity in three channels for the fluorophores FAM, HEX, and ROX (Table 1). Table 1 - Result of PCR amplification of DNA from a clinical sample obtained manually

[0066] When performing GGPR amplification on the prototype, fluorescence growth began at cycle 21 in the FAM and HEX channels, which corresponds to reference values. The analysis time on the device was 2 hours and 15 minutes.

[0067] Thus, the use of an analyzer with built-in LED UV sources and an ultrasonic emitter, tightly adjacent to the wall of the cartridge, which contains filters that isolate the cells with biomaterial, allows us to achieve the stated technical result - the identification of M. tuberculosis safely for the operator in automatic mode.

Claims

CLAUSES OF THE INVENTION 1. A device for the automatic extraction, purification and amplification of Mycobacterium tuberculosis DNA sections with recording of results in real time, consisting of a disposable cartridge, an ultrasonic generator and emitter and an analyzer, which is a device capable of interacting with a disposable cartridge and controlling the processes of DNA extraction and subsequent PNR with detection of a fluorescent signal in real time inside a closed volume of the cartridge, characterized in that • the ventilation well of the cell of the disposable cartridge, where the analyzed sample is loaded, contains a filter element to retain the pathogen; • the analyzer includes at least one UV radiation source located inside the Analyzer, ensuring disinfection of the internal cavities of the Analyzer.

2. The device according to item 1, characterized in that the filter element protects the ventilation well of the remaining cells of the cartridge.

3. The device according to item 1, characterized in that LED lamps are used as sources of UV radiation.

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