KR20240108803A - Rapid and Point-of-Care Testing Biochip and Multiplex Molecular Diagnosis of Infectious Respiratory Disease Using the Same - Google Patents

Abstract

The present invention provides a biochip for rapid on-site diagnostic testing for respiratory infectious diseases, and the multi-molecular diagnostic device using the biochip according to the present invention is optimized for isothermal amplification reactions such as RT-LAMP, allowing for rapid and accurate on-site testing for respiratory infectious diseases such as coronaviruses. It has the advantage of enabling diagnostic testing.

Description

Rapid and Point-of-Care Testing Biochip and Multiplex Molecular Diagnosis of Infectious Respiratory Disease Using the Same}

The present invention relates to a biochip for rapid point-of-care diagnostic testing and a multi-molecular diagnosis method for respiratory infectious diseases using the same.

This invention is related to the results of research conducted with support from the “Regional Specialized Industry Development + (R&D, Project No. S3090125)” project of the Ministry of SMEs and Startups and the Korea Institute for Advancement of Technology.

Following the global spread of the novel coronavirus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2), rapid and accurate diagnosis of viral infection is considered one of the key response strategies for respiratory infectious diseases, including reverse transcription polymerase chain reaction. Transcription polymerase chain reaction (RT-PCR) is widely used as a standard diagnostic technique due to its high diagnostic accuracy.

However, since the general PCR method amplifies and detects the target genetic material by adjusting the reaction temperature dozens of times or more, a temperature control device that stably implements temperature transitions between high and low temperatures within tens of seconds is required, which reduces the time required for diagnosis and is economical. The human burden is pointed out as a major disadvantage.

An alternative to this is isothermal amplification, which allows amplification of target genetic material at a single temperature (room temperature or high temperature below 65°C) without the need to change the reaction temperature, so an expensive temperature control device is required. It has the advantage of being easy for point-of-care test (POCT), which is difficult to implement in general PCR (Literature [Yan, Lei, et al. "Isothermal amplified detection of DNA and RNA." Molecular BioSystems 10.5 (2014): 970-1003.] etc.).

However, in order to effectively implement the isothermal amplification method in point-of-care diagnostic tests, it is necessary to develop an effective biochip or microfluidic chip that allows detection and analysis of trace amounts of samples with just one sample injection without contamination (document [Giuffrida, Maria Chiara , and Giuseppe Spoto. “Integration of isothermal amplification methods in microfluidic devices: Recent advances.” Biosensors and Bioelectronics 90 (2017): 174-186.], etc.

Republic of Korea Patent Publication No. 10-2018-0101084 Republic of Korea Patent Publication No. 10-2021-0065460

Thompson, Dorian, and Yu Lei. “Mini review: Recent progress in RT-LAMP enabled COVID-19 detection.” Sensors and Actuators Reports 2.1 (2020): 100017. Zhou, Ling, et al. “Microfluidic-RT-LAMP chip for the point-of-care detection of emerging and re-emerging enteric coronaviruses in swine.” Analytica chimica acta 1125 (2020): 57-65. Giuffrida, Maria Chiara, and Giuseppe Spoto. “Integration of isothermal amplification methods in microfluidic devices: Recent advances.” Biosensors and Bioelectronics 90 (2017): 174-186. Yan, Lei, et al. “Isothermal amplified detection of DNA and RNA.” Molecular BioSystems 10.5 (2014): 970-1003.

One object of the present invention is to provide a biochip for rapid point-of-care diagnostic testing.

Another object of the present invention is to provide a multi-molecular diagnostic method for respiratory infectious diseases using the biochip.

In one aspect, the present invention provides a biochip for rapid point-of-care diagnostic testing, wherein the biochip includes an injection port through which a sample from a patient suspected of having a respiratory infectious disease is injected; A filter for filtering nucleic acid samples from the specimen injected into the injection port; a plurality of outlets through which nucleic acid samples passing through the filter are discharged; a multi-molecular diagnostic reaction chamber in which nucleic acid samples discharged from the outlet are stored; a first channel connecting the injection port and the filter to move the sample; a second channel connecting the filter and the outlet to move the nucleic acid sample; The first and second channels include one or more openings and closing parts that are opened by pressure when a sample is injected, but close the reverse fluid flow, and the second channel is connected to a plurality of outlets in a one-to-one manner through which the nucleic acid sample that has passed through the filter is branched. Make it possible; The nucleic acid sample is characterized in that it contains target RNA for respiratory infectious diseases.

Specifically, the specimen in the biochip is characterized in that the biological sample isolated from the patient has been pretreated with a lysis buffer.

More specifically, in the biochip, the filter selectively passes nucleic acids in the dissolved sample tissue and concentrates the target RNA in the nucleic acid sample.

More specifically, the filter in the biochip is characterized by having a porous structure containing at least one trillion selected from the group consisting of hydrogel, agarose, and paraffin.

Alternatively, the biochip may be made of polymethyl methacrylate, polycarbonate, cycloolefin copolymer, polyamide, polyethylene, polypropylene, polyphenylene ether, polystyrene, polyoxymethylene, polyether ether ketone, polytetraproporoethylene, poly Vinyl chloride, polyvinylidene fluoride, polybutylene terephthalate, fluorinated ethylene propylene, perfluoroalkoxyalkane, polydimethylsiloxane, cycloolefin copolymer, polymethyl methacrylate, polycarbonate, polypropylene carbonate, poly It is characterized by being based on one or more materials selected from the group consisting of ethersulfone and polyethylene terephthalate.

Alternatively, the biochip may be contacted with a multi-molecular diagnostic device targeting the nucleic acid sample within the reaction chamber.

Specifically, the multi-molecular diagnostic device in the biochip is characterized by performing an isothermal amplification reaction of a nucleic acid sample.

More specifically, the isothermal amplification reaction in the biochip is a Reverse transcriptional Loop-mediated Isothermal Amplicifation (RT-LAMP) reaction.

Alternatively, in the biochip, the respiratory infectious disease is selected from the group consisting of coronavirus, influenza virus, RSV (respiratory syncytial virus), adenovirus, enterovirus, PIV (parainfluenza virus), MPV (metapneumovirus), boca virus, and rhinovirus. It is characterized in that it contains one or more types of viral infectious diseases, and specifically, the viral infectious disease is a coronavirus including SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2).

In another aspect, the present invention provides the biochip for rapid point-of-care diagnostic testing described above; An isothermal amplification reaction device for nucleic acid samples that have passed through the biochip; and a detection system for detecting a signal generated from the amplification product after the isothermal amplification reaction.

Specifically, in the above multiple molecular diagnostic equipment, the isothermal amplification reaction is a reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction.

In another aspect, the present invention provides a method for multiple molecular diagnosis of respiratory infectious diseases using the above-described equipment for multiple molecular diagnosis of respiratory infectious diseases.

Specifically, the multi-molecular diagnostic method for respiratory infectious diseases includes pretreating biological samples isolated from patients with a lysis buffer; Injecting the sample pretreated with the dissolution buffer into the inlet of the biochip for rapid point-of-care diagnostic testing described above; To the nucleic acid sample discharged into the multi-molecular diagnostic reaction chamber of the biochip, a primer set for target RNA of respiratory infectious diseases, reverse transcriptase, and nucleic acid polymerase are added to target sequence through reverse transcription ring-mediated isothermal amplification (RT-LAMP) reaction. amplifying; and detecting the product of the amplification step.

In another aspect, the present invention provides a kit for multiple molecular diagnosis of respiratory infectious diseases, including the biochip for rapid point-of-care diagnostic testing described above.

The biochip according to the present invention is not only miniaturized to improve user convenience for rapid on-site diagnostic testing, but also prevents contamination due to sample mixing by selectively concentrating target RNA in samples derived from samples of patients suspected of respiratory infectious diseases and preventing backflow. Contamination due to sample overflow can be minimized by controlling the water level of the opening and closing part, so it can be used as a reliable biochip for on-site diagnostic testing. In addition, multiple molecular diagnosis using the biochip according to the present invention can be usefully used as a diagnostic tool for respiratory infectious diseases optimized for rapid point-of-care diagnostic testing by maximizing the advantages of isothermal amplification reaction.

1 shows a biochip according to an embodiment of the present invention;
2 and 3 are enlarged views of the filter portion of the biochip according to one embodiment of the present invention;
Figures 4 and 5 are enlarged views of the opening and closing portion of the biochip according to one embodiment of the present invention.

Hereinafter, the present invention will be described in detail with reference to the attached drawings. The following description should be understood as describing the present invention with specific examples, and the technical idea of the present invention is not limited to the following description. The attached drawings are provided to aid understanding of the present invention, and the technical idea of the present invention is not limited to the attached drawings. Additionally, in the drawings, the thickness or size of each member may be exaggerated, omitted, or schematically shown for convenience of explanation.

In the description of the structure of the present invention described in this specification, positional relationships and directions are based on the drawings attached to this specification, unless specifically mentioned.

In the description of the structure of the present invention described in this specification, the description of space or positional relationship refers to the relative positions between the components that make up the present invention. Additionally, unless specifically mentioned, another component may exist in the space between one component and another component. For example, when this specification refers to another component being located “on top” or “on” one component, it refers to another component being located directly on top of one component, as well as referring to one component. This includes cases where another component is located between the element and other components.

In the present invention, molecular diagnosis refers to a diagnostic technique that evaluates various molecular-level changes that occur within cells through numerical values or images. It detects molecular-level changes in DNA or RNA containing genetic information such as cells or viruses, thereby causing disease. It means diagnosing etc.

In the present invention, specimen refers to one or more types selected from blood, sputum, bacteria, semen, cell tissue, saliva, hair, urine, aspirate sample, swab sample, and cell culture medium. For example, those obtained by swabbing or nasal lavage from the nasopharynx or oropharynx, bronchial lavage or aspirate, intertracheal aspirate and bronchial biopsy, sputum, bronchoalveolar lavage, saliva, blood, urine, feces, sweat. etc. can be used.

The specimen or biological sample may be pretreated to increase the efficiency of the amplification reaction. In the present invention, pretreatment refers to preliminary treatment by applying chemical and physical actions prior to molecular diagnosis based on specific base sequences and gene detection. Specifically, it refers to the process of separating interfering substances contained in the sample before biochip injection, nucleic acid extraction and amplification. Pretreatment may include diluting 10-, 50-, 100-, 200-, or 400-fold with water, followed by heat treatment with proteinase K, anion exchange chromatography, affinity chromatography, size-specific exclusion chromatography, Genes may be extracted from biological samples or purified to a certain level using commonly known methods such as liquid chromatography, continuous extraction, centrifugation, or gel electrophoresis, or using commercially available kits.

In one embodiment, the pretreatment is lysis buffer containing 50mM Tris-HCl (pH8.0), 150mM NaCl, 5mM EDTA, and 1% NP-40, Brij-35 0.1% ~ 0.5w/w%, Tris- Sample sample as a solution containing HCl 10mM to 300mM (pH7.0 to 8.0), and CaCl2 1mM to 10mM, specifically Brij-35 0.3w/w%, Tris-HCl 100mM (pH7.5), and CaCl2 5mM. It may mean processing, and specifically, it may mean obtaining only genes from the sample by treatment with the above solution and purification by a conventional method, but is not limited to this. The combination of the above pretreatment solutions allows genes to be easily extracted from the sample and can improve the isothermal amplification reaction efficiency of the primer set of the present invention.

In another embodiment, chemicals for pretreatment include sodium hydroxide (NaOH), sodium bicarbonate, N-acetyl-L-cysteine (NALC), dithiothreitol (DTT), etc. for liquefying the sample and dissolving the surface of cells and viruses. Surfactants such as Triton X-100, Triton Octyl glucoside, Octyl thioglucoside, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), 3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO), sodium dodecyl sulfate (SDS), protein decomposition substances such as sputum proteases, Serine Proteases, Proteinase K, Tissue proteases, etc., and at least one of ethanol, isopropanol, chaotropic agents (sodium iodide, sodium perchlorate, guanidinium thiocyanate, guanidinium hydrochloride), and silica particles for nucleic acid extraction. may be used, but is not limited to this example.

In the present invention, the biochip may be a lab-on-a-chip or a microfluidic chip. Lab-on-a-Chip miniaturizes the components of biology and chemistry laboratories and integrates them with microfluidic-based technology on a single chip, performing all steps from pre-processing of small amounts of samples to mixing, reaction, separation, and analysis on a single chip. It refers to a device implemented to enable this. Therefore, it has been used interchangeably as a micro total analysis system (μTAS) or a microfluidic device. Lab-on-a-Chip, which refers to a laboratory that can be placed on the palm of your hand, is used as a universal term that intuitively expresses the properties of technology. A microfluidic device consisting of a microvalve, micropump, microfilter, and mixer, a biofilter and channel to control the movement of analytes such as antigens, target proteins, and nucleic acids, and a sensor to analyze and detect samples, and this microfluidic device. Actuators to drive and control are integrated on the chip through the MEMS processing process. The microfluidic system used in lab-on-a-chip is based on the physical characteristics of the fluid applied in the micro environment, unlike the macro environment. First, in the microfluidic environment, more predictable laminar flow (a phenomenon in which fluids flow in layers without mixing) dominates instead of turbulent flow. Second, the surface and interstitial tension, which lowers the free energy by reducing the contact surface between air and fluid or two types of immiscible fluids (oil and water), separates proteins and cells or causes fluid to separate due to surface energy difference. It is used to cause passive drive of the enemy. Third, capillary force acts to induce the flow of fluid in micro-scale pipes. Lab-on-a-chip-based technology enables rapid analysis from seconds to minutes, and utilizes extremely small amounts of samples and reagents in the micro (10-6) to femto (10-15) units, enabling various types of samples with high sensitivity and improved accuracy. allows simultaneous analysis. In addition, it has the potential as a high-throughput analysis platform because it can obtain reproducible quantitative information.

In the present invention, the biochip includes several subassemblies including a fluid subassembly, a pneumatic subassembly, a valve subassembly, a bulk fluid handling subassembly, and a separation and detection subassembly. Not all subassemblies are required for a given biochip; for example, if bulk fluid sample volumes and on-chip reagents are not required, bulk fluid subassemblies are not required. Nonetheless, the biochip of the present invention will generally have at least one CNC-machined, embossed, extruded or injection molded layer. A single layer will contain both the fluidic features and control features necessary to move fluid from one area of the biochip to another. In many cases, separate fluid layers and control layers are desirable, allowing for increased processing complexity. The layer or layers may have features on one or both sides; The advantages of double-sided features are increased processing complexity, feature density, and sample number, reduced fabrication cost, and ease of assembly. If multiple layers are used, they can be joined using a number of techniques known in the art, such as thermal bonding, solvent bonding, adhesive bonding, and ultrasonic welding. Considering the number of layers within a microfluidic biochip, it is recommended to use the fewest number of layers that allow the desired complexity to be achieved. Minimizing the number of layers reduces the complexity of fabrication and assembly, and ultimately the cost of the biochip system.

The biochip of the present invention is a closed system in which the sample is inserted into the biochip and sealed, and there is no liquid leakage from the biochip (i.e., the processing reagents contained within the biochip at the beginning of sample processing and contained within the biochip at the end of sample processing are When the biochip is separated, it is separated from the machine). Air from the pneumatic guide line enters the biochip, and air exits the biochip through the vent membrane. The closed system biochip of the present invention simplifies machinery in that it does not expose operators to samples or compounds and important safety features, and no unused or spent reagents need to be stored within the machine.

Injection molding of microfluidic features presents many challenges due to the large number of microscopic features that must be present in a complex, fully integrated biochip. These microfluidic features are often present on parts provided with bulk fluidic features, which further increases the difficulty of injection molding. Although the inventors present herein specific solutions for designing considerations for specific types of biochips, those skilled in the art will be able to adapt the specific solutions to fabricate multiple types of biochips for specific functions. Design considerations for the above example are: geometry, such as shrinkage and shape stability in correct injection molding, flatness of the biochip, bowing of the biochip, minimum feature size, feature density, and appropriate aspect ratio. The design features of the injection molded components of the biochip of the present invention will be described in relation to the injection molding process. Injection molding is a process for manufacturing plastic parts in which a mold is clamped under pressure to accommodate the molten plastic injection and cooling process. Plastic granules (small granulated resin) are fed into the injection molding machine, followed by appropriate colorants. The resins are fed into the injection barrel, where they are heated to melting point and injected into the mold via a reciprocating screw or ramming device. Molten plastic is contained within a mold, and hydraulic or mechanical pressure is applied to ensure that all cavities within the mold are filled. The plastic cools in the mold, the mold is opened, and the plastic part is ejected through an ejection pin. The entire treatment is cyclical, with the number of cycles ranging from 10 to 100 seconds depending on the cooling time required.

As shown in Figure 1, the biochip 100 for rapid point-of-care diagnostic testing according to one embodiment of the present invention includes an injection port 110 through which a sample of a patient suspected of having a respiratory infectious disease is injected; A filter 140 that filters nucleic acid samples from the specimen injected into the injection port; A plurality of outlets 120 through which nucleic acid samples passing through the filter are discharged; a multi-molecular diagnostic reaction chamber 130 in which nucleic acid samples discharged from the outlet are stored; A first channel 160 that connects the injection port and the filter to move the sample; A second channel 170 connecting the filter and the outlet to move the nucleic acid sample; The first and second channels include one or more openings and closing portions 150 that are opened by pressure when a sample is injected into the first and second channels, but close the reverse fluid flow, and the second channel branches the nucleic acid sample that has passed through the filter into a plurality of outlets. It is characterized by one-to-one connection.

The type of substrate of the biochip 100 is not particularly limited as long as it is made of a material that facilitates the flow of biological samples and functions as a filter and opening/closing unit, but is preferably polymethyl methacrylate, polycarbonate, or cycloolefin. Copolymer, polyamide, polyethylene, polypropylene, polyphenylene ether, polystyrene, polyoxymethylene, polyether ether ketone, polytetrapropoethylene, polyvinyl chloride, polyvinylidene fluoride, polybutylene terephthalate, Composed of polymer materials such as fluorinated ethylene propylene, perfluoroalkoxyalkane, polydimethylsiloxane, cycloolefin copolymer, polymethyl methacrylate, polycarbonate, polypropylene carbonate, polyether sulfone, polyethylene terephthalate, or combinations thereof. do. In this case, it may be formed as inserted between the base substrate, which is the lower substrate, and the cover substrate, which is the upper substrate, and may be formed of the same or different plastic materials.

A plurality of reaction chambers and a plurality of channels are formed inside the biochip 100 to provide a structure that enables multiple molecular diagnosis for each reaction chamber (FIG. 1). At this time, the sample injected into the injection port 110 is evenly discharged through the outlet 120 and simultaneously stored in a plurality of reaction chambers 130, and can be used in the subsequent isothermal amplification reaction. The inlet 110 and outlet 120 can be visualized as the same size as Figure 1, but can be formed in various shapes depending on the purpose of multimolecular diagnosis.

If necessary, a heat source that applies heat while the nucleic acid sample moves from the inlet 110 to the outlet 120 may be installed, and this may be appropriately selected depending on the purpose of multimolecular diagnosis. The heat source unit is not limited to a particular type, and may employ, for example, a copper heating block, thin film heater, infrared device, halogen lamp, microwave, platinum resistor, or induction heating device.

In addition, in order to control the physical properties of the biochip 100, plastic films may be stacked in the vertical direction, such as polyethylene terephthalate (PET), polyethylene (PE), polypropylene (PP), polyimide (PI), Polystyrene (PS), polycarbonate (PC), polyurethane, polyvinylidene fluoride, nylon, polymethyl methacrylate (PMMA), polyvinyl chloride (PVE), polyether sulfone (PES), etc. can be used appropriately. . The bonding of each film can be accomplished by a mechanical bonding method using a normal bonding member, or by a method using an adhesive. At this time, the adhesive is sufficient as long as it has sufficient adhesiveness to bond each film to each other to form a space for the flow of the sample, and is not limited to a specific type of adhesive. Examples of types of adhesives include rubber adhesives, acrylic resin adhesives, silicone adhesives, optical adhesives, and heatable adhesives. Additionally, examples of the types of adhesives include pressure-sensitive adhesive tapes, heat-activated adhesive tapes, chemically-activated adhesive tapes, and light-activated adhesive tapes.

Referring to Figures 2 to 5, the sample injected into the injection port passes through the filter 140, and at this time, the filter passes the dissolved tissue nucleic acid (e.g., target RNA) and filters out impurities such as other particles. Perform. The sample filtered from the outside is filtered through the filter once more, thereby improving the overall reliability of the biochip. The type of the filter is not particularly limited as long as it is a material that performs the function of selectively concentrating nucleic acids such as target RNA, but is preferably composed of hydrogel, agarose, and paraffin. It may have a porous structure containing one or more cells selected from the group.

The biochip 100 includes one or more stoppers that are opened by pressure when a sample is injected into the first channel 160 and the second channel 170, but close the reverse fluid flow. The opening/closing unit 150 is in the form of a valve and functions to prevent contamination due to sample mixing by preventing backflow. Through the filter 140 and the opening/closing unit 150, the biochip 100 of the present invention can function as a reliable multi-molecular diagnostic device in rapid point-of-care diagnostic testing.

The biochip 100 of the present invention may include a microfluidic chip controller, where the controller includes at least one rotary electrical contact couple configured to couple a stationary controller and an electrical circuit for supply of control signals, data, and power. May include a ring. The microfluidic chip controller may be configured to measure the characteristics of the chip or the liquid contained therein by further including a sensor or actuator mounted on the main body and/or the chip. The sensor or actuator may be connected to an electrical circuit and may be configured to feed back information about the location of the fluid within the chip or to change the location of the fluid within the chip. The sensor or actuator may be comprised of a chemical, physical, or electrical sensor or actuator, and may include a temperature sensor or regulator, a fluid dynamic sensor or regulator, an optical sensor or optical emitter, etc.

In the biochip 100 of the present invention, the additional chambers 180 and 190 serve as a dam and prevent sample overflow by controlling the water level when more than an appropriate amount of sample is administered, thereby minimizing contamination. Perform.

The biochip 100 of the present invention is installed in contact with the multimolecular diagnostic device 300, so that the sample injected into the injection port 110 of the biochip 100 is effectively transmitted as a sample for multimolecular diagnostics. In this case, the multimolecular diagnostic device 300 in contact with the reaction chamber 130 may be an isothermal amplification reaction device 400, and the nucleic acid sample is equally delivered to the plurality of reaction chambers 130, and the isothermal amplification reaction device 400 is prepared in advance. An effective isothermal amplification reaction can be initiated by mixing with the amplification reaction sample. A detection system 500 may be installed to detect the signal formed from the reaction product during or after the isothermal amplification reaction. In this case, the biochip 100, the isothermal amplification device 400, and the detection system 500 By forming a complete device for rapid on-site diagnostic testing, there is an advantage in enabling rapid diagnostic testing by doctors or non-experts in the field where rapid diagnosis of respiratory infectious diseases is required.

As an example, the detection system 500 may be based on a colorimetric diagnostic method, and may include a sample pad for receiving a sample; a buffer pad disposed separately from the sample pad and containing a rehydration buffer; a first connection pad disposed on the sample pad and connecting the sample pad and the reaction pad; an initiator pad disposed on an upper portion of the buffer pad and connecting the buffer pad and the reaction pad; a reaction pad disposed below the first connection pad and the initiator pad, including a primer capable of specifically binding to a target nucleic acid and a reagent for an isothermal amplification reaction (LAMP), where the isothermal amplification reaction occurs; A blocking pad disposed on the reaction pad and maintaining the reaction temperature and blocking evaporation of the sample; a second connection pad disposed on the reaction pad and on which gold nanoparticles are fixed; A detection pad disposed below the second connection pad and acquiring a target nucleic acid amplified from the isothermal amplification reaction product bound to the gold nanoparticles; and an absorption pad disposed on a side of the detection pad to absorb remaining sample, and may include a heating pad disposed below the sample pad, the initiator pad, the reaction pad, and the second connection pad. In this case, the gold nanoparticles may include streptavidin on the surface, but are not limited thereto.

The biochip 100 may include a single or multiple valves in addition to the above-described devices, and may be appropriately disposed at the inlet or outlet portion to transfer the specimen or nucleic acid sample in the forward direction or block the forward transfer to the reaction chamber. (130) It can help in the process of effective capture. A syringe pump containing a sample can be connected to the injection port 110 to inject the sample.

The reaction chamber 130 is formed to have a microchannel to which an appropriate heat source (e.g., 60 to 70°C temperature range) for isothermal amplification reaction can be attached, and additional samples for isothermal amplification reaction, such as reaction buffer, dNTPs, and nucleic acid polymerization. Enzymes and primers may be included, and other conventional isothermal amplification reaction (e.g., RT-LAMP) reagents may be added. By pre-loading the isothermal amplification reaction reagent, the usability of the biochip 100 in field diagnosis can be improved.

The isothermal amplification reaction detection system 500 can perform genetic analysis using an externally located optical module once amplification of the target nucleic acid is completed. The optical module includes a light source for emitting light (e.g., a laser) toward the sample collected in the amplification and reaction chamber 130 (containing a label such as a fluorescent substance for analyte detection), and receiving reflected light to receive the sample. It may include a CCD camera that detects fluorescent signals and converts them into digital signals for image processing. Since the detection system is made of a light-transmitting material, there is no obstacle to analyzing target nucleic acids using this optical module. The read sample may be discharged through additional transfer channels and/or outlets.

The channel and fluid transfer formed in the biochip 100 described above can be formed in, for example, a straight pattern, a curved pattern (spiral, serpentine, zigzag, etc.), or a random pattern, using the shape shown in FIG. 1 as an example. there is. At this time, a valve member may be disposed in each disconnected section as needed to form a fluid bypass passage for each disconnected section to control the movement of the fluid, but is not limited to this.

Control of fluid movement and fluid movement speed (flow rate) within the biochip 100 can be achieved through appropriately arranged ports. These ports can each be connected to a pressure regulator. If the pressure of the forward direction side passage is less than the pressure of the backward direction side passage based on the fluid inside the biochip 100, the fluid moves in the forward direction. The larger the pressure difference, the faster the fluid will move. In the opposite case, the fluid will move backwards. Therefore, the fluid movement and flow rate within the biochip 100 can be controlled through the pressure control device connected to each port. As the pressure control device, a device with a normal air supply function and air suction function can be used, so detailed description is omitted.

As described above, the biochip according to embodiments of the present invention concentrates the specimen within one chip, forms a filter for extracting the target nucleic acid, and a reaction chamber that collects the separated nucleic acid and reads it through an isothermal amplification reaction. It allows sample concentration, isothermal amplification reaction, and diagnosis to all be performed on one chip. Accordingly, the presence of the respiratory leaf disease virus can be quickly confirmed even in the field without specialized personnel.

The types of respiratory infectious diseases that can be diagnosed through the biochip of the present invention and the multimolecular diagnostic equipment using the same are not particularly limited, but are preferably coronavirus, influenza virus, RSV (respiratory syncytial virus), adenovirus, enterovirus, and PIV. It may include one or more viral infectious diseases selected from the group consisting of (parainfluenza virus), MPV (metapneumovirus), boca virus, and rhinovirus. Preferably, the viral infectious disease may be a coronavirus such as SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2).

Coronaviruses are enveloped viruses and their positive-strand RNA genomes, the largest of all RNA viruses, encode up to 16 non-structural proteins (NSPs), 4 major structural proteins and up to 8 accessory proteins. Many of these proteins provide essential, often enzymatic, functions during the viral life cycle and therefore represent attractive targets for antiviral intervention. Antiviral strategies are mainly proposed to target coronavirus entry and essential enzymatic functions, such as coronavirus protease or RNA-dependent-RNA polymerase (RdRp) activity. For example, the spike (S) protein mediates the binding of different CoVs to specific cellular receptors, an event associated with preferential viral tropism toward ciliated or non-ciliated cells of the airway epithelium. The S protein also mediates fusion between the lipids of the viral envelope and the host cell plasma membrane or the membrane of the phagocytosis vesicle, facilitating the transfer of viral genomic RNA into the cytoplasm.

Virus binding and cell entry events can be inhibited by antibodies directed against the S protein, antibodies or small molecules that interfere with the viral receptor, or synthetic peptides derived from the fusion-inducing heptad repeat region of the S protein. After virus entry, the positive sense coronavirus genome, a capped and polyadenylated RNA strand, is directly translated to synthesize the coronavirus replacase gene-encoded nsp. Coronavirus NSPs have two large proteolytic enzymes, papain-like and chymotrypsin-like proteinases, that extensively process the coronavirus polyprotein to liberate up to 16 NSPs (nsp 1-16). Translated as a polyprotein. This proteolytic function is considered essential for coronavirus replication, and as a result, many candidate drugs have been reported to inhibit coronavirus polyprotein processing. Likewise, it is known that coronavirus RdRp activity in nsp8 and nsp12 is essential for coronavirus replication. Additionally, coronaviruses encode a series of RNA-processing enzymes that represent additional candidate targets. These are the helicase activity related to the NTPase activity of nsp13, the 3'-5'-exonuclease activity related to the N7-methyltransferase activity of nsp14, the endonuclease activity of nsp15 and the 2'-O-methyltransferase activity of nsp16. Contains lase activity.

Like all positive-strand RNA viruses, coronaviruses undergo this critical phase of the viral life cycle in a special environment rich in replicating virus and host cell factors and at the same time protected from antiviral host defense mechanisms. synthesizes RNA. There is now increasing knowledge about the involvement, rearrangement and necessity of cell membranes for RNA synthesis in many positive-strand RNA viruses, including coronaviruses. Three coronavirus nsps, nsp3, nsp4, and nsp6, are thought to participate in the formation of this site for viral RNA synthesis. In particular, these proteins contain multiple transmembrane domains that are thought to anchor the coronavirus replication complex through the recruitment of intracellular membranes into complex membranes interconnected through an outer membrane with a rough ER and variants containing double-membrane vesicles (DVMs). and often form a reticulovesicular network (RVN) of paired membranes.

In the multimolecular diagnostic method of the present invention, suitable reactants and reagents for performing the isothermal amplification reaction may include, for example, additional primers, dNTPs, and enzymes (DNA polymerase or reverse transcriptase), and the reaction For efficiency, it may further contain inorganic salts or surfactants such as potassium chloride, magnesium sulfate, and ammonium sulfate. In one embodiment, the reactants and reagents are 10 to 20mM Tris-HCl, 1 to 10mM (NH4)2SO4, 10 to 50nM KCl, 0.1 to 2mM MgSO4, and 0.01 to 0.1% Tween-20, more specifically, based on the final reaction solution. It may be a solution of pH 7 to 9, preferably pH 8.8, containing 20mM Tris-HCl, 10mM (NH4)2SO4, 50nM KCl, 2mM MgSO4, and 0.1% Tween-20. The reactant or reagent may further include 40 to 50 mM sucrose, 0.001 to 0.01% Triton X-100, and 0.1 w/w% to 0.3 w/w% of glycerol. Trehalose can be used instead of the sucrose. The combination of sucrose, Triton X-100, and glycerol helps maintain reaction efficiency by increasing the storage stability of virus diagnostic compositions and kits. In one embodiment, to improve the clarity of diagnosis of respiratory infectious diseases and to visually confirm reaction results, reagents such as colorimetric analysis substances, fluorescent analysis substances, or detection labeling compounds may be further included. Such reagents may be commonly known or commercially available, but are not limited thereto. In order for the reaction to occur efficiently, these reagents may include Tris-HCl, (NH4)2SO4, KCl, MgSO4, and Tween-20 as mentioned above, and amplification such as DNA polymerase and reverse transcriptase. Enzymes for performing the reaction may be further included.

In addition, methods for confirming virus detection results include, but are not limited to, electrophoresis and radioactivity measurement using detection labels.

The isothermal amplification method can detect target genetic material quickly and easily without complicated auxiliary equipment, so it is being actively researched in various fields such as medical diagnosis and environmental sensors. As the demand for on-site diagnosis increases, more accurate and faster diagnosis and easier use are being conducted. Development of devices is actively taking place.

Isothermal amplification reactions that can be applied to the biochip of the present invention include, for example, TMA (transcription mediated amplification), NASBA (nucleic acid sequence-based amplification), SMART (signal mediated amplification of RNA technology), SDA (strand displacement amplification), and RCA. (rolling circle amplification), LAMP (loop-mediated isothermal amplification), IMDA (isothermal multiple displacement amplification), HAD (helicase dependent amplification), RPA (recombinase polymerase amplification), single primer isothermal amplification (SPIA), etc. may be mentioned, preferably LAMP.

LAMP or loop-mediated isothermal amplification creates a loop structure (stem-loop DNA) at the primer binding site from 4 primers that are combined by selecting 6 parts from the target DNA strand. It is a nucleic acid amplification method that amplifies DNA through chain displacement reaction. In this case, amplification can be achieved by binding to the target ssDNA at an isothermal temperature of 60-65°C. This method cannot be applied to targets that are too short, but is possible for targets consisting of a few hundred bases. Even if there are DNA strands that interfere with complementary binding, the amount of sample can be amplified more than 100 times within one hour.

In the present invention, ring-mediated isothermal amplification refers to a method that can perform an amplification reaction under isothermal conditions, unlike the existing polymerase chain reaction (PCR) method. Basically, 4 types of primers (F3, B3, FIP, BIP) are required for the LAMP reaction, and to improve the reaction speed, 2 types of primers (LF, LB) are added to produce 6 different base sequences. Oligonucleotide primers are required for the reaction.

The above four types of basic primers consist of two types of outer primers and two types of inner primers, and the outer primers include two types of forward outer (F3) primer and reverse outer (B3) primer. It is composed of and plays a role in unwinding DNA double strands during the non-cyclic step of the reaction. The internal primer consists of two types, the forward inner primer (FIP) and the reverse inner primer (BIP), and is attached to the forward and reverse base sequences to create a loop essential for the ring-mediated isothermal amplification reaction. It is composed of the corresponding nucleotides. The additional two types of primers are composed of a forward loop (LoopF) primer and a backward loop (LoopB) primer, and attach to a base sequence that the inner primer does not bind to, thereby carrying out a loop-mediated isothermal amplification reaction. Accelerates (Patent No. 10-2313941, etc.).

In one embodiment, when the respiratory infectious disease to be diagnosed in the present invention is SARS-CoV-2, a coronavirus, GenBank accession no. NC_045512.2; Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, SEQ ID NO. 1, specific to the 201st to 500th base sequence, which is the non-mutating site of the gene encoding the nucleocapsid protein (N gene, SEQ ID NO. 7) in the complete genome A primer set for RT-LAMP consisting of oligonucleotides from 6 to 6 may be used, but is not limited thereto.

In the primer set consisting of oligonucleotide primers of SEQ ID NO: 1 to 6 of the present invention, the primer of SEQ ID NO: 1 is F3, a forward external primer, the primer of SEQ ID NO: 2 is B3, a reverse external primer, and the primer of SEQ ID NO: 3 is FIP, a forward internal primer, the primer of SEQ ID NO: 4 is BIP, a reverse internal primer, the primer of SEQ ID NO: 5 is LoopF, a forward loop primer, and the primer of SEQ ID NO: 6 is LoopB, a reverse loop primer.

The primer of the present invention may include an oligonucleotide consisting of a segment of 15 or more, 16 or more, and 17 or more consecutive nucleotides in SEQ ID NOs: 1, 2, 5, and 6, depending on the sequence length of each primer, It may include an oligonucleotide consisting of a segment of 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, and 37 or more consecutive nucleotides in SEQ ID NOs: 3 and 4. For example, the primer (20 oligonucleotides) of SEQ ID NO: 1 includes oligonucleotides consisting of segments of 15 or more, 16 or more, 17 or more, 18 or more, 19 or more nucleotides in the sequence of SEQ ID NO. can do.

In the present invention, a primer refers to a single-stranded oligonucleotide sequence complementary to the nucleic acid strand to be replicated, and can serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primers should allow for initiation of synthesis of the extension product. The specific length and sequence of the primer will depend on the complexity of the DNA or RNA target desired as well as the conditions under which the primer is used, such as temperature and ionic strength.

In the present invention, oligonucleotides used as primers may also include nucleotide analogues, such as phosphorothioates, alkylphosphorothioates or peptide nucleic acids, or May contain an intercalating agent.

Additionally, each primer of the present invention may be modeled with a detection label to increase convenience of detection. The label for detection may be a compound, biomolecule, or biomolecule analog that can be linked, bound, or attached to a primer to check the density, concentration, amount, etc. of the amplification product in a conventional manner, but is not limited to FAM, VIC, etc. , TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, TYE563, BIOTIN, DIGOXIGENIN and NED, etc. can be used.

When the kit of the present invention is for Direct RT-LAMP, the kit further includes a lysis buffer, and the lysis buffer preferably contains 0.1 to 1.5% (v/v) of Triton X-100, pH 6.0. It may be 30-500mM Tris at pH 7.0, more preferably 300-500mM Tris at pH 6.5, containing 0.8-1.2% (v/v) Triton may be, but is not limited to, 400 mM Tris at pH 6.5, containing 1% (v/v) Triton X-100.

Additionally, in the kit of the present invention, reagents for performing the reverse transcription ring-mediated isothermal amplification reaction may include reverse transcriptase, DNA polymerase, dNTPs, and buffer. The DNA polymerase according to one embodiment of the present invention may be Bst polymerase, but is not limited thereto, and is mainly used in the existing ring-mediated isothermal amplification (LAMP) method for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Instead of the existing Bst polymerase, GspSSD DNA polymerase, which has faster amplification ability and stronger reverse transcriptase ability, can be used, but is not limited to this.

In addition, the kit of the present invention may additionally include a user guide describing optimal reaction performance conditions. The guide is a printed material that explains how to use the kit, for example, how to prepare the buffer, and the suggested reaction conditions. Instructions include information leaflets in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. Additionally, the guide includes information disclosed or provided through electronic media such as the Internet.

<Example>

In one embodiment of the present invention, multiple diagnosis using the biochip of the present invention using LAMP primers (SEQ ID NOs. 1 to 6) designed to target the coronavirus SARS-CoV-2 can be performed using RT-LAMP. You can.

In this case, total RNA is extracted from 50㎕ of the sample obtained by nasal swab, then introduced into the biochip for rapid point-of-care diagnostic testing provided as an embodiment of the present invention, and then subjected to RT-LAMP reaction for the RNA in the reaction chamber. can be performed.

For 2㎕ of template RNA, RT-LAMP premix (1㎕ of Bst polymerase (8U), 2.5㎕ of 10×buffer, 1.5㎕ of 25mM dNTPs, 1㎕ of 10mM MgSO4, 5㎕ of 5M betaine, 0.1㎕ (10U) of Reverse Transcriptase), 1㎕ of each of the six primers (5pmol each for F3 and B3, 40pmol each for FIP and BIP, and 10pmol each for LoopF and LoopB) was mixed with 1㎕ of 25X SYBR Green I and distilled water (up to 25㎕) at 50°C. After reacting for 5 minutes, isothermal amplification reaction is performed at 68°C for 30 minutes using a BioRad CFX 96 Real time PCR device, and the fluorescence signal can be measured every 20 seconds.

Above, the technical idea of the present invention has been described in detail. However, those of ordinary skill in the technical field to which the present invention pertains will understand that this invention includes simple design changes, omission of some components, simple changes in use, etc. according to specific application of the technology within the scope of the technical idea of the present invention described in the claims. It is obvious that the invention can be modified in various ways, and that such modifications are also included within the scope of the rights of the present invention.

100: biochip 110: injection port
120: outlet 130: reaction chamber
140: Filter 150: Opening/closing part
160: 1st channel 170: 2nd channel
180: Additional chamber 190: Additional chamber
200: Biochip substrate 300: Multi-molecular diagnostic device
400: Isothermal amplification reaction device 500: Detection system

Sequence list electronic file attached

Claims (15)

An injection port through which samples from patients suspected of having respiratory infectious diseases are injected;
A filter for filtering nucleic acid samples from the specimen injected into the injection port;
a plurality of outlets through which nucleic acid samples passing through the filter are discharged;
a multi-molecular diagnostic reaction chamber in which nucleic acid samples discharged from the outlet are stored;
a first channel connecting the injection port and the filter to move the sample;
a second channel connecting the filter and the outlet to move the nucleic acid sample;
Comprising one or more openings and closing parts that open by pressure when injecting a sample into the first and second channels but close the reverse fluid flow;
The second channel allows the nucleic acid sample that has passed through the filter to be branched and connected one-to-one to multiple outlets;
A biochip for rapid point-of-care diagnostic testing, wherein the nucleic acid sample contains target RNA for respiratory infectious diseases. The biochip for rapid point-of-care diagnostic testing according to claim 1, wherein the specimen is a biological sample isolated from a patient and is pretreated with a lysis buffer. The biochip for rapid point-of-care testing according to claim 2, wherein the filter selectively passes nucleic acids in the dissolved sample tissue and concentrates the target RNA in the nucleic acid sample. The biochip for rapid point-of-care testing according to claim 3, wherein the filter has a porous structure containing at least one trillion selected from the group consisting of hydrogel, agarose, and paraffin. . The method of claim 3, wherein polymethyl methacrylate, polycarbonate, cycloolefin copolymer, polyamide, polyethylene, polypropylene, polyphenylene ether, polystyrene, polyoxymethylene, polyetheretherketone, polytetraproporoethylene, Polyvinyl chloride, polyvinylidene fluoride, polybutylene terephthalate, fluorinated ethylene propylene, perfluoroalkoxyalkane, polydimethylsiloxane, cycloolefin copolymer, polymethyl methacrylate, polycarbonate, polypropylene carbonate, A biochip for rapid on-site diagnostic testing, characterized in that it is based on one or more materials selected from the group consisting of polyethersulfone and polyethylene terephthalate. The biochip for rapid point-of-care testing according to claim 1, wherein the biochip is in contact with a multi-molecular diagnostic device targeting the nucleic acid sample in the reaction chamber. The biochip for rapid point-of-care diagnostic testing according to claim 6, wherein the multiple molecular diagnostic device performs an isothermal amplification reaction of a nucleic acid sample. The biochip for rapid point-of-care testing according to claim 7, wherein the isothermal amplification reaction is a Reverse transcriptional Loop-mediated Isothermal Amplicifation (RT-LAMP) reaction. The method of claim 1, wherein the respiratory infectious disease is selected from the group consisting of coronavirus, influenza virus, respiratory syncytial virus (RSV), adenovirus, enterovirus, parainfluenza virus (PIV), metapneumovirus (MPV), boca virus, and rhinovirus. A biochip for rapid point-of-care diagnostic testing, characterized in that it contains one or more types of viral infectious diseases. The biochip for rapid point-of-care diagnostic testing according to claim 9, wherein the viral infectious disease is a coronavirus, including SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). A biochip for rapid point-of-care testing according to any one of claims 1 to 10;
An isothermal amplification reaction device for nucleic acid samples that have passed through the biochip; and
A detection system that detects a signal generated from the amplification product after the isothermal amplification reaction;
Including, equipment for multiple molecular diagnosis of respiratory infectious diseases. The device for multiple molecular diagnosis according to claim 11, wherein the isothermal amplification reaction is a reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction. Multiple molecular diagnosis method for respiratory infectious diseases using the equipment of Article 12. Pretreating a biological sample isolated from a patient with a lysis buffer;
Injecting the sample pretreated with the dissolution buffer into the inlet of the biochip for rapid point-of-care diagnostic testing according to any one of claims 1 to 10;
To the nucleic acid sample discharged into the multi-molecular diagnostic reaction chamber of the biochip, a primer set for target RNA of respiratory infectious diseases, reverse transcriptase, and nucleic acid polymerase are added to target sequence through reverse transcription ring-mediated isothermal amplification (RT-LAMP) reaction. amplifying; and
Detecting the product of the amplification step
Multiple molecular diagnosis method for respiratory infectious diseases including. A kit for multiple molecular diagnosis of respiratory infectious diseases, comprising the biochip for rapid point-of-care testing according to any one of claims 1 to 10.