KR20240052014A - High-throughput isothermal amplification method using an automated molecular diagnostic system - Google Patents

Abstract

The present disclosure relates to a high throughput isothermal amplification method using an automated molecular diagnostic system. Since the method according to the present disclosure can quickly detect target nucleic acids from a large number of samples in a short period of time, it can be usefully applied in large hospitals, contract testing organizations, research institutes, etc. that require molecular diagnosis of large amounts of samples.

Description

High-throughput isothermal amplification method using an automated molecular diagnostic system

The present disclosure relates to a high throughput isothermal amplification method using an automated molecular diagnostic system.

Nucleic acid amplification technology is a technology mainly used in the fields of molecular biology and biotechnology and is a method of detecting and analyzing a small amount of target nucleic acid present in a sample. PCR (Polymerase chain reaction) technology is most commonly used to amplify nucleic acids, which is a series of processes in which double-stranded DNA is denatured into single-stranded DNA, primers are annealed to it, and the annealed primers are extended by polymerase. Repeat.

The real-time PCR method using a fluorescent substance is a method of detecting an increase in fluorescence intensity due to nucleic acid amplification during the PCR process. The real-time PCR method has the advantage of enabling multiplex detection by using different fluorescent dyes for each target, but has the disadvantage of requiring expensive equipment and taking a long time to detect.

As an alternative to the PCR method, an isothermal amplification method has been developed that does not require expensive real-time PCR equipment and can detect target nucleic acids in a faster time than PCR. Examples of isothermal amplification methods include rolling circle amplification (RCA, M. M. Ali, F. Li, Z. Zhang, K. Zhang, D.-K. Kang, J. A. Ankrum, X. C. Le and W. Zhao, Chem. Soc. Rev. , 2014, 43, 3324-3341.), loop-mediated isothermal amplification (LAMP, Y. Mori, H. Kanda and T. Notomi, J. Infect. Chemother., 2013, 19, 404-411), recombinase polymerase amplification (RPA, J. Li, J. Macdonald and F. von Stetten, Analyst, 2018, 144, 31-67), nucleic acid sequence based amplification (NASBA, A. Borst, J. Verhoef, E. Boel and A. C. Fluit, Clin. Lab., 2002, 48, 487-492), strand displacement amplication (SDA, B. J. Toley, I. Covelli, Y. Belousov, S. Ramachandran, E. Kline, N. Scarr, N. Vermeulen, W. Mahoney , B. R. Lutz and P. Yager, Analyst, 2015, 140, 7540-7549), helicase dependent amplification (HAD, M. Vincent, Y. Xu and H. Kong, EMBO Rep., 2004, 5, 795-800), transcription mediated amplification (TMA, L. Comanor, Am. J. Gastroenterol., 2001, 96, 2968-2972), etc.

Among the isothermal amplification methods, the LAMP method is the most widely used. LAMP was discovered in 2000 by Notomi et al (T. Notomi, H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino and T. Hase, Nucleic Acids Res., 2000, 28, E63). It was first developed and then optimized by Nagamine et al (K. Nagamine, T. Hase and T. Notomi, Mol. Cell. Probes, 2002, 16, 223-229) by using additional primers to accelerate amplification. .

The standard detection method for LAMP is turbidity measurement by precipitation of magnesium pyrophosphate (Y. Mori, K. Nagamine, N. Tomita and T. Notomi, Biochem. Biophys. Res. Commun., 2001, 289, 150-154). , Other methods include gel electrophoresis, metal indicators for calcium, colorimetric LAMP, Coffee-Ring effect on colloidal-crystal matrices, paper-based rapid detection of LAMP magnetic bead aggregates, melting and annealing curve analysis, and interferometers such as SYBR green. These include collating fluorescent dyes, bioluminescence through pyrophosphate conversion, or electrochemiluminescence. Recently, a method for detecting fluorescence specific to a target nucleic acid amplification sequence using an assimilating probe (PCT/US2011/041540) was developed.

In general, isothermal amplification methods such as the LAMP method can amplify genes at one temperature within a short period of time (usually within 1 hour) and do not require expensive equipment compared to real-time PCR, so they have been applied for rapid on-site diagnosis.

However, rapid on-site diagnosis using this isothermal amplification method is disadvantageous compared to the real-time PCR method in situations that require analysis of large quantities of samples, such as the recent coronavirus pandemic.

The present inventors developed a high-throughput isothermal amplification method using an automated molecular diagnosis system that can process a large amount of samples in a short time as a method to replace the conventional molecular diagnosis by real-time PCR.

Numerous references and patent documents are referenced and cited throughout this specification. The disclosures of the cited documents and patents are incorporated herein by reference in their entirety to more clearly explain the content of the present invention and the level of technical field to which the present invention pertains.

The present inventors attempted to develop a high-throughput isothermal amplification method to replace conventional molecular diagnosis by real-time PCR. As a result, the present inventors applied direct lysis, used a 96-well plate as a reaction vessel, and used an automated molecular diagnostic system including a sample preparation device including a liquid dispensing module, thereby achieving It was confirmed that target nucleic acid can be detected from a large number of samples.

Therefore, the purpose of the present disclosure is to provide a high throughput isothermal amplification method using an automated molecular diagnostic system.

According to one aspect, a high throughput isothermal amplification method using an automated molecular diagnostic system is provided comprising the following steps:

(a) mounting a sample collected from a subject into a sample preparation device within an automated molecular diagnosis system; The automated molecular diagnosis system includes a sample preparation device, a reaction vessel transfer device, and a sample analysis device, and the sample preparation device includes a direct lysis buffer installed therein and an isothermal amplification for detecting target nucleic acid. Contains reagents;

(b) in the sample preparation device,

(b-1) mixing the sample directly with the lysis buffer;

(b-2) incubating the mixture at 15°C to 35°C for 3 to 5 minutes to obtain a lysate; and

(b-3) mixing the lysate with an isothermal amplification reagent in a reaction vessel to complete sample preparation;

The sample preparation device includes a liquid handling module for receiving the sample, direct lysis buffer, and isothermal amplification reagent into a reaction vessel, wherein the liquid handling module has 8 to 96 pipetting channels. ) and;

(c) moving the reaction vessel containing the mixture to a sample analysis device with the help of a reaction vessel transfer device;

(d) in the sample analysis device,

(d-1) reacting the mixture in the reaction vessel at a temperature selected from 50°C to 75°C for 10 to 20 minutes to amplify the target nucleic acid in the sample;

(d-2) Detecting the amplified product.

Steps (b) to (d) are automatically performed by a control module within the automated molecular diagnosis system.

In one embodiment, the sample derived from the subject is a swab, saliva, or a mixture thereof.

In one embodiment, the swab is a nasopharyngeal swab, a nostril swab, an oropharyngeal swab, an oral swab, or a saliva swab. , genital swab, rectal swab, or a mixture of two or more of these.

In one embodiment, the sample preparation device and the sample analysis device are each individually operated stand-alone devices.

In one embodiment, at least one of the sample analysis device and/or the reaction vessel transport device is located inside a spatially enclosed enclosure.

In one embodiment, the sample preparation device and the closed structure form a defined passage through which the reaction vessel is transported, and the reaction vessel transfer device uses the defined passage to transport the reaction vessel.

In one embodiment, the sample preparation device is located above or below the sample analysis device.

In one embodiment, the reaction vessel is a 96-well plate.

In one embodiment, the automated molecular diagnosis system additionally includes an automated container sealer for sealing the upper surface of the reaction vessel.

In one embodiment, the automatic vessel sealer is located in the sample preparation device.

In one embodiment, the automatic container sealer is located in the closure structure.

In one embodiment, the target nucleic acid is a viral nucleic acid.

In one embodiment, the target nucleic acid is an RNA viral nucleic acid.

In one embodiment, the target nucleic acid is a nucleic acid of SARS-CoV-2.

In one embodiment, the nucleic acid of SARS-CoV-2 is selected from the group consisting of the E gene, N gene, RdRP gene, S gene, and combinations thereof.

In one embodiment, the isothermal amplification reagent for detecting a target nucleic acid includes an oligonucleotide containing a fluorescent molecule and an oligonucleotide containing a quencher molecule.

In one embodiment, the isothermal amplification reagent additionally includes oligonucleotides for amplifying and detecting an internal control.

In one embodiment, the internal control is RNase P.

In one embodiment, the isothermal amplification reagent for detecting a target nucleic acid is an isothermal amplification reagent for detecting a plurality of target nucleic acids.

In one embodiment, the plurality of target nucleic acids is 2 to 5.

In one embodiment, the method further comprises the step of moving the reaction vessel to an automatic vessel sealer with the aid of a reaction vessel transfer device and sealing the upper surface of the reaction vessel prior to step (c).

In one embodiment, sealing the top surface of the reaction vessel is performed by heat or adhesive.

In one embodiment, the temperature of step (d-1) is any temperature selected from 60°C to 65°C.

In one embodiment, step (d-2) is performed at regular time intervals while performing step (d-1).

In one embodiment, the method takes 18 to 60 minutes per 96 samples.

In one embodiment, step (b) takes between 7 and 36 minutes per 96 samples.

In one embodiment, step (b-1) takes 6 to 13 minutes per 96 samples.

In one embodiment, step (b-2) takes 5 minutes per 96 samples.

In one embodiment, step (b-3) takes between 1 and 18 minutes per 96 samples.

In one embodiment, step (c) takes between 1 and 4 minutes per 96 samples.

In one embodiment, step (d) takes 15 to 20 minutes per 96 samples.

The features and advantages of the present invention are summarized as follows:

(a) Since the method of the present invention can detect target nucleic acids from 96 samples within 18 to 60 minutes, it can be usefully applied in large hospitals, contract testing organizations, research institutes, etc. that require molecular diagnosis of large quantities of samples.

(b) By combining direct lysis and isothermal amplification, the method of the present invention can maintain high sensitivity even while omitting the purification process when extracting target nucleic acid from a sample.

(c) The method of the present invention can process 96 samples simultaneously by using a system capable of accommodating a 96-well plate.

(d) The method of the present invention can shorten the time required to dispense the liquid required for the reaction by using a system including a liquid dispensing module with 8 to 96 pipetting channels and reduces operator intervention and resulting errors. The risk of occurrence can be prevented.

(e) Since the method of the present invention is automatically performed by a control module within an automated molecular diagnosis system, test reproducibility and operator fatigue can be improved.

1 is a front view showing an integrated system according to the present disclosure.
2 is a right side view showing an integrated system according to the present disclosure.
Figure 3 is an exemplary diagram showing the operational connection of an integrated system according to one implementation.
4 is an internal front view showing the integrated system of the present disclosure.
Figure 5 is a perspective view showing a stand-alone sample preparation device according to one embodiment.
Figure 6 is a perspective view showing a closed structure according to one embodiment.
Figure 7 is an exemplary diagram showing an operating state of a second opening of a closed structure according to an embodiment.
8 is an internal perspective view showing a closed structure of an integrated system of an integrated system according to one implementation.
Figure 9 is a perspective view showing a reaction vessel transfer device according to one embodiment.
Figure 10 is a perspective view showing the lifting module of the reaction vessel transfer device according to one embodiment.
Figure 11 schematically shows an example of a loop-mediated isothermal amplification (LAMP) method.
Figure 12 schematically shows an example of a method for detecting a target nucleic acid using an assimilating probe.
Figure 13 shows the results of high-throughput isothermal amplification by the automated molecular diagnosis system according to the present disclosure using the RT-LAMP reagent (1) containing the fluorescent dye prepared in Example 1-2.
Figure 14 shows the results of high-throughput isothermal amplification by the automated molecular diagnosis system according to the present disclosure using the RT-LAMP reagent (2) containing the assimilated probe prepared in Example 1-2.

Best mode for carrying out the invention

The present invention provides a high throughput isothermal amplification method using an automated molecular diagnostic system comprising the following steps:

(a) mounting a sample collected from a subject into a sample preparation device within an automated molecular diagnosis system; The automated molecular diagnosis system includes a sample preparation device, a reaction vessel transfer device, and a sample analysis device, and the sample preparation device includes a direct lysis buffer installed therein and an isothermal amplification for detecting target nucleic acid. Contains reagents;

(b) in the sample preparation device,

(b-1) mixing the sample directly with the lysis buffer;

(b-2) incubating the mixture at 15°C to 35°C for 3 to 5 minutes to obtain a lysate; and

(b-3) mixing the lysate with an isothermal amplification reagent in a reaction vessel to complete sample preparation;

The sample preparation device includes a liquid handling module for receiving the sample, direct lysis buffer, and isothermal amplification reagent into a reaction vessel, wherein the liquid handling module has 8 to 96 pipetting channels. ) and;

(c) moving the reaction vessel containing the mixture to a sample analysis device with the help of a reaction vessel transfer device;

(d) in the sample analysis device,

(d-1) reacting the mixture in the reaction vessel at a temperature selected from 50°C to 75°C for 10 to 20 minutes to amplify the target nucleic acid in the sample;

(d-2) Detecting the amplified product.

Steps (b) to (d) are automatically performed by a control module within the automated molecular diagnosis system.

Below, the present invention is explained in more detail according to each step:

Step (a): Mount the sample on the sample preparation device within the automated molecular diagnostic system

In step (a) of the present disclosure, a sample collected from a subject is mounted on a sample preparation device within an automated molecular diagnosis system. Here, the automated molecular diagnosis system includes a sample preparation device, a reaction vessel transfer device, and a sample analysis device, and the sample preparation device includes a direct lysis buffer installed therein and an isothermal amplification reagent for detecting target nucleic acid. do.

As used herein, the term “subject” refers to an individual suspected of containing a target nucleic acid (eg, a specific pathogen) to be detected using the method of the present disclosure. Examples of the subject include, but are not limited to, mammals such as dogs, cats, rodents, primates, and humans, especially humans.

As used herein, the term “sample” may refer to any analyte that contains or is suspected of containing nucleic acids to be detected. For example, the samples include biological samples (e.g., cells, tissues, and body fluids) and non-biological samples (e.g., food, water, and soil), and the biological samples include, for example, viruses, bacteria, tissues, cells, blood (whole blood). , including plasma and serum), lymph, bone marrow fluid, saliva, sputum, swab, aspiration, milk, urine, feces, ocular fluid, semen, brain extract, spinal fluid, joint fluid, thymus It may be fluid, bronchial lavage fluid, ascites, or amniotic fluid, but is not limited thereto.

The sample may be used interchangeably with 'specimen' herein.

In one embodiment, the sample may be obtained from a subject, particularly a mammal, more particularly a human, for example a swab, saliva, sputum, aspiration, bronchoalveolar tissue. It may be bronchoalveolar lavage (BAL), gargle, or blood, but is not limited thereto.

In certain embodiments, the sample derived from the subject is a swab, saliva, or a mixture thereof.

In one embodiment, the swab is nasopharyngeal swab, nostril swab, oropharyngeal swab, oral swab, or saliva swab. ), genital swab, rectal swab, or a mixture of two or more of these.

As used herein, the term “nucleic acid,” “nucleic acid sequence,” or “nucleic acid molecule” refers to a deoxyribonucleotide or ribonucleotide polymer in single-stranded or double-stranded form, wherein the nucleotides are naturally occurring nucleotides and It may include derivatives of natural nucleotides, non-natural nucleotides or modified nucleotides that can function in the same way.

As used herein, the term “target nucleic acid”, “target nucleic acid sequence” or “target sequence” refers to the nucleic acid sequence to be detected. The target nucleic acid sequence may hybridize with some primers or probes included in the isothermal amplification reagent used in the method of the present invention.

In one embodiment, the target nucleic acid can be a human, animal, plant, or microbial nucleic acid. Microorganisms may be fungi, protozoa, bacteria, viruses, or algae.

In one embodiment, the target nucleic acid may be a viral nucleic acid, and specifically, the target nucleic acid may be an RNA viral nucleic acid.

In one embodiment, the target nucleic acid may be a respiratory virus nucleic acid. For example, influenza virus nucleic acid, respiratory syncytial virus (RSV) nucleic acid, adenovirus nucleic acid, enterovirus nucleic acid, parainfluenza virus nucleic acid, metapneumovirus (MPV) nucleic acid, bocavirus nucleic acid, It may include rhinovirus nucleic acid and/or coronavirus nucleic acid.

In one embodiment, the target nucleic acid may be a SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) virus nucleic acid.

In one embodiment, the nucleic acid of the SARS-CoV-2 virus may be selected from the group consisting of the E gene, N gene, RdRP gene, S gene, and combinations thereof.

In one embodiment, the sample can be obtained from the subject using various sample collection devices.

As used herein, the term “sampling device” is used to encompass means, tools, etc. necessary to collect a sample and maintain the collected sample.

In one embodiment, the sampling device includes a container capable of receiving a sample.

In another embodiment, the sample collection device includes a transport medium. The transport medium refers to a transport medium for preservation that can maintain sample integrity, such as cell integrity of viruses or bacteria. The transport medium may be based on a saline solution, such as phosphate buffered saline (PBS) or normal saline, or based on a balanced salt solution, such as Hank's Balanced Salt Solution (HBSS).

In one embodiment, the transport medium is not an inactivating transport medium. That is, the transport medium does not contain a material that inactivates organisms, such as viruses, that is, a material for lysis. In the present disclosure, the term “material for lysis” refers to a component used to dissolve a specimen (i.e., cells) by chemical cell lysis and may include, for example, a chaotropic material. Examples of such chaotropic substances include, but are not limited to, guanidinium or guanidine or similar chemicals.

Examples of such transport media include, but are not limited to, the commercially available Copan universal transport medium (UTM), viral transport medium (VTM), or Clinical Virus Transport Medium (CTM) from Noble Biosciences, Inc. No.

In one embodiment, the transport medium may be a liquid medium.

In one embodiment, the sample collection device may additionally include a sample collection swab tool.

The sample collected from the subject is mounted on a sample preparation device within an automated molecular diagnosis system. The “automated molecular diagnosis system” includes a sample preparation device, a reaction vessel transfer device, and a sample analysis device.

As used herein, “automated molecular diagnostic system” refers to a system including a plurality of devices in which the process of preparing and analyzing samples for molecular diagnosis, especially isothermal amplification (e.g., LAMP), can be performed automatically without operator intervention. It is used for.

In one embodiment, the automated molecular diagnosis system is a fully automated system that cannot individually perform sample preparation, sample analysis, etc., as is widely known in the art. Examples of commercially available fully automated systems include Roche's Cobas 6800/Cobas 8800, Hologic's Panther/Panther Fusion, Abbott's Alinity, and Qiagen's QIAsymphony.

In certain embodiments, our automated molecular diagnostic system is an all in one system. Herein, the automated molecular diagnosis system is described based on an integrated system throughout the detailed description, but those skilled in the art will recognize that the fully automated system described above can be applied in addition to the integrated system.

The sample preparation device and sample analysis device can be applied as an integrated system while being licensed as a stand-alone device. Alternatively, for application as an integrated system, some changes to the housing, etc. can be made and partial approval for this can be obtained.

Therefore, the sample preparation device and sample analysis device are stand-alone devices and approved reagents can be used as is.

In one embodiment, the integrated system includes one or more passthrough cavities and a reaction vessel transfer device for operatively connecting a sample preparation device and a sample analysis device, respectively. Includes.

As used herein, the term “sample preparation device” refers to a device used to prepare an analytical sample that contains or is presumed to contain an analyte. In one embodiment, the sample preparation device is a stand-alone device that is individually operated.

Herein, the sample preparation device includes a direct lysis buffer loaded therein and an isothermal amplification reagent for detecting target nucleic acids.

The sample preparation device processes the sample to an optimal state for application to the analysis device, and performs this automatically using a microrobot. The sample preparation process in the present disclosure includes extracting nucleic acids from a sample (e.g., specimen), preparing an isothermal amplification reagent, and combining the extracted nucleic acids with the isothermal amplification reagent.

In one embodiment, the sample preparation device includes a housing, and the sample preparation device may be located within a separate hexahedron-shaped enclosure.

The sample preparation device may provide an internal reaction vessel to the sample analysis device.

In one embodiment, the sample preparation device may provide the internal reaction vessel to the sample analysis device through one or more openings formed in the housing.

In another embodiment, the sample preparation device may form one or more openings in the housing to provide an internal reaction vessel to the sample analysis device.

In another embodiment, where the sample preparation device is located inside the closed structure and the sample analysis device is located outside the closed structure, the closed structure has one or more openings to provide the reaction vessel of the sample preparation device to the sample analysis device. It can be.

In one embodiment, the sample preparation device includes a nucleic acid extraction module.

The nucleic acid extraction module directly mixes an aliquot of a sample (e.g., a sample containing transport medium) with an aliquot of a lysis buffer (e.g., a buffer mounted on a sample preparation device), and the mixture The incubation process can be performed.

Additionally, the sample preparation device may perform a process of mixing the incubated mixture with an aliquot of the isothermal amplification reagent.

In the sample preparation device, the sample preparation process is implemented through a control device (not shown) for controlling the sample preparation device, and the operation of each sample preparation process is achieved by the control device controlling each component.

The control device may be configured to be embedded in the sample preparation device, or may be provided as a separate device and connected to the sample preparation device through a network.

The control device according to the present invention is software controlled. The control method of the sample preparation device may be controlled by software. Methods implemented as software or algorithms may be stored on a computer-readable recording medium as computer-readable codes or program instructions executable on a processor.

Here, computer-readable recording media include magnetic storage media (e.g., ROM (read-only memory), RAM (random access memory), floppy disk, hard disk, etc.) and optical read media (e.g., CD-ROM). , DVD (Digital Versatile Disc), etc. The computer-readable recording medium is distributed among networked computer systems, so that computer-readable code can be stored and executed in a distributed manner. The media may be readable by a computer, stored in memory, and executed by a processor.

The sample preparation device according to the invention is an automated liquid handling device. An automated liquid handling device can automatically and programmatically aspirate and/or dispense desired quantities of reagents, samples or other liquids from designated containers for automation of a chemical or biochemical laboratory. Various configurations of automated liquid handling devices are known to those skilled in the art.

All components of the sample preparation device are designed as an integrated device and located within the system housing.

In one embodiment, the sample preparation device may use a product such as “Microlab VANTAGE”, “Microlab STAR”, “Microlab NIMBUS”, or “Microlab Prep” from Hamilton (https://www.hamiltoncompany.com/automated- (see liquid-handling/platforms).

As used herein, the term “sample analysis device” refers to a device used to subject a sample to isothermal amplification, particularly LAMP.

As used herein, sample analysis includes detecting the presence of a target nucleic acid in a sample.

A sample analysis device may refer to a device that amplifies a target nucleic acid having a nucleotide sequence of interest and detects the amplified nucleic acid.

The sample analysis device may include a nucleic acid amplification device and a nucleic acid detection device. The nucleic acid detection device may include an optical device including a light source and a light detector.

Additionally, the sample analysis device may include a temperature control device capable of maintaining the temperature of the sample at isotherm.

The sample analysis device may be an isothermal amplification device (eg, a LAMP device) or a commercially available real-time PCR device, such as a thermal cycler. If the sample analysis device is a real-time PCR device, the sample analysis device may be set to maintain a predetermined temperature, i.e., to achieve isothermal conditions.

In the sample analysis device, the target nucleic acid can be amplified by a conventionally known isothermal amplification method (eg, LAMP method) or a modification thereof.

Meanwhile, the nucleic acid detection device included in the sample analysis device of the present invention is a device for detecting target nucleic acids in a sample amplified through a nucleic acid amplification device.

In one embodiment, the nucleic acid detection device includes an optical module that detects emission light emitted from a fluorescent material in the presence of a target nucleic acid.

The optical module is an optical mechanism that analyzes (or monitors) the amplification reaction performed in the nucleic acid amplification device in real time. In one embodiment, the optical module may be composed of a plurality of components such as a light source, an optical filter, a convex lens, a beam splitter, and an optical detector, and detects fluorescence generated from the nucleic acid amplification reaction occurring in the optical module in real time. can do.

In one implementation, the sample analysis device is a real-time detection device.

In one embodiment, the sample analysis device is a real-time nucleic acid detection device.

In one embodiment, the sample analysis device is a real-time isothermal amplification device.

In one embodiment, the sample analysis device is a real-time PCR device.

In one embodiment, the sample analysis device is a LAMP device, particularly a real-time LAMP device.

The sample analysis device herein is a stand-alone device that is operated individually.

In one embodiment, the sample analysis device includes a housing, and the sample analysis device may be located within a separate hexahedron-shaped enclosure.

A sample analysis device can receive a reaction vessel containing a sample from a sample preparation device.

The sample analysis device does not directly receive the reaction vessel from the sample preparation device, but may receive the reaction vessel transferred from the reaction vessel transfer device.

As used herein, the term “enclosure” is a housing-type structure formed of one or more closed spaces that can be environmentally/spatially separated from the outside.

In one embodiment, the closed structure may have a hexahedron shape.

In another embodiment, the closed structure may have a shape in which a plurality of hexahedrons are connected. At this time, the interior may be connected into one space or divided into multiple spaces.

In one embodiment, when the sample preparation device is located outside the closed structure and the sample analysis device is located inside the closed structure, the reaction vessel transfer module may be located inside the closed structure.

In other embodiments, where the sample analysis device is located outside the closed structure and the sample preparation device is located inside, the reaction vessel transfer module may be located outside the closed structure.

In another embodiment, when the sample preparation device and the sample analysis device are located inside the closed structure, the reaction vessel transfer module may be located inside the closed structure.

An automated container sealer may be located in the closed structure.

When a sample preparation device and/or a sample analysis device are located inside the closed structure, the closed structure may include environmental control means therein to implement an environment in which each device operates as a stand-alone device.

The environmental control means is intended to control temperature, humidity, contamination, etc. inside the closed structure and may include a heating device, a cooling device, a humidity control device, a fan, a filter, etc.

As used herein, the term “passthrough cavity” is a configuration for connecting an environmentally/spatially separated sample preparation device and a sample analysis device. The opening is a passage through which the reaction vessel prepared by the sample preparation device is moved to the sample analysis device or the sample analysis device within the closed structure so as to spatially connect the adjacent sample preparation device and the sample analysis device.

In one embodiment, the opening may be comprised of a first opening (1st passthrough cavity) formed in the sample preparation device and a second opening (2nd passthrough cavity) included in the closed structure.

In another implementation, the first opening and/or the second opening may include a door device capable of blocking a state in which they are spatially connected to each other. The opening and closing device may be included in the first opening and may be included in the second opening. Alternatively, it may be included in both the first opening and the second opening.

The opening may be operated to open when the reaction vessel prepared in the sample preparation device is moved to the sample analysis device or to the sample analysis device within the closed structure, and to be closed after the movement is completed.

The first opening is formed in the lower part of the sample preparation device, and may preferably be formed in the deck of the sample preparation device. The first opening formed in the deck of the sample preparation device may be located at the bottom to provide a reaction vessel to the sample analysis device or to the sample analysis device located in the closed structure.

The second opening may be formed in the top, side, or bottom of the closed structure to allow entry of a reaction vessel that is moved in the first opening of the sample preparation device.

Additionally, the closed structure may include a reaction vessel transfer device for transferring the reaction vessel from the first opening through the second opening.

As used herein, the term "reaction vessel transfer device" refers to the ability to move a reaction vessel used in an integrated system from a sample preparation device to a sample analysis device or a sample analysis device within a closed structure, and to be transported and mounted on each component within the closed structure. It is a device that has

In one embodiment, a reaction vessel transfer device for moving a reaction vessel from a sample preparation device to a sample analysis device may include at least one robot module.

In one implementation, the robotic module includes a lift module. The lifting module is a robot module for moving the reaction vessel up and down, and can move the reaction vessel of the sample preparation device into a closed structure.

In another implementation, the robot module includes a crane module. The crane module can transport the reaction vessel moved into the closed structure within the closed structure and mount it on the component.

In another implementation, the robotic module includes robot arms. The robot arm can move the reaction vessel to a desired location through one or more joint movements.

The robot module may operate in the up/down, forward/backward, and left/right directions, but in one embodiment, the lifting module operates in the up/down and left/right directions, and the crane module operates in the up/down, forward/backward, and left/right directions. It operates in left/right directions and can be rotated.

As used herein, the term “vessel” refers to a space containing materials used in a sample preparation device and sample analysis device. The substance generally contains a solution. In this specification, a space containing materials used in a sample preparation device and a sample analysis device may be used as a “container” or “carrier.” There is no special distinction between “vessel”, “container”, and “carrier”. However, it can be used selectively depending on the device used, its form, or the material contained within.

As used herein, the term “reaction vessel” refers to a vessel containing a sample (sample mixed with an isothermal amplification reagent) for amplification of a target nucleic acid.

Reaction vessel herein refers to a 96-well plate, which is used in sample preparation devices and sample analysis devices. The 96-well plate herein can process 96 samples (including controls) in one reaction, i.e. simultaneously.

FIG. 1 is a front view showing an integrated system according to an embodiment, and FIG. 2 is a right side view showing an integrated system according to an embodiment. 1 and 2, in one implementation, an all in one system (1000) includes a sample preparation device (1100) and an enclosure (1300).

In one implementation, sample preparation device 1100 may be configured to be located on top of closed structure 1300. In another implementation, sample preparation device 1100 may be configured to be located on the side of closed structure 1300. In another implementation, sample preparation device 1100 may be configured to be located at the rear of closed structure 1300. In another implementation, sample preparation device 1100 may be configured to be located in the front of closed structure 1300. In another implementation, sample preparation device 1100 may be configured to be located inside the enclosed structure 1300. In another implementation, sample preparation device 1100 may be configured to be located at the bottom of closed structure 1300.

The closed structure 1300 is a closed space in the shape of a hexahedron.

The closed structure 1300 may accommodate at least one of a sample preparation device 1100, a sample analysis device 1200, and a reaction vessel transfer device 1400.

In one implementation, the enclosed structure 1300 can accommodate one or more sample analysis devices 1200 and reaction vessel transfer devices 1400. In other implementations, the enclosed structure 1300 can accommodate one or more sample analysis devices 1200. In another implementation, enclosure structure 1300 can accommodate reaction vessel transfer device 1400. In another implementation, the enclosed structure 1300 can accommodate the sample preparation device 1100 and the reaction vessel transfer device 1400. In another implementation, enclosed structure 1300 can accommodate sample preparation device 1100.

Enclosed structure 1300 may provide an operative connection between one or more devices housed therein and devices located externally.

In one implementation, enclosure structure 1300 closes a single space. When the closed structure 1300 is closed into a single space, at least one of the sample preparation device 1100, the sample analysis device 1200, and the reaction vessel transfer device 1400 is one device. It can be located in the space of

In another implementation, enclosure structure 1300 closes a plurality of spaces. When the closed structure 1300 is closed with a plurality of spaces, at least one of the sample preparation device 1100, the sample analysis device 1200, and the reaction vessel transfer device 1400 is It may be located in one of a plurality of spaces, or it may be located in any two or more spaces among a plurality of spaces. For example, in a closed structure 1300 having a single space, a sample analysis device 1200 and a reaction vessel transfer device 1400 may be located in the same space. For example, in the closed structure 1300 having a plurality of spaces, the sample analysis device 1200 and the reaction vessel transfer device 1400 may be located in different spaces.

In one embodiment, the sample analysis device 1200 and the reaction vessel transfer device 1400 may be located inside the closed structure 1300. At this time, the sample preparation device 1100 is located outside the closed structure 1300.

The closed structure 1300 is a second defined passage through which the reaction vessel transfer device 1400 is moved to transfer the reaction vessel 1500 to be provided from the sample preparation device 1100 to the sample analysis device 1200. An opening (2nd passthrough cavity, 1310) is formed.

The second opening 1310 of the closed structure 1300 is a defined passage through which the reaction vessel 1500 is transferred, and the reaction vessel transfer device 1400 is provided from the first opening 1130 of the sample preparation device 1100. The reaction vessel 1500 may be provided to the sample analysis device 1200 through the second opening 1310 of the closed structure 1300.

The second opening 1310 of the closed structure 1300 may be located on any one of the outer surfaces of the closed structure 1300. As shown in FIG. 6, when the closed structure 1300 has a hexahedral shape, the second opening 1310 may be formed on the upper surface of the closed structure 1300, but is not limited to this.

The reaction vessel transfer device 1400 passes the reaction vessel 1500 through a path formed between the first passthrough cavity 1130 of the sample preparation device 1100 and the second opening 1310 of the closure structure 1300. Can be programmed to transport.

Therefore, it is preferable that each opening is located at a distance within a range within which the reaction vessel transfer device 1400 can move the reaction vessel 1500.

The closed structure 1300 may be implemented in the shape of a hexahedral cabinet, locker, or box/case. The closed structure 1300 is closed on the front/back/left/right/top/bottom sides and is provided with at least one closed structure door (door 1320). The closed structure door 1320 is installed front/rear, left/right, etc. to allow the user to access devices and components located inside.

The closed structure 1300 is not sealed, and an air vent 1370 may be formed.

The closed structure 1300 may be located at least one of a sample preparation device 1100, a sample analysis device 1200, and/or a reaction vessel transfer device 1400.

Additionally, an automated container sealer (1700) for sealing the upper surface inlet of the reaction vessel (1500) may be located in the closed structure (1300).

Additionally, a liquid waste collection bin 1330 may be located in the closed structure 1300 to recover various solutions used to prepare analysis samples in the sample preparation device 1100.

Additionally, a reaction vessel retrieval container 1360 may be located in the closed structure 1300 to retrieve the reaction vessel 1500 on which analysis has been completed in the sample analysis device 1200.

In one implementation, the reaction vessel recovery bin can be located inside the enclosed structure 1300. In other implementations, the reaction vessel recovery bin may be located external to the enclosed structure 1300.

The reaction vessel recovery box located on the outside transports the reaction vessel 1500 from the inside to the outside through a recovery opening (retrieval passthrough cavity, 1340) connecting the inside and outside of the closed structure 1300. It can be recovered.

In order to move the reaction vessel 1500, a conveyor 1350 may be installed in the recovery opening 1340 that connects the inside and outside of the closed structure 1300 to be interconnected. When the reaction vessel transfer device 1400 places the reaction vessel 1500 on the inner conveyor 1350 of the closed structure 1300, the reaction vessel 1500 moves by the conveyor 1350 and moves on the inner conveyor 1350 of the closed structure 1300. It can be accommodated in a reaction vessel recovery box located externally.

In one implementation, the conveyor 1350 forms an inclined surface where the outside is lower than the inside. The conveyor 1350 is provided with rollers on an inclined surface, so that the reaction vessel 1500 can be discharged to the outside of the closed structure 1300. The reaction vessel 1500 discharged to the outside may be accommodated in a reaction vessel recovery box.

In other implementations, conveyor 1350 may be powered. Power can be used to rotate the belt included in the conveyor 1350, thereby discharging the reaction vessel 1500 placed on the conveyor 1350 to the outside of the closed structure 1300. The reaction vessel 1500 discharged to the outside may be accommodated in a reaction vessel recovery box.

The reaction vessel recovery box may have one or more reaction vessels 1500 accommodated therein emptied by a user.

The recovery opening 1340, where the inside and outside of the closed structure 1300 are connected by the conveyor 1350, may include an opening and closing module (not shown). The opening/closing module may be opened when the reaction vessel 1500 is moved to the reaction vessel recovery box, but is preferably closed in other cases. The opening and closing module can protect the interior of the closed structure 1300 from external contamination.

In one implementation, the closed structure 1300 is not completely sealed and is implemented to allow ventilation. To this end, the closed structure 1300 includes an environmental control means, and the environmental control means may include a ventilation port, an exhaust port, a fan, a temperature control device, a humidity control device, an air filter, etc.

In Figures 6 and 8 of the present invention, an air vent (1370) or an exhaust vent (1370) for air circulation within the closed structure 1300, and/or a fan (1380) for discharging internal air ) indicates that one or more is formed.

Figure 3 is an exemplary diagram showing the operational connection of an integrated system according to one implementation. As shown in Figure 3, sample preparation device 1100 is located on top of closed structure 1300.

The sample preparation device 1100 may be installed and operated independently for the preparation of samples for analysis, and, according to one embodiment, may be operatively connected to the closed structure 1300 and used as an integrated system 1000.

According to one embodiment, when the sample preparation device 1100 is combined with the closed structure 1300 and used as an integrated system 1000, the sample preparation device 1100 is an elevating module provided on the closed structure 1300 ( A first opening 1130 may be formed so that 1410 can move inward (see FIG. 5). The first opening 1130 is an empty space and is a defined passage through which the reaction vessel 1500 is moved by the lifting module 1410.

When the lifting module 1410 is moved into the interior of the sample preparation device 1100, the sample preparation device 1100 includes a reaction vessel (1500) accommodating the analysis sample or a plate (1500) accommodating the reaction vessel (1500). A plate (not shown) is mounted on the lifting module 1410. The lifting module 1410 moves the mounted reaction vessel 1500 or plate into the interior of the closed structure 1300.

According to one embodiment, when the sample preparation device 1100 is located on the closed structure 1300, the sample preparation device 1100 and the closed structure 1300 are coupled through a coupling mechanism (not shown). It can be.

In one embodiment, when at least one of the sample preparation device 1100 and the sample analysis device 1200 is operatively connected to the closed structure 1300 and used as an integrated system 1000, the sample preparation device ( 1100) and the sample analysis device 1200 use the same power module used as a stand-alone device.

In one embodiment, when at least one of the stand-alone sample preparation device 1100 and the stand-alone sample analysis device 1200 is operatively connected to the closed structure 1300 and used as an integrated system 1000. , the stand-alone sample preparation device 1100 and the stand-alone sample analysis device 1200 are already commercially available devices and/or previously licensed devices.

Accordingly, even if it is operatively connected to the closed structure 1300 and used as an integrated system 1000, no separate approval or permission is required or partial modifications may be made.

Figure 4 is an internal front view showing an integrated system of one implementation. As shown in FIG. 4, sample analysis devices 1200-a and 1200-b are located below the closed structure 1300.

The closed structure 1300 will be described with reference to FIG. 8 as follows.

8 is an internal perspective view showing a closed structure of an integrated system according to one implementation.

In one embodiment, the closed structure 1300 includes a reaction vessel transfer device 1400 for providing the reaction vessel 1500 prepared in the sample preparation device 1100 to the sample analysis devices 1200-a and 1200-b. do.

The reaction vessel transfer device 1400 is a robot module, and is particularly composed of a lift module (1410) and a crane module (1430).

In one implementation, the reaction vessel transfer device 1400 includes an elevating module 1410 and a crane module 1430.

In another implementation, the reaction vessel transfer device 1400 includes an elevating module 1410.

In another embodiment, the reaction vessel transfer device 1400 includes a crane module 1430.

In another implementation, the reaction vessel transfer device 1400 includes a robotic arm (not shown).

In another embodiment, the reaction vessel transfer device 1400 may include a mechanical device capable of transferring the reaction vessel 1500.

In one implementation, the lifting module 1410 and the crane module 1430 may be located inside the enclosed structure 1300.

The lifting module 1410 and crane module 1430 included in the closed structure 1300 are robot modules. The robot module moves the reaction vessel 1500 under the control of a control module (not shown) included in the integrated system 1000.

An automated container sealer (1700) for sealing the inlet of the reaction vessel (1500) may be located in the closed structure (1300).

At least one sample analysis device (sample analysis device, 1200-a, 1200-b) for analyzing an analysis sample accommodated in the reaction vessel 1500 may be located in the closed structure 1300.

A liquid waste collection bin 1330 may be located in the closed structure 1300 to recover various solutions used to prepare analysis samples in the sample preparation device 1100.

A reaction vessel retrieval container 1360 may be located in the closed structure 1300 to retrieve the reaction vessel 1500 on which analysis has been completed in the sample analysis devices 1200-a and 1200-b. .

Enclosed structure 1300 includes a control module (not shown) that transmits and/or receives data from a plurality of devices operatively connected to integrated system 1000.

In one embodiment, the control module is operatively connected to at least one of a sample preparation device 1100, a sample analysis device 1200, a reaction vessel transfer device 1400, and/or an automatic vessel sealer 1700. A communication channel may be connected for this purpose. For example, communication channels may be wireless and/or wired.

In one implementation, the control module may receive a signal that the analysis sample is ready from the sample preparation device 1100 using a communication channel.

Additionally, a control signal for moving the reaction vessel 1500 of the sample preparation device 1100 to the sample analysis device 1200 may be provided to the reaction vessel transfer device 1400 using a communication channel.

Additionally, the control module may receive a signal from the sample analysis device 1200 that analysis of the analysis sample has been completed using a communication channel.

Additionally, the control module may provide a control signal for moving the reaction vessel 1500 of the sample analysis device 1200 to the reaction vessel recovery box to the reaction vessel transfer device 1400 using a communication channel.

The closed structure 1300 has a second opening (2nd passthrough cavity, 1310) formed on its upper surface through which the lifting module 1410 moves to the sample preparation device 1100 to receive the reaction vessel 1500.

According to one implementation, the second opening 1310 can be described as follows with reference to FIGS. 6 and 7. Figure 6 is a perspective view showing a closed structure according to one embodiment. Figure 7 is an exemplary diagram showing an operating state of a second opening of a closed structure according to an embodiment. As shown in FIGS. 6 and 7 , the second opening 1310 is formed on the upper surface of the closure structure 1300.

The second opening 1310 is sized so that a vertical motion guide 1413 and a reaction vessel rack 1416 included in the lifting module 1410 can pass through.

The second opening 1310 is formed to be vertically connected to the first opening 1130 formed in the deck 1110 of the sample preparation device 1100.

In one embodiment, the second opening 1310 is formed on the right side in the upper surface of the enclosure structure 1300, and the first opening 1130 is formed on the right side in the plane of the deck 1110. In another implementation, the second opening 1310 may be formed on the left side in the upper surface of the enclosure structure 1300, and the first opening 1130 may be formed on the left side in the plane of the deck 1110. . In another embodiment, the second opening 1310 may be formed on the upper side of the closed structure 1300, and the first opening 1130 may be formed on the upper side in the plane of the deck 1110. there is. In another embodiment, the second opening 1310 may be formed on the lower side of the upper surface of the enclosure structure 1300, and the first opening 1130 may be formed on the lower side in the plane of the deck 1110. there is.

In one implementation, the second opening 1310 is a passage for a portion of the lifting module 1410 located inside the closed structure 1300 to move into the sample preparation device 1100 (see FIG. 6).

In another embodiment, the second opening 1310 is an open passage for a portion of the elevating module 1410 inside the closed structure 1300 to move into the interior of the sample preparation device 1100, and the portion of the elevating module 1410 If it does not move to the sample preparation device 1100, the open second opening 1310 can be closed through an open/close module 1311 provided in the second opening 1310 (see FIG. 7). .

The opening/closing module 1311 is provided to block contaminants outside the closed structure 1300.

In one implementation, the opening and closing module 1311 is implemented in a hinge manner and can be opened and closed to the upper or lower side of the upper surface of the closed structure 1300. In another implementation, the opening and closing module 1311 may be implemented in a sliding manner and may be opened and closed by moving on the upper surface of the closed structure 1300. In another implementation, the opening/closing module 1311 may be of any type as long as it can open and close the second opening 1310 in a manner other than a hinge method or a sliding method.

As shown in (a) of FIG. 7, when the lifting module 1410 is located inside the closed structure 1300, the opening/closing module 1311 of the second opening 1310 is closed.

As shown in (b) of FIG. 7, when the lifting module 1410 moves to the sample preparation device 1100, the opening/closing module 1311 of the second opening 1310 is opened. And, the opening/closing module 1311 of the second opening 1310 is closed after the lifting module 1410 moves into the closing structure 1300.

According to one implementation, the lifting module 1410 can be described with reference to FIGS. 9 and 10 as follows. FIG. 9 is a perspective view showing a reaction vessel transfer device according to an embodiment, and FIG. 10 is a perspective view showing a lifting module of the reaction vessel transfer device according to an embodiment.

The lifting module 1410 is configured to receive the reaction vessel 1500 from the sample preparation device 1100. The lifting module 1410 represents an operating form of an elevator that can ascend to the sample preparation device 1100 at the top of the enclosed structure 1300 to receive an analysis sample from the sample preparation device 1100.

The lifting module 1410 includes a vertical fixed guide 1411 in the form of a pillar fixed inside the closed structure 1300. The vertical fixed guide 1411 includes a fixed guide connector 1412 that moves up and down. The lifting module 1410 includes a vertical motion guide (1413) coupled to the fixed guide connector 1412 of the vertical fixed guide 1411.

The vertical motion guide 1413 includes a motion guide connector 1414 that moves up and down. The vertical motion guide 1413 includes a rack guide 1415 coupled to the motion guide connector 1414.

The lifting module 1410 includes a reaction vessel rack (analytical sample vessel rack 1416) that receives reaction vessels on top of a rack guide 1415.

The lifting module 1410 includes an actuator 1417 that provides power to move the vertical motion guide 1413 in the up and down directions.

The vertical fixing guide 1411 is coupled and fixed to at least one of the top, bottom, and/or sides of the closed structure 1300. The vertical fixed guide 1411 is coupled to the fixed guide connector 1412. The vertical fixed guide 1414 can move the coupled fixed guide connector 1412 in the up/down direction.

The vertical fixed guide 1411 may use power provided by the driving device 1417 to move the fixed guide connector 1412 in the up/down direction.

The size of the vertical fixing guide 1411 is equal to or smaller than the internal height of the closed structure 1300. As the vertical fixed guide 1411 moves the fixed guide connector 1412 upward, the vertical motion guide 1413 coupled to the fixed guide connector 1412 passes through the second opening 1310 and enters the sample preparation device 1100. can be moved to

The fixed guide connector 1412 coupled to the vertical fixed guide 1411 couples the vertical motion guide 1413. The fixed guide connector 1412 moves the vertical motion guide 1413 according to the up/down movement provided by the vertical fixed guide 1411.

The vertical motion guide 1413 may move in the up/down direction according to the driving of the vertical fixed guide 1411.

In one implementation, vertical motion guide 1413 is coupled with motion guide connector 1414. The vertical motion guide 1413 can move the coupled motion guide connector 1414 in the up/down direction.

The vertical motion guide 1413 may use power provided by the driving device 1417 to move the motion guide connector 1414 in the up/down direction.

In another implementation, the vertical motion guide 1413 does not move the motion guide connector 1414 and can be coupled in a fixed configuration.

In one implementation, the motion guide connector 1414 that is coupled to the vertical motion guide 1413 and provides up/down movement may be coupled to the rack guide 1415.

A rack guide 1415 is coupled to the upper part of the motion guide connector 1414, the vertical fixed guide 1411 moves the vertical motion guide 1413 upward, and the vertical motion guide 1413 moves the rack guide 1415. When moving upward, the rack guide 1415 is moved inside the sample preparation device 1100.

In another implementation, the motion guide connector 1414 may be coupled with the rack guide 1415.

When the vertical fixed guide 1411 moves the fixed guide connector 1412 upward, the rack guide 1415 moves inside the sample preparation device 1100 by the movement of the vertical motion guide 1413 and the motion guide connector 1414. can be moved to

The vertical motion guide 1413 uses power provided from the driving device 1417 or a separate driving device (not shown) to move the rack guide 1415 in the up and down directions.

The rack guide 1415 is coupled to the motion guide connector 1414, and a reaction vessel rack 1416 capable of holding the reaction vessel 1500 may be located at the top.

The reaction vessel rack 1416 is a space in the sample preparation device 1100 where the reaction vessel 1500 containing the analysis sample is located. The reaction vessel rack 1416 may be used as another expression for a holder, such as pedestal, cradle, or holder.

The reaction vessel rack 1416 can be moved into the sample preparation device 1100 by moving the motion guide connector 1414 together with the rack guide 1415 and receive the reaction vessel 1500.

The rack guide 1415 performs extension movement of the reaction vessel rack 1416 connected to the top in the horizontal direction.

The vertical fixed guide 1411 of the lifting module 1410 uses power provided by the driving device 1417 to move the fixed guide connector 1412 in the up/down direction. A portion of the fixed guide connector 1412 is coupled to the vertical fixed guide 1411, and the other portion is coupled to the vertical motion guide 1413.

The vertical motion guide 1413 may move inside the sample preparation device 1100 as the coupled fixed guide connector 1412 moves upward. Additionally, the vertical motion guide 1413 can move the motion guide connector 1414 coupled to the other side in the up/down direction. The motion guide connector 1414, which moves upward by the operation of the vertical motion guide 1413, can move the rack guide 1415 connected to the top into the interior of the sample preparation device 1100.

When the rack guide 1415 is moved inside the sample preparation device 1100, the rack guide 1415 can horizontally extend and move the reaction vessel rack 1416 located at the top a predetermined distance.

The transfer module (not shown) provided in the sample preparation device 1100 picks up and moves the prepared reaction vessel 1500 and places it on the upper part of the horizontally extended and moved reaction vessel rack 1416, thereby forming the reaction vessel 1500. so that it can be moved to the closed structure 1300.

When the reaction vessel 1500 is mounted on the upper part of the reaction vessel rack 1416, the rack guide 1415 horizontally moves the horizontally extended reaction vessel rack 1416 back to its original position.

When the reaction vessel 1500 is ready to be moved to the closed structure 1300, the vertical fixed guide 1411 and/or vertical motion guide 1413 are coupled to the fixed guide connector 1412 and/or the motion guide connector ( 1414) is moved to the bottom. The reaction vessel 1500 is moved inside the closed structure 1300.

The reaction vessel rack 1416 includes a coupling guide (not shown) corresponding to the reaction vessel 1500 mounted on the top. The coupling guide prevents the reaction vessel 1500 from being separated from the reaction vessel rack 1416 during movement.

In one implementation, drive device 1417 can provide power for horizontal movement of reaction vessel rack 1416.

In another implementation, the rack guide 1415 may be powered by another driving device to horizontally move the reaction vessel rack 1416.

The driving device 1417 may provide power to move the lifting module 1410 within the closed structure 1300. One or more driving devices 1417 may be provided and located in one or more places.

In one implementation, drive device 1417 may use a hydraulic motor. In another implementation, drive device 1417 may use an electric motor. In another implementation, the driving device 1417 may use a combination of hydraulic motors and electric motors. In another implementation, drive device 1417 may use a device capable of generating power other than hydraulic motors and electric motors. In another implementation, drive device 1417 may use hydraulic motors, electric motors, and devices capable of generating power.

One or more driving devices 1417 are provided and can provide power to at least one of the lifting module 1410, the crane module 1430, and/or the reaction vessel rack 1416 within the closed structure 1300. .

The crane module 1430 performs an operation for the lifting module 1410 to move the reaction vessel 1500 received from the sample preparation device 1100 to each component of the closed structure 1300.

In one implementation, the crane module 1430 includes a horizontal stationary guide, a horizontal motion guide, a gripper lift, a gripper rotation module, and a gripper on top within the enclosed structure 1300, each guide and gripper rotation module. ), the gripper can move and rotate in the X, Y, and Z axes. In another embodiment, the crane module 1430 includes a horizontal fixed guide, a guide connector, a horizontal motion guide, and a gripper lift on top within the enclosed structure 1300. ), a gripper motion guide, and a gripper, and the gripper can move in the X, Y, and Z axes by each guide and gripper lift.

The horizontal fixed guide is coupled to the horizontal motion guide by a fixed guide connector.

The horizontal fixed guide may be provided in the form of one or more movable rails. The horizontal motion guide is moved in the X-axis direction by being combined with a horizontal fixed guide in the form of a rail and a fixed guide connector.

The horizontal fixed guide is provided in the form of two rails to stably move the horizontal motion guide, and the horizontal motion guide is connected to the two rails and can be moved in the X-axis direction. Among the two rails of the horizontal fixed guide, any one rail can move the combined horizontal motion guide in the X-axis direction.

In one implementation, power for the horizontal fixed guide to move the horizontal motion guide may be provided by the driving device 1417. In another implementation, power for the horizontal fixed guide to move the horizontal motion guide may be provided by a separately provided driving device (not shown).

The horizontal motion guide is combined with a gripper lift.

The horizontal motion guide is provided in the form of a rail. The gripper lift is coupled to a horizontal motion guide in the form of a rail and moves in the Y-axis direction. A horizontal motion guide providing rail-type movement can move the combined gripper lift in the Y-axis direction.

In one embodiment, the power for the horizontal motion guide to move the gripper lift may be provided by a drive device.

In another implementation, power for the horizontal motion guide to move the gripper lift may be provided by a separately provided driving device (not shown).

The gripper lift is connected to a gripper motion guide and coupled to the gripper. The gripper movement guide is coupled to a gripper lift that moves in the up/down direction and moves in the Z-axis direction. The gripper list can move the combined gripper in the Z-axis direction.

The gripper lift is a combination of two modules.

One is a fixed module that is coupled to a horizontal motion guide and moves in the Y axis. The other is a movement module that is combined with the gripper movement guide and moves the gripper in the up/down direction. The gripper movement guide is coupled with the gripper, and the gripper is moved in the Z-axis direction by the movement provided in the up and down directions of the gripper lift.

The gripper can be moved to the position of the reaction vessel 1500 and pick up the reaction vessel 1500 by the operation of the gripper lift. The gripper detects the pressure of gripping the reaction vessel 1500 using a pressure sensor, etc., and can hold the reaction vessel 1500 to prevent it from being damaged.

In one implementation, the gripper can rotate the picked reaction vessel 1500.

The gripper movement guide coupled to the gripper lift is coupled with the gripper and the gripper rotation module. The gripper rotation module can rotate the gripper. The gripper rotation module consists of a rotation motor.

In one implementation, the gripper rotation module can rotate the gripper through a rotation angle of 90 degrees. In another implementation, the gripper rotation module can rotate the gripper to any rotation angle.

One or more drive devices used in the lifting module 1410 and/or the crane module 1430 may be operated using various driving forces.

In one embodiment, at least one of the driving devices may use a hydraulic motor. In other implementations, at least some of the drive devices may use electric motors. In another embodiment, at least one of the driving devices may be a combination of a hydraulic motor and an electric motor. In another embodiment, at least one of the driving devices may use a device capable of generating power other than a hydraulic motor and an electric motor. In another embodiment, at least one of the driving devices may use a hydraulic motor, an electric motor, or a device capable of generating power.

The crane module 1430 may move the gripper to the upper position of the reaction vessel rack 1416 of the lifting module 1410 in order to move the reaction vessel 1500. The crane module 1430 can lift the reaction vessel 1500 after sending down the gripper located at the top of the reaction vessel rack 1416.

In one implementation, the crane module 1430 moves the gripper to the top of the reaction vessel rack 1416 based on pre-stored position coordinates.

The integrated system 1000 stores all positions where the crane module 1430 can move within the closed structure 1300 as coordinate information. The crane module 1430 may move according to coordinate information of the position to be moved.

In another implementation, the operation of the crane module 1430 to move the gripper to the upper part of the reaction vessel rack 1416 moves by pre-stored position coordinates, and additionally, the position sensor module (not shown) provided within the closed structure 1300 (time) so that it can stop in the correct position. The position sensor module transmits and receives signals between the gripper and the reaction vessel rack 1416 based on optical signals, allowing the gripper to stop at a designated position.

The position sensor module is a reaction vessel rack 1416, an automatic vessel sealer 1700, a sample analysis device (1200-a, 1200-b), a reaction vessel recovery bin, or a conveyor, which are components through which the gripper moves the reaction vessel 1500. (1350), etc. may be provided.

Among the reaction vessel transfer devices 1400 included in the closed structure 1300, the lifting module 1410 and the crane module 1430 are provided with power to move the reaction vessel 1500.

The lifting module 1410 receives power from a vertical fixed guide 1411, a vertical motion guide 1413, and/or a rack guide 1415.

Crane module 1430 may be powered by horizontal stationary guides, horizontal motion guides, gripper lifts, and/or grippers.

Each component that receives power from the lifting module 1410 and the crane module 1430 can perform operations through the following driving method.

In one implementation, the lifting module 1410 and/or the crane module 1430 may move the reaction vessel 1500 by providing belt-type movement. In another implementation, the lifting module 1410 and/or the crane module 1430 may provide a chain type of movement to move the reaction vessel 1500. In another embodiment, the lifting module 1410 and/or the crane module 1430 may move the reaction vessel 1500 by providing screw type or jackscrew type movement. . In another implementation, the lifting module 1410 and/or the crane module 1430 may move the reaction vessel 1500 by providing a cylinder type movement. In another implementation, the lifting module 1410 and/or the crane module 1430 may move the reaction vessel 1500 by providing a hoist type of movement. In another embodiment, the lifting module 1410 and/or the crane module 1430 may move the reaction vessel 1500 through a driving method other than the method described above.

The crane module 1430 can move the reaction vessel 1500 picked up from the reaction vessel rack 1416 so that it is mounted on the automatic vessel sealer 1700. The automatic vessel sealer 1700 is a device for automatically sealing the upper surface of the reaction vessel 1500.

An automated container sealer (1700) can seal the inlet of the reaction vessel (1500) containing the analysis sample, and can be implemented as follows.

In one embodiment, the reaction vessel 1500 is a multi-well plate, and an analysis sample is accommodated in each of the multi-well plates formed with a plurality of wells with closed bottoms.

The automatic vessel sealer 1700 can prevent mixing of analysis samples and contamination from the outside by sealing the upper surface of the reaction vessel 1500, which is a multi-well plate.

In another embodiment, the reaction vessel 1500 is a tube-shaped vessel inserted into each well of a multi-well plate. A plurality of connected or individually separated tubes may be inserted into each well of a multi-well plate.

The automatic vessel sealer 1700 can prevent mixing of analysis samples and contamination from the outside by sealing the upper surface of one or more reaction vessels 1500 inserted into each well of a multi-well plate.

The automatic container sealer 1700 can heat-seal the inlet of the reaction container 1500 using a transparent film. Alternatively, adhesion may be performed using adhesive.

In one embodiment, the automatic container sealer 1700 may use Hamilton's Plate Sealer product (see https://www.hamiltoncompany.com/automated-liquid-handling/small-devices/hamilton-plate-sealer).

The automatic container sealer 1700 can be positioned in a variety of ways within the closure structure 1300.

In one implementation, the automatic container sealer 1700 may be positioned between the first sample analysis device 1220-a and the second sample analysis device 1220-b.

When the automatic vessel sealer 1700 is located between the first sample analysis device (1220-a) and the second sample analysis device (1220-b), the reaction vessel 1500 is horizontally rotated 90 degrees to use the automatic vessel sealer ( 1700).

A 90-degree horizontal rotation of the reaction vessel 1500 may be performed by a gripper rotation module.

In order to mount the reaction vessel 1500 on the automatic vessel sealer 1700, the crane module 1430 horizontally rotates the reaction vessel 1500 by 90 degrees using a gripper rotation module.

In other implementations, the automatic container sealer 1700 may be implemented to be located at another location inside the closure structure 1300.

In another implementation, the automatic container sealer 1700 may be implemented to be located in the sample preparation device 1100.

The crane module 1430 may move the reaction vessel 1500 sealed by the automatic vessel sealer 1700 to be mounted on any one of the plurality of sample analysis devices 1220-a and 1220-b. The sample analysis devices 1220-a and 1220-b are devices for automatically analyzing one or more analysis samples contained in the reaction vessel 1500.

The sample analysis device 1200 according to one embodiment is a stand-alone device. That is, the sample analysis device 1200 may be installed and operated independently to analyze an analysis sample.

The sample analysis device 1200 is a device for automatically analyzing one or more analysis samples contained in the reaction vessel 1500.

According to one implementation, the sample analysis device 1200 may be operatively connected to the sample preparation device 1100 and/or the enclosed structure 1300 to be used as an integrated system 1100.

The sample analysis device 1200 may include a nucleic acid amplifier for amplifying nucleic acids and/or an optical module for detecting the amplified nucleic acids.

In one implementation, sample analysis device 1200 includes a nucleic acid amplifier and an optical module.

In another implementation, sample analysis device 1200 includes a nucleic acid amplifier. In another implementation, sample analysis device 1200 includes an optical module.

In one implementation, one sample analysis device 1200 can be operatively connected and applied to the integrated system 1000. In another implementation, a plurality of sample analysis devices 1200 may be operably connected and applied to an integrated system 1000.

The sample analysis device 1200 may be equipped with a reaction vessel 1500 that accommodates the analysis sample prepared in the sample preparation device 1100.

In one embodiment, the sample analysis device 1200 may be equipped with a reaction vessel 1500 whose upper surface is sealed by an automatic vessel sealer 1700. To this end, the reaction vessel 1500 can be moved from the sample preparation device 1100 to the automatic container sealer 1700 and mounted on the sample analysis device 1200 after sealing of the upper surface is completed.

The sample analysis device 1200 is provided with a sample holder in which the reaction vessel 1500 is accommodated.

The sample analysis device 1200 may be equipped with a sample holder and a cover to protect the reaction vessel 1500 accommodated in the sample holder. In one implementation, the sample analysis device 1200 is uncovered prior to receiving the reaction vessel 1500. In another embodiment, the sample analysis device 1200 is capped after the reaction vessel 1500 is housed.

When the sample analysis device 1200 is operated as a stand-alone device, the cover can be opened or closed by a user's command input.

When operatively connected to the integrated system 1000, the sample analysis device 1200 can have its cover opened or closed by a control module of the integrated system 1000.

In one embodiment, the reaction vessel 1500 may be provided in the form of a multi-well plate for amplification containing a plurality of analysis samples to be analyzed in each of a plurality of wells. In this case, the sample holder can accommodate one multi-well plate for amplification. A well plate includes n A well plate in the present disclosure may include 96 wells of 8×12.

In another embodiment, the reaction vessel may be equipped with one or more independent reaction vessels. In this case, the sample holder can accommodate one or more respective reaction vessels.

In another embodiment, the reaction vessel may be provided in the form of a strip tube in which two or more sample vessels are connected. In this case, the sample holder can accommodate one or more reaction vessels in the form of a strip tube.

In one embodiment, at least one sample analysis device 1200 that receives the reaction vessel 1500 sealed by the automatic vessel sealer 1700 may be provided in the closed structure 1300. That is, referring to FIG. 4, two sample analysis devices 1200-a and 1200-b may be provided inside the closed structure 1300.

In one embodiment, when two sample analysis devices are configured within the closed structure 1300, the sample preparation device 1100 sequentially prepares analysis samples for analysis. When the first reaction vessel is prepared in the sample preparation device 1100, one of the sample analysis devices in the closed structure 1300 moves the first reaction vessel to perform analysis of the first reaction vessel.

When the second reaction vessel is prepared in the sample preparation device 1100, another sample analysis device in the closed structure 1300 moves the second reaction vessel to perform analysis of the second reaction vessel.

In another embodiment, when two sample analysis devices are configured within the closed structure 1300, the sample preparation device 1100 simultaneously or sequentially prepares analysis samples for analysis. When the first reaction vessel and the second reaction vessel are prepared in the sample preparation device 1100, each reaction vessel 1500 is sequentially moved to the closed structure 1300, but the first reaction vessel is connected to one of the sample analysis devices. When mounted on 1200-a, the second reaction vessel is mounted on another sample analysis device 1200-b.

Figure 5 is a perspective view showing a stand-alone sample preparation device according to one embodiment. As shown in FIG. 5, the sample preparation device 1100 is a stand-alone device. That is, the sample preparation device 1100 may be installed and operated independently to prepare analysis samples.

According to one implementation, the sample preparation device 1100 may be operatively connected to the sample analysis device 1200 and/or the enclosed structure 1300 to be used as an integrated system 1000.

The sample preparation device 1100 may include a nucleic acid extraction module and/or a liquid handling module for extracting nucleic acids from the analyte.

In one implementation, sample preparation device 1100 includes a nucleic acid extraction module and a liquid dispensing module.

In another implementation, sample preparation device 1100 includes a nucleic acid extraction module.

In another implementation, sample preparation device 1100 includes a liquid dispensing module.

In the present disclosure, the nucleic acid extraction module and/or liquid dispensing module included in the stand-alone sample preparation device 1100 may be operably connected to the integrated system 1000 without modification.

Additionally, when the stand-alone sample preparation device 1100 is operatively connected to the integrated system 1000, the sample preparation device 1100 can use a previously used reagent container in the form of a tube.

In one embodiment, when the sample preparation device 1100 is used as an integrated system 1000, the first opening 1130 formed in the sample preparation device 1100 is used to transport the reaction vessel 1500 by the reaction vessel transfer device 1400. ) can be used as a defined passage through which it is transported.

As shown in FIG. 5, the first opening 1130 is formed on the bottom of the sample preparation device 1100, but depending on the type of the sample preparation device 1100, the first opening 1130 is located on the side (front/rear). /including left/right) or may be already formed on the upper surface.

In another embodiment, when the sample preparation device 1100 is used as an integrated system 1000, the sample preparation device 1100 may be configured as a first defined passageway through which the reaction vessel may be transported by the reaction vessel transfer device 1400. An opening 1130 may be formed.

The first opening of the sample preparation device 1100 may be formed on any of the top, bottom, or sides (including front/back/left/right).

The first opening 1130 is sized so that the reaction vessel 1500 and the reaction vessel transfer device 1400 that transfers the reaction vessel can be moved.

In one embodiment, when the sample analysis device 1200 used in the integrated system 1000 is provided as one, the sample preparation device 1100 sequentially prepares the analysis samples one by one and analyzes them in the sample analysis device 1200. After this is completed, the prepared sample can be provided for analysis.

In another implementation, when a plurality of sample analysis devices 1200 used in the integrated system 1000 are provided, the sample preparation device 1100 sequentially prepares each analysis sample to be provided to each sample analysis device, , the prepared analysis sample is provided to any one sample analysis device. Thereafter, the next analysis sample prepared in the sample preparation device can be provided to another sample analysis device.

In another implementation, when a plurality of sample analysis devices 1200 used in the integrated system 1000 are provided, the sample preparation device 1100 prepares each analysis sample to be provided to each sample analysis device. Prepare the same number or less. Thereafter, the prepared plurality of analysis samples can be provided to each corresponding sample analysis device.

The sample preparation device 1100 includes a deck 1110 that can hold various types of instruments and containers for preparing analysis samples. The deck 1110 is configured to allow components included in the sample preparation device 1100 to be mounted and fixed.

In one implementation, the deck 1110 provides a guide, and the guide guides the components of the sample preparation device 1100 to be inserted into the interior of the sample preparation device 1100 in a sliding manner and positioned on top of the deck 1110. do.

The guide is one implementation of the deck 1110, and may be provided in the form of other implementations.

In one implementation, one or more components located on the deck 1110 may be fixed during operation of the sample preparation device 1100 by protrusions and/or grooves formed on the lower portion of each component.

Sample preparation device 1100 may include a planar loading tray 1120 extending from deck 1110. The loading tray 1120 is installed to extend from the deck 1110 so that components mounted on the sample preparation device 1100 can be easily moved into the sample preparation device 1100. The loading tray 1120 is formed with a guide that extends or is connected to the guide of the deck 1110. Components mounted on the sample preparation device 1100 can be easily moved and mounted by the guide of the deck 1110 and the guide of the loading tray 1120.

In one embodiment, the sample preparation device 1100 includes a pipette module (not shown) including pipette arms for dispensing liquid and one or more pipetting channels connected to the pipette arms. It is provided. The pipette module is configured on the upper side inside the sample preparation device 1100.

In addition, a transport module (not shown) for transporting various containers used for sample preparation, including the reaction vessel, is configured on one side of the sample preparation device 1100. .

In another implementation, the transfer module is configured on the upper side inside the sample preparation device 1100 along with the pipette module.

In another embodiment, the transport module is implemented by a pipetting channel of the pipette module and a gripper (not shown) coupled to the pipetting channel.

Each component located on the deck 1110 of the sample preparation device 1100 is placed at a designated position for preparation of analysis samples.

The deck 1110 is formed with a first opening 1130. The first opening 1130 is a space where the reaction vessel transfer device 1400 is moved to receive the reaction vessel 1500 prepared in the sample preparation device 1100. The first opening 1300 is sized so that the reaction vessel transfer device 1400 can receive and move the reaction vessel 1500.

In one implementation, each component located on deck 1110 can be described as follows. The components described below are generally included in the sample preparation device 1100 and used to prepare samples for analysis, but depending on the type of stand-alone device operated individually, one or more components may not be included. . Or it can be used as a separate device.

All components of sample preparation device 1100 are designed as an integrated device. The sample preparation device 1100 includes a nucleic acid extraction module for extracting nucleic acids from a sample and various components for amplification reaction setup (eg, PCR setup).

The interior of the sample preparation device 1100 according to one embodiment includes a pipette tip adapter, a container carrier, a nucleic acid extraction module, and a multi-well plate adapter. ), a scanner, a waste liquid inlet, a transfer module, and a pipette module.

A pipette tip adapter accommodates one or more pipette tips coupled to a pipetting channel. The pipette tip is coupled to the pipetting channel and can aspirate and dispense solutions such as samples or reagents contained in the container.

One or more pipette tips accommodated in the pipette tip adapter may be provided in different sizes and dispensing amounts depending on the preparation environment, such as the size of the container and the volume of the solution to be dispensed.

In one embodiment, the pipette tip adapter of the sample preparation device is plural to accommodate tips of various volumes, such as 1 mL, 500 μL, 300 μL, 250 μL, 200 μL, 150 μL, 100 μL, and/or 50 μL. It can be provided as a dog. Additionally, one or more tips among tips with various capacities may be a piercing tip.

Each pipette tip adapter can accommodate one or more pipette tips, and the pipette module places the pipetting channel on the top of the pipette tip adapter and then moves it in the direction of the pipette tip so that the pipetting channel can couple the pipette tip. do.

The number of pipette tip adapters and the capacity and size of each pipette tip accommodated in the pipette tip adapter may be modified or changed according to various embodiments of the present disclosure.

The vessel carrier contains various vessels that hold various types of solutions used in the sample preparation device. The sample preparation device can prepare an analysis sample containing extracted nucleic acids using a nucleic acid extraction module. Various types of containers are used in the operation of the sample preparation device, and containers other than the well plate can be inserted into the container carrier.

The container carrier may be provided in various forms so that each container can be easily inserted and fixed depending on the capacity and/or size of the inserted container.

In one embodiment, the inserted container includes a container accommodating a sample, a container accommodating an extraction reagent, and a container accommodating a reaction reagent. The container carrier can insert containers in series or in parallel.

A multi-well plate adapter is a structure in which a reaction vessel accommodating a sample on which detection is to be performed can be located, and the reaction vessel can be mounted on a sample holder of a sample analysis device.

The multi-well plate adapter can load a reaction vessel (multi-well plate), and samples for detection can be dispensed into the reaction vessel (multi-well plate) located in the multi-well plate adapter. The multi-well plate adapter can load two or more multi-well plates used in a sample preparation device. In one embodiment, the term “multi-well plate” may be used as a reaction vessel adapter.

At this time, one of the plurality of multi-well plates provided in the multi-well plate adapter can be moved to the starting position and used for preparing the analysis sample. The multi-well plate is moved to the starting position by the transfer module.

Various containers can be mounted on the fixed frame directly or through adapters, etc. The container may accommodate an analysis sample or extraction reagent. The container is equipped with a cap, and the cap may be a pierceable cap by a pipette tip. The penetrating part of the cap may be made of materials such as rubber, silicone, and plastic.

The pipette tip pierces the upper part of the cap by a downward motion, performs suction or dispensing of the solution, and then moves back to the upper part. When the pipette tip moves upward from the container, the container needs to be secured because the container can be lifted upward together by the pipette tip inserted into the perforated portion of the cap. The holding frame can secure a vessel using a pierceable cap such that the vessel is not moved by the pipette tip.

The first opening 1130 is a defined passageway through which the reaction vessel transfer device 1400 moves to the sample preparation device 1100 to receive the reaction vessel 1500.

In one implementation, the first opening 1130 is an empty space. The reaction vessel transfer module 1400 is moved from the bottom of the sample preparation device 1100 to the inside of the sample preparation device 1100. Therefore, the first opening 1130, which is an empty space, is formed in the deck 1110, which is the lower surface of the sample preparation device 1100, or is preferably formed.

In another implementation, the first opening 1130 includes an open/close module (not shown). The opening and closing module is provided to block the open space into which the reaction vessel transfer device 1400, which is the first opening 1130, enters. The reaction vessel transfer device 1400 is moved to the sample preparation device 1100 to receive the reaction vessel prepared in the sample preparation device 1100. The sample preparation device 1100 may block the first opening 1130 using an opening/closing module to close the open space when the reaction vessel transfer device 1400 is not moving.

The waste portion includes a waste liquid inlet and/or a waste pipette tip collecting unit. The recovery solution inlet may collect the solution used for the preparation of the analysis sample to be discarded, and the pipette tip recovery part may collect the pipette tip used for the preparation of the analysis sample to be discarded.

In one embodiment, the recovery solution inlet is connected to a separately located liquid waste collection bin (not shown). The waste solution of the sample preparation device 1100 is moved through the recovery solution inlet to be accommodated in the solution recovery box.

Additionally, pipette tips collected through the pipette tip recovery unit may be moved to a waste container and stored. In one implementation, a waste container may be located on deck 1110 of sample preparation device 1100. In another implementation, a waste container may be located on the bottom of sample preparation device 1100. In another implementation, the waste container may be located external to sample preparation device 1100.

When the waste container is located on the deck 1110, the waste container may be separated into an area where analysis samples are prepared, a partition, etc.

The transfer module is a mechanical device in the form of grippers for moving a reaction vessel, etc. within the sample preparation device 1100. The transfer module is operated by the control device of the sample preparation device 1100.

In one implementation, the transfer module is located on the inner rear of sample preparation device 1100. The transfer module is configured to move the reaction vessel, etc. up and down, left and right, forward and backward, and rotation.

In another implementation, the transfer module is located on the top inside of sample preparation device 1100. The transfer module is configured to move the reaction vessel up and down, left and right, forward and backward, and rotationally through an operation similar to a pipette module.

In another embodiment, the transfer module is configured to move the reaction vessel up and down, left and right, forward and backward, and rotationally, using a gripper coupled to at least two pipetting channels among the pipetting channels of the pipette module.

The transfer module can move components necessary for preparation of analysis samples, such as reaction vessels, reagent vessels, adapters, cartridges, and multi-well plates, within the sample preparation device 1100.

In one implementation, the transfer module may move the reaction vessel in which the analysis sample has been set up to the reaction vessel transfer device 1400. The reaction vessel transfer device 1400 is moved through the first opening 1130 of the sample preparation device 1100 to receive the prepared reaction vessel. The transfer module moves the reaction vessel to the reaction vessel transfer device 1400, and the reaction vessel transfer device 1400 can move the reaction vessel to the outside of the sample preparation device 1100 through the first opening 1130.

The scanner can read identifying codes displayed on samples, reagents, reaction solutions, etc. An identification code is a mark containing information such as a barcode or matrix code. The scanner can recognize the identification code and receive information such as the type of solution contained in the container and the volume of the solution.

In one implementation, a plurality of scanners may be provided, and any one scanner may be configured as needed. Additionally, the scanner may be configured as a barcode scanner and/or a 2D scanner. This configuration is preferably provided to recognize different types of identification codes displayed on reaction vessels, etc.

In one implementation, the scanner is capable of recognizing 1D and/or 2D barcodes. The scanner can obtain information such as the type and/or volume of the solution contained in the container by recognizing the identification code printed or attached to the side of the container. Containers include reaction vessels, reagent vessels, sample vessels, etc. used in sample preparation devices. The scanner can recognize the identification code of the container inserted into the deck 1110. The scanner can sequentially recognize at least one container identification code inserted into the deck 1110. The scanner can recognize the identification code on the side of the container by moving to the position where the container or the carrier containing the container is coupled to the deck 1110.

In another implementation, the scanner is a 2D barcode scanner (not shown). Matrix (2D) codes can be recognized, and identification codes printed or attached to the bottom of the container can be recognized. Containers include containers used in sample preparation devices, such as reaction containers, reagent containers, and specimen containers.

Accordingly, when a plate formed to have an opening in all or part of the bottom of each well is mounted on the scanner, the scanner can recognize the identification code printed or attached to the bottom of the container inserted into the plate. The scanner can recognize the codes of multiple containers inserted into the plate at once.

In one embodiment, the plane on which the vessel is mounted in the scanner is made of a transparent material. The scanner can recognize the identification code on the bottom of the container using an optical signal that passes through a transparent material. Additionally, the scanner can recognize the identification code of the container by photographing the bottom of the container. The scanner is designed to accommodate a multi-well plate so that it can recognize the identification code located on the bottom of the container inserted into the multi-well plate.

The 2D scanner according to one embodiment may use Hamilton's "easyCode Carrier" product (see https://www.hamiltoncompany.com/automated-liquid-handling/small-devices/easycode-carrier).

Although not shown in the drawing, the pipette module is located on the upper inner side of the sample preparation device 1100. The pipette module, which is a solution fractionator, includes a pipette arm and a pipetting channel, and the pipetting channel can automatically move up and down, left and right, and forward and backward by a control device.

The pipette arm may include one or more independently or dependently moving pipetting channels. In one embodiment, a pipette tip or needle is coupled to the end of the pipetting channel and can be used to aspirate and dispense a solution.

In another embodiment, a gripper may be coupled to the end of the pipetting channel, and the gripper may be used to move containers (including reaction containers) used in the sample preparation device 1100, such as reaction containers. Can be used as a module.

The pipette arm moves one or more pipetting channels to a pipette tip for fractionation, fixes the pipette tip for fractionation to the pipetting channel, and moves the pipette tip with the pipetting channel fixed to a certain location. Actions such as inserting a pipette tip for fractionation into a container at a certain depth can be performed.

The pipette arm is located at the top inside the sample preparation device 1100, and the pipetting channel is operated within the sample preparation device 1100 by the pipette arm. One or more pipetting channels couple a pipette tip inserted into a pipette tip adapter to an end of the pipetting channel.

The pipette arm can be moved so that the pipetting channel is positioned at the top of the container containing the solution to be dispensed. The pipetting channel is positioned to descend toward the container from the moved position to accommodate the solution in the pipette tip, and then rise again. The pipetting channel may move to the top of another container to be dispensed by the pipette arm, descend to dispense the solution contained in the pipette tip, and then rise again to end dispensing.

A plurality of pipetting channels can be operated simultaneously, and the number of containers that can be dispensed simultaneously can be determined depending on the number of pipetting channels.

After dispensing of the pipette arm and pipetting channel is completed, the pipette tip coupled to the end can be removed. The combined pipette tip can be removed from the pipette tip recovery unit located in the waste unit and disposed of in a waste container.

In one embodiment, in order for at least one of the sample preparation device 1100, the sample analysis device 1200, and/or the reaction vessel transfer device 1400 to be operatively connected to the closed structure 1300, each device must be located accurately. For this purpose, the closed structure 1300 and each device include positioning means.

Since the sample preparation device 1100, sample analysis device 1200, and/or reaction vessel transfer device 1400 must be coupled to the closed structure 1300 to prevent external deformation, the positioning means for each device is It is preferred to use ready-made components in the device.

At least one of the sample preparation device 1100, the sample analysis device 1200, and/or the reaction vessel transfer device 1400 is spatially closed, and a closed structure 1300 is used to spatially close it. .

At least one of the sample preparation device 1100, the sample analysis device 1200, and/or the reaction vessel transfer device 1400 is configured to be located inside the closed structure 1300 in order to operatively connect at least one of the sample preparation device 1100, the sample analysis device 1200, and/or the reaction vessel transfer device 1400. do.

Sample analysis devices 1200-a and 1200-b may be located in the closed structure 1300. At this time, in the sample analysis devices 1200-a and 1200-b, the reaction vessel 1500 containing the analysis sample in the sample preparation device 1100 located outside the closed structure 1300 is provided by the reaction vessel transfer device 1400. can be moved through.

In particular, the crane module 1430 of the reaction vessel transfer device 1400 provides the reaction vessel 1500 to the sample analysis devices 1200-a and 1200-b. The crane module 1430 is a robot module that moves to a designated location, lifts the reaction vessel 1500, and sets the reaction vessel 1500 down at the designated location.

If the positions of the sample analysis devices 1200-a and 1200-b deviate even slightly from the designated positions, appropriate analysis cannot proceed because the reaction vessel 1500 cannot be placed in the correct position. Therefore, the sample analysis devices 1200-a and 1200-b must be accurately positioned or coupled to a designated position within the closed structure 1300. For this purpose, the closed structure 1300 provides positioning means.

Positioning means may be located in the sample analysis device 1200 and the closed structure 1300.

In one implementation, the first positioning means of the sample analysis device 1200 may be a fixing means located at the bottom. Additionally, the second positioning means of the closed structure 1300 may be a structure formed at a location where the sample analysis device 1200 is located.

The first positioning means and the second positioning means may be formed in a shape that can be coupled to each other.

According to one embodiment, even if the first positioning means and the second positioning means do not have separate fastening means, the sample analysis device 1200 can be fixed to the exact position of the closed structure 1300.

According to another embodiment, the first positioning means and the second positioning means are provided with a mutually fastening means (not shown), so that when the sample analysis device 1200 is fixed to the correct position of the closed structure 1300, the fastening means Each positioning means can be fastened.

Although the positioning means between the sample analysis device 1200 and the closed structure 1300 has been described above, the closed structure 1300 such as the reaction vessel transfer device 1400 and the automatic vessel sealer 1700 according to one embodiment It is desirable for each device located inside to be provided with positioning means matching the closed structure.

The integrated system can position at least one of a sample preparation device, a sample analysis device, and/or a reaction vessel transport device within a spatially closed, enclosed structure.

In one embodiment, the integrated system is configured to have a sample analysis device inside the closed structure and a sample preparation device located on top of the closed structure.

The integrated system includes a control module. The control module controls the sample preparation device to prepare an analysis sample. The control module controls the closed structure to analyze the prepared analysis sample.

How the control module of the integrated system controls the sample preparation device and the closure structure to perform preparation and analysis of the analysis sample is as follows.

The control module controls the sample preparation device so that an analysis sample is prepared in a reaction vessel provided in the sample preparation device.

The sample preparation device prepares solutions such as specimens, nucleic acid extraction reagents, and amplification reaction reagents to prepare analysis samples, and prepares analysis samples using internal components.

The sample preparation device includes: preparing the analysis sample by dispensing at least one of a specimen and a reagent; A process of accommodating the analysis sample into the reaction vessel; Alternatively, at least one preparation may be performed during the process of extracting nucleic acid from the specimen expected to contain pathogens.

The control module controls the lifting module within the closed structure to move the reaction vessel prepared in the sample preparation device into the closed structure.

In this step, the lifting module moves into the interior of the sample preparation device through a second opening formed in the closure structure and a first opening formed in the deck of the sample preparation device. The sample preparation device mounts the reaction vessel on a moved elevating module.

When the reaction vessel is mounted on the lifting module, the control module controls the lifting module to move to the closed structure.

The first opening is a defined passage through which the reaction vessel can be transported.

In one embodiment, the closed structure may be provided with a plurality of sample analysis devices for analyzing an analysis sample.

The control module controls the sample preparation device so that analysis samples to be analyzed can be prepared simultaneously or sequentially using a plurality of sample analysis devices.

The control module controls the elevating module so that the elevating module moves the reaction vessel containing the analyte samples that are prepared simultaneously or sequentially in the sample preparation device into the closed structure.

In the above step, when the lifting module is moved to the sample preparation device to receive the reaction vessel, the control module controls the lifting module to extend movement in the horizontal direction to easily receive the reaction vessel.

The control module controls the crane module so that the reaction vessel moved to the closed structure is moved to the location where analysis is performed.

The crane module can perform movement operations to move the reaction vessel moved to the closed structure in the up/down/left/right/forward/backward directions. The crane module may perform a rotation operation to horizontally rotate the reaction vessel.

In one embodiment, the automatic container sealer is contained within the closure structure.

When an automatic vessel sealer is provided in the closed structure, the control module controls the crane module to move the reaction vessel moved from the sample preparation device to the automatic vessel sealer. An automatic vessel sealer can seal the top of a reaction vessel.

The control module controls the crane module so that the reaction vessel sealed in the automatic vessel sealer is moved to the location where analysis of the analysis sample is performed. The location where the analysis is performed is the sample analysis device.

In another embodiment, an automatic vessel sealer may be included within the sample preparation device.

If an automatic vessel sealer is provided in the sample preparation device, the control module moves the prepared reaction vessel from the sample preparation device to the automatic vessel sealer. The control module controls the automatic vessel sealer to seal the upper surface of the moved reaction vessel.

If an automatic vessel sealer is provided in the sample preparation device, the lifting module can receive a reaction vessel that has been sealed.

In one embodiment, when a plurality of sample analysis devices are provided and a plurality of reaction vessels are sequentially received from the sample preparation device, the control module operates a crane module to move the sequentially moved analysis sample containers to each sample analysis device. Control.

The control module controls the closed structure so that the analysis sample contained in the reaction vessel within the closed structure is analyzed.

In this step, the reaction vessel can be moved to the sample analysis device by a crane module. The sample analysis device analyzes the analysis sample of the moved reaction vessel and generates a result.

In this step, the closed structure is a sample analysis device, in which the control module controls the closed structure such that analysis of the analysis sample is performed.

The sample analysis device is a process that performs isothermal amplification, especially LAMP; Alternatively, at least one of the processes of analyzing reaction results may be performed.

A sample analysis device is provided at the location where analysis is performed.

The sample analysis device includes a thermal cycler and an optics module. The process of performing a polymerase chain reaction in a sample analysis device uses a thermal cycler, and the process of measuring the reaction results uses an optical module.

The sample analysis device may be equipped with a cover for analysis of the analysis sample. The control module may provide a control signal to the sample analysis device to open a cover of the sample analysis device to which the reaction vessel is to be moved. Thereafter, when the crane module provides a reaction vessel to the sample analysis device, the control module may provide a control signal to close the cover of the sample analysis device.

The control module controls the crane module to remove the reaction vessel on which analysis has been completed from the location where analysis is performed.

In this step, the control module controls the crane module so that the reaction vessel for which analysis has been completed can be moved to the reaction vessel recovery box.

According to one aspect, the present invention includes a memory, at least one processor configured to access the memory, and one or more program codes stored in the memory and configured to be executed by the processor, when the automated molecular diagnosis system is executed by the processor, A sample preparation device, a sample analysis device, a reaction vessel transfer device, and an enclosure, each comprising an opening for operably connecting the sample preparation device and the enclosure, wherein an elevating module is located in the enclosure. A system that allows a reaction vessel to be moved to the sample analysis device by moving to the sample preparation device through the opening causes a control module to move the reaction vessel containing the analysis sample from the sample preparation device to the sample analysis device. controlling the reaction vessel transfer device to be transferred to the analysis device; The sample preparation device and the sample analysis device are stand-alone devices; The control module includes controlling the reaction vessel transfer device so that the reaction vessel in which analysis of the analysis sample has been completed is removed from the sample analysis device, wherein at least one of the sample analysis device and/or the reaction vessel transfer device One device is located inside a spatially enclosed enclosure, wherein the sample preparation device and the enclosure define a defined passage through which the reaction vessel is transported, the reaction vessel The transfer device includes instructions to perform transfer of the reaction vessel using the confirmed passage.

Since each component described in the above-described aspect of the present invention overlaps with the above-described content, its description is omitted.

According to one aspect, the present invention provides a non-transitory computer-readable storage medium that, when executed by one or more processors, stores instructions performed by the one or more processors, wherein the instructions include a sample preparation device, a sample analysis device, and a reaction device. Comprising a vessel transfer device and a closure structure, each opening for operatively connecting the sample preparation device and the closure structure, wherein an elevating module located in the closure structure moves into the sample preparation device through the opening. When the integrated system that allows a reaction vessel to be moved to the sample analysis device is executed by the one or more processors, the control module of the automated molecular diagnosis system is configured to perform a reaction in which the analysis sample is received. controlling the reaction vessel transfer device to transfer the vessel from the sample preparation device to the sample analysis device; The sample preparation device and the sample analysis device are stand-alone devices; The control module includes controlling the reaction vessel transfer device so that the reaction vessel in which analysis of the analysis sample has been completed is removed from the sample analysis device, wherein at least one of the sample analysis device and/or the reaction vessel transfer device One device is located inside a spatially enclosed enclosure, wherein the sample preparation device and the enclosure define a defined passage through which the reaction vessel is transported, the reaction vessel The transfer device is configured to transfer the reaction vessel using the confirmed passage.

Since each component described in the above-described aspect of the present invention overlaps with the above-described content, its description is omitted.

Step (b): Complete sample preparation in the sample preparation device

Step (b) of the present disclosure includes, in a sample preparation device, (b-1) mixing the sample mounted in step (a) directly with a lysis buffer, and (b-2) mixing the mixture at 15°C to 35°C for 3 hours. After incubating for 1 to 5 minutes to obtain a lysate, (b-3) the obtained lysate is mixed with an isothermal amplification reagent to complete sample preparation.

Step (b) is performed by the automated molecular diagnostic system described above in step (a). Accordingly, the description of common content between steps (a) and (b) is omitted to avoid excessive complexity of the specification.

According to the present disclosure, the sample preparation device includes a liquid handling module for receiving sample, direct lysis buffer, and isothermal amplification reagents into a reaction vessel.

The method according to the present disclosure allows the sample to be directly mixed with the lysis buffer, then incubated for 3 to 5 minutes without a separate nucleic acid purification step, and an isothermal amplification reaction can be performed immediately, thereby extracting and purifying nucleic acids from the sample. It has the advantage of being faster than conventional isothermal amplification reactions that require steps.

(b-1) Mixing the sample with lysis buffer directly

Samples are directly mixed with lysis buffer.

As used herein, the term “direct lysis buffer” refers to a composition that acts or has the activity to enable nucleic acids to be extracted directly from various samples containing nucleic acids. Specifically, when a sample extracted from a subject is directly contacted with the lysis buffer, the tissues of organisms present in the sample, such as animal or plant cells, yeast, bacteria, viruses, etc., are destroyed, thereby destroying the tissue contained therein. A lysate in which the target nucleic acid is eluted may be generated.

One feature of the present invention is to obtain nucleic acids sufficient to perform a simple nucleic acid amplification reaction directly using a lysis buffer without performing complex nucleic acid extraction and purification processes from the sample.

Conventional nucleic acid extraction methods require lysis of the organism, binding of the nucleic acid to the substrate, washing of the nucleic acid, and purification and elution of the nucleic acid. Specifically, according to the conventional nucleic acid extraction method, the collected sample is incubated in a lysis buffer to destroy the cell wall and cell membrane to expose the nucleic acid, and then the exposed nucleic acid is bound to a specific structure such as a membrane or bead. Afterwards, pure nucleic acid is obtained by separating the nucleic acid from the structure using a step-by-step buffer. As such, the conventional nucleic acid extraction method requires equipment, reagents, and tools for each step, and a lot of time and money is consumed as each process is performed while moving the sample. For example, in the case of existing PCR molecular diagnosis, modules for nucleic acid extraction and nucleic acid amplification are provided, and additional tools such as reagents and pipettes for nucleic acid extraction are required.

When extracting nucleic acids using this conventional method, it is possible to extract highly pure nucleic acids, but the extraction process takes a long time and the procedure is somewhat complicated, making it unsuitable for urgent situations or situations requiring large-scale diagnosis. .

In contrast, the direct lysis buffer used in the method according to the present disclosure allows direct extraction of nucleic acids from cells in the sample without the need to discard the transport medium or exchange it for another buffer. Therefore, it is possible to save time and cost by allowing the sample to be directly input into the nucleic acid amplification step without going through a complicated nucleic acid purification process.

The direct lysis buffer mixed with the sample in step (b-1) of the present disclosure may be a conventionally known buffer, and may be a commercially available buffer or a buffer prepared in-house.

In one embodiment, the direct dissolution buffer may include a buffer component and a nonionic surfactant.

Examples of such buffer components include, but are not limited to, tris(hydroxymethyl)aminomethane (Tris for short) and acid salts of tris(hydroxymethyl)aminomethane (e.g., Tris-HCl).

Examples of such nonionic surfactants include polyethylene glycol based nonionic surfactants such as polyethylene glycol octylphenyl ether (commercially available as Triton x-100). Additional examples of suitable nonionic surfactants include polysorbate surfactants, such as polyoxyethylene (20) sorbitan monolaurate (commercially available as Polysorbate 20 or Tween® 20).

Commercially available direct lysis buffers are included in the Swab:Direct™ Extraction Kit (Cat. No. 9799140107; Genesystem), KGIC™ DRX solution (Cat. No. KGIC DRX02-15ML; KGIC), and Asan Easy Test COVID-19 Ag. buffer (Asan pharm. Co. Ltd), buffer included in the STANDARD Q COVID-19 AG test (SD Biosensor), CellAmp Direct Lysis set (Cat. No. 3738A), Luna Cell Ready Lysis Module (Cat. No. E3032S) ; NEB), XpressAmp Direct Amplification Reagent (Cat. No. A8882; Promega), etc., but is not limited thereto.

In one embodiment, the direct lysis buffer may be 30-500 mM Tris at pH 6.0-7.0, containing 0.1-1.5% (v/v) of Triton X-100, specifically, 0.8-1.2% ( v/v) of Triton It may be Tris, but is not limited thereto.

In another embodiment, the direct lysis buffer is urea; Monovalent salts containing monovalent cations; A secondary salt containing a divalent cation; and a phosphate-based buffer, wherein the first salt may be any one selected from the group consisting of LiCl, NaCl, KCl, Kl, NaBr, NaNO ₃ , KNO ₃ and combinations thereof, and the second salt is It may be any one selected from the group consisting of MgCl ₂ , CaCl ₂ , NiCl ₂ , CaCO ₃ , MgBr ₂ , CaSO ₄ , MgSO ₄ and combinations thereof, and the phosphate-based buffer may be phosphate buffer, PBS (phosphate buffered saline), It may be any one selected from the group consisting of DPBS (Dulbecco's phosphate buffered saline), NaH ₂ PO ₄ , Na ₂ HPO ₄ , KH ₂ PO ₄ , K ₂ HPO ₄ , K ₃ PO ₄ , and combinations thereof.

In one embodiment, in step (b-1), the sample (including transport medium) mounted in step (a) and the direct lysis buffer are respectively 2 μl to 1,000 μl, 2 μl to 900 μl, and 2 μl to 2 μl. 800 μl, 2 μl to 700 μl, 2 μl to 600 μl, 2 μl to 500 μl, 2 μl to 400 μl, 2 μl to 300 μl, 2 μl to 200 μl, 2 μl to 100 μl, 2 μl to 5 0 ㎕ may be mixed with each other, specifically 5 ㎕ to 300 ㎕, 5 ㎕ to 200 ㎕ or 5 ㎕ to 100 ㎕ may be mixed with each other, more specifically, 20 ㎕ to 300 ㎕, 20 ㎕ to 200 ㎕ or 20 μl to 100 μl may be mixed with each other, and more specifically, 30 μl to 70 μl may be mixed with each other. In one specific embodiment, the amount of sample is 50 μl and the amount of direct lysis buffer is 50 μl.

In one embodiment, the mixing of the sample and direct lysis buffer in step (b-1) is performed in a vessel separate from the reaction vessel in step (b-3), which will be described below.

The vessel used in step (b-1) may be the same type or a different type from the reaction vessel in step (b-3). This means that the vessel used in step (b-1) is the same or different type from the reaction vessel in step (b-3). It means having the same or different number and arrangement of wells compared to the reaction vessel.

The container used in step (b-1) may be provided in the form of a well plate having a plurality of wells that can directly accommodate a plurality of samples to be analyzed and a lysis buffer. The well plate includes n The well plate may have a rectangular shape with n x m wells arranged in rows and columns. For example, it represents 16 wells of 4

Various examples of well plates include:

Well plates can be 4 wells of 2 It may include 64 wells, etc. Additionally, well plates can be 8 wells of 2 x 4, 18 wells of 3 x 6, 32 wells of 4 It may include 16 128 wells. Additionally, the well plate can be 12 wells of 2 x 6, 27 wells of 3 x 9, 48 wells of 4 It may include 24 192 wells, etc. Additionally, the well plate can be 16 wells of 2 x 8, 36 wells of 3 x 12, 64 wells of 4 It may include 32 256 wells. Additionally, the well plate may include 96 wells of 8 x 12, 192 wells of 12 x 16, or 384 wells of 16 x 24.

In one embodiment, the vessel used in step (b-1) may be a plate with the same or more wells compared to the vessel used in step (b-3). As an example, if the vessel used in step (b-3) is a 96-well plate, the vessel used in step (b-1) may be a 96-well plate, a 192-well plate, or a 384-well plate. You can.

The mixing in step (b-1) is performed by a liquid dispensing module having 8 to 96 pipetting channels.

(b-2) Incubation of mixture

In step (b-2) of the present disclosure, the mixture is incubated at 15°C to 35°C for 3 to 5 minutes to obtain a lysate.

The incubation temperature of the mixture can be adjusted according to test results such as lysate yield by those skilled in the art, and can be any temperature selected from 15°C to 35°C, any temperature selected from 15°C to 25°C, and 15°C to 25°C. It may be any temperature selected from 20°C, any temperature selected from 20°C to 30°C, or any temperature selected from 25°C to 30°C, or specifically, it may be room temperature.

The incubation time of the mixture may be adjusted according to test results such as lysate yield by those skilled in the art, and may be any time selected from 3 to 5 minutes, or specifically 5 minutes.

During the incubation in step (b-2), the sample is directly contacted with the lysis buffer to destroy organisms, such as pathogens, contained in the sample, thereby producing a lysate in which the target nucleic acid has been eluted.

In one embodiment, the incubation of step (b-2) may be performed after completing step (b-1) described above. For example, the sample can be directly mixed with the lysis buffer in step (b-1) and then incubated the mixture in step (b-2).

In another embodiment, the incubation of step (b-2) may be performed during step (b-1) described above and/or during step (b-3) described below. This especially applies when there are multiple samples and the number of pipetting channels is less than the number of samples so that the entire sample cannot be directly mixed with the lysis buffer at once. For example, among n samples, there is a time interval after the first sample is directly mixed with the dissolution buffer until the second to nth samples are directly mixed with the dissolution buffer. In addition, after directly mixing the lysis buffer for the n-th sample, there is a time interval until the lysates of the 1st to n-1th samples are mixed with the isothermal amplification reagent in the next step (b-3). If this time interval corresponds to the 3 to 5 minutes required in step (b-2) of the present disclosure, each sample may be considered to have been incubated during the interval and no separate incubation may be performed. For example, after mixing the 1st sample with the direct lysis buffer, incubation of the 1st sample can be carried out for the time it takes to mix the remaining 2 to n samples with the direct lysis buffer, and then mix the nth sample with the direct lysis buffer. After mixing, incubation of the n-th sample may be performed for the time required to mix the lysate of the 1st to n-1th samples with the isothermal amplification reagent in the next step (b-3).

(b-3) Mixing of lysate and isothermal amplification reagent

In step (b-3) of the present disclosure, sample preparation is completed by mixing the lysate with an isothermal amplification reagent in a reaction vessel.

In this step, an isothermal amplification reaction is prepared by mixing the lysate obtained from the sample with an isothermal amplification reagent.

The method according to the present disclosure can be performed by various isothermal amplification methods. For example, rolling circle amplication (RCA, M. M. Ali, F. Li, Z. Zhang, K. Zhang, D.-K. Kang, J. A. Ankrum, X. C. Le and W. Zhao, Chem. Soc. Rev., 2014, 43 , 3324-3341.), loop-mediated isothermal amplication (LAMP, Y. Mori, H. Kanda and T. Notomi, J. Infect. Chemother., 2013, 19, 404-411), recombinase polymerase amplication (RPA, J Li, J. Macdonald and F. von Stetten, Analyst, 2018, 144, 31-67), nucleic acid sequence based amplication (NASBA, A. Borst, J. Verhoef, E. Boel and A. C. Fluit, Clin. Lab. , 2002, 48, 487-492), strand displacement amplication (SDA, B. J. Toley, I. Covelli, Y. Belousov, S. Ramachandran, E. Kline, N. Scarr, N. Vermeulen, W. Mahoney, B. R. Lutz and P. Yager, Analyst, 2015, 140, 7540-7549), helicase dependent amplication (HAD, M. Vincent, Y. Xu and H. Kong, EMBO Rep., 2004, 5, 795-800), transcriptionmediated amplication (TMA) , L. Comanor, Am. J. Gastroenterol., 2001, 96, 2968-2972), etc., but are not limited thereto.

In one embodiment, the isothermal amplification reaction may be LAMP (Loop-mediated isothermal amplification).

Throughout the detailed description herein, LAMP is mainly described as an example of an isothermal amplification reaction, but those skilled in the art will recognize that various isothermal amplification reactions in addition to LAMP can be applied.

In one embodiment, when the target nucleic acid is RNA, the isothermal amplification reaction may include a reverse transcription reaction step. For further details, see Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and Noonan, K. F. et al., Nucleic Acids Res. 16:10366 (1988).

Reverse transcription is a reaction that synthesizes cDNA from RNA by reverse transcription using reverse transcriptase.

As used herein, the term “reverse transcriptase” is also called RNA-dependent DNA polymerase and refers to an enzyme that synthesizes complementary DNA using RNA as a template.

The reverse transcriptase in the present disclosure may use reverse transcriptase enzymes originating from a variety of sources, for example, Avian Myeloblastosis Virus-derived Reverse Transcriptase (AMV RTase), Murine Leukemia Virus-derived Reverse transcriptase (Murine Leukemia Virus-derived Reverse Transcriptase; MuLV RTase) and Rous-Associated Virus 2 Reverse Transcriptase (RAV-2 RTase) may be used, but are not limited to these.

Reverse transcriptase requires a primer to synthesize cDNA from an RNA template. There are three main types of primers used in reverse transcription reactions: (i) oligo dT primers that anneal to the poly A tail of mRNA and synthesize cDNA from the 3'-end, (ii) random nucleotides of 6 to 9 nt in size. (iii) a random primer that initiates synthesis in all regions of RNA and (iii) a target specific primer that synthesizes only the target cDNA.

In one embodiment, the isothermal amplification reagent for detecting the target nucleic acid may include a reagent for reverse transcription reaction.

In one embodiment, the reagent for reverse transcription reaction may include reverse transcription polymerase and/or reverse transcription primer.

In one embodiment, the reverse transcription primer may be an oligo dT-primer, a random primer, or a target-specific primer.

In one embodiment, the reverse transcription primer may be a target-specific primer.

In one embodiment, the target-specific primer is used to synthesize cDNA in a reverse transcription reaction, and then forms a primer pair or primer set with another primer in a target nucleic acid sequence amplification reaction to be used as a primer pair for amplification of the obtained cDNA. You can.

In the present disclosure, the reagent for the reverse transcription reaction may optionally include reagents necessary for performing the reverse transcription reaction, such as buffer and dNTPs (deoxyribonucleotide triphosphates). The optimal amount of reagent to be used in a particular reaction can be readily determined by one skilled in the art having the benefit of the present disclosure. The components of the composition for reverse transcription reaction may be present in individual containers or a plurality of components may be present in one container.

In one embodiment, the isothermal amplification reaction may be RT-LAMP (Loop-mediated isothermal amplification).

In one embodiment, the isothermal amplification reagent may be a LAMP reagent, and according to a specific embodiment, it may be an RT-LAMP reagent.

Loop-Mediated Isothermal Amplification (LAMP) is a nucleic acid amplification method that can amplify target nucleic acids with high sensitivity and specificity under isothermal conditions (Notomi, T. et al. 2000. Loop-Mediated Isothermal Amplification of DNA. Nucleic Acids Res 28, E63). The LAMP method uses a DNA polymerase with strand displacement activity and a set of at least 4 to 6 primers specifically designed at various sites of the target nucleic acid.

As used herein, the term "primer" refers to a single-stranded oligonucleotide used in an amplification reaction and refers to a nucleic acid molecule that is extended by adding nucleotides to its 3' end by covalent bond in a nucleic acid amplification or synthesis reaction using a polymerase. .

Four types of primers (F3, B3, FIP, BIP) are required for the LAMP reaction, and two additional primers (LF, LB) can be used to improve the reaction speed. As explained with reference to FIG. 11, the four types of primers consist of two types of outer primers and two types of inner primers, and the outer primers include a forward outer (F3) primer and a reverse outer (backward) primer. outer, B3) It consists of two types of primers 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 nucleotides corresponding to the forward and reverse base sequences to create the loop essential for the LAMP reaction. It consists of The additional two types of primers are composed of a forward loop (LoopF) primer and a backward loop (LoopB) primer, and accelerate the LAMP reaction by attaching to a base sequence that the inner primer does not bind to.

Primer sets used in LAMP are designed for six distinct regions (F3, F2, F1, B1c, B2c, and B3) on the target nucleic acid, with F3 and B3 being the two outer primers that determine the size of the amplified product. FIP and BIP are two inner primers. FIP is composed of F1c sequence and F2 sequence, and BIP is a hybrid primer composed of B1c and B2 sequence.

The LAMP reaction largely includes an initiating structure production step and a cycling amplification step, and unlike PCR, it does not include a step of denaturing it into a single strand. In the initiation structure production stage, F2 of FIP binds to the F2c region to initiate elongation. BIP proceeds in the same way. The F3 primer binds to the F3c region and initiates DNA synthesis by strand displacement, and is released as the extended DNA strand is replaced in the FIP. The single strand released in this way forms a ring at its 5' end through F1/F1c bond, and the synthesis reaction proceeds in the same way for BIP and B3 primers, resulting in a dumbbell-shaped single-stranded nucleic acid molecule with rings formed at both ends. is formed. Next, DNA synthesis begins from the 3' end of the F1 region, and the cycling step proceeds.

The LB or LF primer, which is an acceleration primer, binds to the single-stranded region of the dumbbell structure described above and binds to the loop between B1 and B2 or the loop between F1 and F2. Using LB or LF primers increases the origin where DNA synthesis begins, ultimately resulting in faster reaction speed. In the LAMP reaction, six rings are usually formed in the process, and only two rings are used as the origin of synthesis, but when LB and LF primers are used, all six rings are used as the origin of synthesis, allowing synthesis for a predetermined time. The amount of DNA produced increases.

A more detailed description of the characteristics and functions of each F3 primer, B3 primer, FIP primer, BIP primer, Loop F primer (LF), and Loop B (LB) primer is given in Notomi et al (T. Notomi, H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino and T. Hase, Nucleic Acids Res., 2000, 28, E63) and Nagamine et al (K. Nagamine, T. Hase and T. Notomi, Mol. Cell. Probes, 2002, 16, 223-229).

In one embodiment, the RT-LAMP reagent for detecting target nucleic acid may include a set of 4 to 6 primers for amplifying the target nucleic acid.

In one embodiment, the RT-LAMP reagent for detecting the target nucleic acid includes a set of six primers for amplifying the target nucleic acid, F3 primer, B3 primer, FIP primer, BIP primer, Loop F primer (LF), and Loop B (LB) ) May include primers.

In one embodiment, the appropriate length of the primer may be determined by considering a number of factors, such as temperature, application field, primer source, and length of the final amplification product. For example, the length of the primer may be at least 10 nucleotides, at least 15 nucleotides for F3 and B3, at least 30 nucleotides for FIP and BIP, and at least 15 nucleotides for LF and LB, but is not limited thereto.

In one embodiment, the primer set is prepared using commercially available primer design software, such as Primer Explorer V5 (Fujitsu, Japan), LAVA (LAMP Assay Versatile Analysis), and LAMP Designer (PREMIER), taking into account the characteristics of the LAMP reaction and the characteristics of the primers. It can be designed using various design software such as Biosoft.

In one embodiment, the RT-LAMP reagent may additionally include reverse transcriptase, DNA polymerase, dNTPs, buffer, and/or magnesium.

In one embodiment, the DNA polymerase that may be included in the RT-LAMP reagent is a polymerase derived from thermophilic microorganisms, and may particularly include a polymerase that lacks the 5'->3' exonuclease function. For example, Bacillus stearothermophilus (Bst) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermusaquaticus (Taq) DNA polymerase, Thermococcus litoralis DNA polymerase; It may include, but is not limited to, Pyrococcus furiosus (Pfu) DNA polymerase, and Bacillus caldotenax DNA polymerase.

In one embodiment, the DNA polymerase may be Bst DNA polymerase, but (i) has strand displacement activity, and (ii) the polymer as a reaction product maintains thermodynamic activity to form a ring structure. It is not limited to Bst DNA polymerase if it satisfies two main requirements for polymerization to occur at a temperature (e.g., between 50°C and 75°C). In addition, for the RT-LAMP reaction, GspSSD DNA polymerase, which has a faster amplification ability and stronger reverse transcriptase activity than the Bst DNA polymerase mainly used in the existing LAMP reaction, can be used, but is not limited to this.

In one embodiment, the RT-LAMP reagent may additionally include nucleotide analogs (analogs) instead of dNTPs. Nucleotide analogs are modified or not found in nature and can be polymerized alone or together with natural nucleotides during template-guided DNA synthesis.

In one embodiment, buffers that may be included in the RT-LAMP reagent include, but are not limited to, sodium phosphate buffer, potassium phosphate buffer, Tris-HCl buffer, or Tricine buffer.

In one embodiment, the magnesium that may be included in the RT-LAMP reagent may be in the form of a salt such as magnesium acetate, magnesium chloride, or magnesium sulfate.

The reaction vessel used in step (b-3) of the present disclosure may be provided in the form of a well plate having a plurality of wells capable of accommodating lysate and isothermal amplification reagent (eg, RT-LAMP reagent). The well plate includes n The well plate may have a rectangular shape with n x m wells arranged in rows and columns. For example, it represents 16 wells of 4

Various examples of well plates include:

Well plates can be 4 wells of 2 It may include 64 wells, etc. Additionally, well plates can be 8 wells of 2 x 4, 18 wells of 3 x 6, 32 wells of 4 It may include 16 128 wells. Additionally, the well plate can be 12 wells of 2 x 6, 27 wells of 3 x 9, 48 wells of 4 It may include 24 192 wells, etc. Additionally, the well plate can be 16 wells of 2 x 8, 36 wells of 3 x 12, 64 wells of 4 It may include 32 256 wells. Additionally, the well plate may include 96 wells of 8 x 12, 192 wells of 12 x 16, or 384 wells of 16 x 24.

In one embodiment, the reaction vessel is a 96-well plate.

The mixing in step (b-3) is performed by a liquid dispensing module having 8 to 96 pipetting channels.

In one embodiment, the liquid dispensing module includes eight pipetting channels.

In another embodiment, the liquid dispensing module includes 96 pipetting channels.

In another embodiment, the liquid dispensing module includes 8 pipetting channels and 96 pipetting channels.

The 8 pipetting channels that can be used herein may be channels whose spacing can be adjusted, while the 96 pipetting channels may be channels whose spacing cannot be adjusted because the spacing is adjusted to a 96-well plate. These 96 pipetting channels may be referred to as pipetting channel heads. The 96 pipetting channels may have channels arranged in an 8

The method according to the present disclosure is a high-throughput isothermal amplification method capable of processing a plurality of samples (eg, 96 samples). This can be achieved by a liquid dispensing module comprising multi-pipetting channels (e.g., 8 to 96 pipetting channels).

Step (c): Transfer of reaction vessel to sample analysis device

In step (c), the reaction vessel containing the mixture prepared in step (b) is moved to the sample analysis device with the help of the reaction vessel transfer device described above.

For the reaction vessel transfer device and movement of the reaction vessel thereby, refer to the description in step (b) described above.

In one embodiment, the step (c) may further include moving the reaction vessel to an automatic vessel sealer with the help of a reaction vessel transfer device and sealing the upper surface of the reaction vessel.

In one embodiment, sealing the upper surface of the reaction vessel may be performed by heat or adhesive.

In one embodiment, the sealing may be performed by the automatic container sealer described above in step (a).

Step (d): Isothermal amplification reaction and detection of target nucleic acid

In step (d), in the sample analysis device, (d-1) reacting the mixture in the reaction vessel at a temperature selected from 50 to 75°C for 10 to 20 minutes to amplify the target nucleic acid in the sample; (d-2) Detect the amplified product.

The above-described step (d) is a process of (d-1) performing an isothermal amplification reaction and (d-2) detecting the amplification product from the isothermal amplification reaction.

In one embodiment, step (d) is a process of (d-1) performing a LAMP reaction and (d-2) detecting an amplification product from the LAMP reaction.

The step (d) is performed in a sample analysis device, and a specific description of the sample analysis device refers to the disclosure of step (a).

The temperature for step (d-1) is a temperature suitable for DNA polymerase activity, which can be easily determined by a person skilled in the art considering the enzyme and target nucleic acid used in the reaction.

In one embodiment, the temperature for step (d-1) is any temperature selected from 50°C to 70°C, any temperature selected from 50°C to 65°C, any temperature selected from 55°C to 75°C, 55°C to 70°C. Any temperature selected from, any temperature selected from 55°C to 65°C, any temperature selected from 60°C to 75°C, any temperature selected from 60°C to 70°C, or any temperature selected from 60°C to 65°C. In certain embodiments, the temperature for step (d-1) is 60°C, 61°C or 62°C, and in more particular embodiments 62°C.

The time for step (d-1) of the present disclosure refers to a sufficient time for significant amplification of the target nucleic acid to be observed by an isothermal amplification reaction, such as a LAMP reaction, which determines the sensitivity of the reaction and is expected to be included in the sample. It can be adjusted considering the amount of target nucleic acid, etc. As an example, the time for step (d-1) can be increased to increase sensitivity.

In one embodiment, the time for step (d-1) is any time selected from 10 to 20 minutes, any time selected from 12 to 20 minutes, any time selected from 15 to 20 minutes, or any time selected from 12 to 18 minutes. It's time. In certain embodiments the time for step (d-1) is 15 minutes or 20 minutes, in more particular embodiments 20 minutes.

In one embodiment, step (d-2) may detect the amplified product by a nucleic acid detection instrument.

In one implementation, step (d-2) may be performed in an end-point manner or may be performed in real-time manner.

In one embodiment, step (d-2) may be performed at regular time intervals while performing step (d-1), for example, step (d-2) is performed while performing step (d-1). This can be done every 30 seconds, 1 minute, 1 minute 30 seconds, 2 minutes, 3 minutes, 4 minutes, or 5 minutes.

In one embodiment, detection of the amplification product is performed by fluorescence measurement using SYBR Green I, color change of an indicator such as phenol red, turbidity, precipitate production, DNA laddering, capillary electrophoresis, DNA chip, gel electrophoresis, and radioactivity measurement. , or may be performed through phosphorescence measurement, but is not limited to this.

In one embodiment, the amplified product can be detected by a color change in the mixture in the reaction vessel following DNA synthesis. In this case, the RT-LAMP reagent may include an appropriate indicator, for example, HNB (hydroxy naphthol blue) can be used as a dye that changes color in response to the magnesium ion concentration in the RT-LAMP reagent. Additionally, the target nucleic acid can be quantitatively or qualitatively analyzed by comparison with the amplified product of positive and/or negative control samples.

In one embodiment, isothermal amplification is confirmed in real time without separate electrophoresis or addition of SYBR Green I after the reaction using a nucleic acid detection device that can detect fluorescent dyes, etc. in real time during the RT-LAMP reaction, and annealing. It is also possible to check whether the target nucleic acid is amplified through peak.

In one embodiment, the amplified product can be detected by direct or indirect methods. In the direct method, a detectably labeled primer (or probe) can be used, and the amplification of the target nucleic acid is detected through a signal emitted through the labeled primer after specific binding to the target nucleic acid, enabling real-time detection. In an indirect detection method, a labeled probe capable of binding to an amplified target nucleic acid can be used. As used herein, detectably labeled means a substance capable of emitting a detectable signal using any suitable method, such as spectroscopic, optical, photochemical, biochemical, enzymatic, electrical and/or immunochemical methods. This means a labeled substance. Such materials may include, for example, fluorescent moieties, chemiluminescent moieties, bioluminescent moieties, magnetic particles, enzymes, substrates, radioactive and chromophore substances.

In one embodiment, labels for detection may include fluorescent labels, luminescent labels, chemiluminescent labels, electrochemical labels, and metal labels. The label may be used as a label itself, such as an intercalating dye. Alternatively, a single label or an interactive dual label comprising a donor molecule and an acceptor molecule may be used in the form of one or more oligonucleotides.

The single label may include a fluorescent label, a luminescent label, a chemiluminescent label, an electrochemical label, and a metal label. The single label provides different signals (eg, different signal intensities) depending on whether it is present on a double strand or a single strand.

In specific embodiments, the single label may be a fluorescent label.

The single label can be linked to an oligonucleotide by various methods. For example, a label is linked to a primer (or probe) through a spacer containing carbon atoms (e.g., 3-carbon spacer, 6-carbon spacer, or 12-carbon spacer).

Interactive labeling systems include signal generation systems in which energy is transferred non-radioactively between a donor molecule (reporter molecule) and an acceptor molecule (quencher molecule).

As a representative example of the interactive labeling system, a fluorescence resonance energy transfer (FRET) labeling system includes a fluorescent reporter molecule (donor molecule) and a quencher molecule (acceptor molecule). In FRET, the energy donor is fluorescent, but the energy acceptor may be fluorescent or non-fluorescent. In another type of interactive labeling system, the energy donor is non-fluorescent, such as a chromophore, and the energy acceptor is fluorescent. In another type of interactive labeling system, the energy donor is luminescent, such as bioluminescent, chemiluminescent, or electrochemiluminescent, and the acceptor is fluorescent.

Interactive dual labeling involves a pair of labels that provide a detectable signal based on contact-mediated quenching (Salvatore et al., Nucleic Acids Research, 2002 (30) no.21 e122 and Johansson et al., J. AM. SOC 2002 (124) pp 6950-6956. The interactive labeling system includes all or any cases involving signal changes due to interaction between at least two molecules (eg, dyes).

Reporter and quencher molecules useful as single or dual labels may include any molecules known in the art. Examples include: Cy2 ^TM (506), YO-PRO ^TM -1 (509), YOYO ^TM -1 (509), Calcein (517), FITC (518), FluorX ^TM (519), Alexa ^TM ( 520), Rhodamine 110 (520), Oregon Green ^TM 500 (522), Oregon Green ^TM 488 (524), RiboGreen ^TM (525), Rhodamine Green ^TM (527), Rhodamine 123 (529), Magnesium Green ^TM (531) , Calcium Green ^TM (533), TO-PRO ^TM -1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPY TMR (568), BODIPY558/568 ( 568), BODIPY564/570 (570), Cy3 ^TM (570), Alexa ^TM 546 (570), TRITC (572), Magnesium Orange ^TM (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange ^TM (576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red ^TM (590), Cy3.5 ^TM (596), ROX (608), Calcium Crimson ^TM (615), Alexa ^TM 594 (615), Texas Red (615), Nile Red (628), YO-PRO ^TM -3 (631), YOYO ^TM -3 (631), R-phycocyanin (642), C-Phycocyanin (648), TO -PRO ^TM -3 (660), TOTO3 (660), DiD DilC (5) (665), Cy5 ^TM (670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536) , Biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM (520) ), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705) and Quasar 705 (610). The number in parentheses is the maximum emission wavelength expressed in nanometers.

Suitable reporter-quencher pairs are described in many publications: Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Color AND Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, editor, indicators (Pergamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949); Haugland, R. P., Handbook of Fluorescent Probes and Research Chemicals, 6th Edition (Molecular Probes, Eugene, Oreg., 1996) U.S. Patent Nos. 3,996,345 and 4,351,760.

The reporter molecule and quencher molecule can each be fluorescent. Additionally, the reporter molecule may be fluorescent but the quencher molecule may be non-fluorescent. For example, non-fluorescent dark quenchers that can quench fluorescence of a broad range of wavelengths or specific wavelengths can be used in the present disclosure. If the quencher molecule is fluorescent, the target nucleic acid sequence can be detected from the signal change of the fluorescent quencher molecule.

Reporter molecules and quencher molecules can be linked to oligonucleotides according to conventional methods. For example, the reporter molecule and the quencher molecule can be linked to the probe through a spacer containing at least 3 carbon atoms (e.g., a 3-carbon spacer, a 6-carbon spacer, a 9-carbon spacer, or a 12-carbon spacer).

In one embodiment, techniques for detecting fluorescence specifically for nucleic acid amplification sequences by LAMP reaction can be used. For example, the assimilation probe method (WO 2011/163425 A1), which measures in real time using the FRET (Fluorescence Resonance Energy Transfer) principle, can be used as a two-stranded probe specifically designed for the nucleic acid amplification sequence, and among the two stranded probes A reporter molecule is attached to the 5' end of one strand, and a quencher molecule is attached to the 3' end of the other strand. Specifically, referring to Figure 12, the assimilation probe is designed as a probe with a reporter molecule attached to the 5' end of one of the loop primers among the six LAMP primers, and a portion of the complementary sequence is designed to be attached to the 3' end. The remaining probes to which the quencher is attached are designed and a total of 7 types of oligonucleotides (i.e., 6 primers and the remaining probe to which the quencher is attached) are used.

In addition, a fluorescence detection method using a probe similar to an assimilation probe is a method of using a pair of probes by designing a fluorophore-labeled primer/probe and a quencher-labeled probe to have complementary sequences (WO 2013/065574 A1), etc. may be used, but are not limited thereto.

In one embodiment, detection of the amplified product can be performed by measuring fluorescence.

In one embodiment, the isothermal amplification reagent for detecting the target nucleic acid may include an oligonucleotide containing a fluorescent molecule and an oligonucleotide containing a quencher molecule.

In one embodiment, the RT-LAMP reagent for detecting the target nucleic acid may include an oligonucleotide containing a fluorescent molecule and an oligonucleotide containing a quencher molecule.

In one embodiment, one of the six LAMP primers included in the RT-LAMP reagent can be used as an oligonucleotide containing the fluorescent molecule or an oligonucleotide containing a quencher molecule, such as FIP, BIP, LF or LB. can be labeled with a fluorescent molecule or a quencher molecule and designed as an oligonucleotide containing the fluorescent molecule or an oligonucleotide containing a quencher molecule.

In one embodiment, the isothermal amplification reagent for detecting target nucleic acids may be an isothermal amplification reagent for detecting a plurality of target nucleic acids. For example, the plurality of target nucleic acids may be 2 to 5, specifically 2 to 4, More specifically, there may be three.

In one embodiment, the isothermal amplification reagent for detecting a plurality of target nucleic acids refers to an isothermal amplification reagent capable of detecting a plurality of target nucleic acids in one reaction vessel.

Throughput of the method of the invention

The method of the present invention can process a large amount of samples in a short time using an automated molecular diagnosis system. Additionally, the method of the present invention can achieve high throughput with the help of a liquid dispensing module comprising 8 to 96 pipetting channels and by using a large capacity vessel such as a 96-well plate as a reaction vessel.

The method of the invention is characterized by having the following throughput:

The method of the present invention may take a total of 18 to 60 minutes per 96 samples. The time required refers to the time from when the sample is mounted on the sample preparation device in the automated molecular diagnostic system until detection of the amplification product is completed. In other words, the time refers to the time required from step (b) to completion of step (d) of the method of the present invention. The time required refers to the time required when the method of the present invention is applied to all 96 samples, and when the number of samples is less than 96, the time required can be shortened.

Herein, the process from step (b) to step (d) may be referred to as one round, and therefore, the time required refers to the time required for one round.

In other words, the method of the present invention may take a total of 18 to 60 minutes per round.

The total time required for the method of the present invention depends on the performance of the sample preparation device, reaction vessel transfer device, and sample analysis device in the automated molecular diagnosis system, the performance of the liquid dispensing module in the sample preparation device, and the number of pipetting channels included in the liquid dispensing module. , may vary from 18 to 60 minutes depending on whether the direct lysis buffer is pre-dispensed into the container, whether the isothermal amplification reagent is pre-dispensed into the container, etc.

For example, the total time required for the method of the present invention can be reduced as the performance of the sample preparation device, reaction vessel transfer device, and sample analysis device in the automated molecular diagnostic system, or the performance of the liquid dispensing module in the sample preparation device is better, , can be reduced as the number of pipetting channels included in the liquid dispensing module increases, and if direct lysis buffer is pre-dispensed into the vessel (e.g., when using a commercial product in which direct lysis buffer is dispensed into each well) ) can be reduced, and can be reduced if the isothermal amplification reagent is pre-dispensed into the vessel (e.g., when using a commercial product in which the RT-LAMP reagent is dispensed into each well).

The total time required for the method of the present invention is 18 minutes to 60 minutes, 20 minutes to 60 minutes, 25 minutes to 60 minutes, 30 minutes to 60 minutes, 35 minutes to 60 minutes, 40 minutes to 60 minutes, and 45 minutes to 60 minutes. , 50 minutes to 60 minutes, 55 minutes to 60 minutes, 18 minutes to 55 minutes, 18 minutes to 50 minutes, 18 minutes to 45 minutes, 18 minutes to 40 minutes, 18 minutes to 35 minutes, 18 minutes to 30 minutes, 18 minutes to 25 minutes, 18 minutes to 20 minutes, 20 minutes to 55 minutes, 20 minutes to 50 minutes, 20 minutes to 45 minutes, 20 minutes to 40 minutes, 20 minutes to 35 minutes, 20 minutes to 30 minutes, 20 minutes to 25 minutes, 25 minutes to 55 minutes, 25 minutes to 50 minutes, 25 minutes to 45 minutes, 25 minutes to 40 minutes, 25 minutes to 35 minutes, 25 minutes to 30 minutes, 30 minutes to 55 minutes, 30 minutes to 50 minutes , 30 to 45 minutes, 30 to 40 minutes, 30 to 35 minutes, 35 to 55 minutes, 35 to 50 minutes, 35 to 45 minutes, 35 to 40 minutes, 40 to 55 minutes, 45 It may be from 50 minutes to 50 minutes, or from 50 minutes to 55 minutes.

In the method of the invention, step (b) may take between 7 and 36 minutes per 96 samples. That is, step (b) takes 7 to 36 minutes per round of the method of the present invention. The time refers to the time required to complete steps (b-1), (b-2) and (b-3).

The total time required for step (b) is determined by the performance of the sample preparation device in the automated molecular diagnosis system, the performance of the liquid dispensing module in the sample preparation device, the number of pipetting channels included in the liquid dispensing module, and the number of pipetting channels included in the liquid dispensing module. Depending on whether or not the isothermal amplification reagent is dispensed in advance in the container, the time may vary from 7 minutes to 36 minutes.

The total time required for step (b) is 7 minutes to 36 minutes, 10 minutes to 36 minutes, 15 minutes to 36 minutes, 20 minutes to 36 minutes, 25 minutes to 36 minutes, 30 minutes to 36 minutes, and 35 minutes to 36 minutes. minutes, 7 minutes to 35 minutes, 7 minutes to 30 minutes, 7 minutes to 25 minutes, 7 minutes to 20 minutes, 7 minutes to 15 minutes, 7 minutes to 10 minutes, 10 minutes to 35 minutes, 10 minutes to 30 minutes, 10 to 25 minutes, 10 to 20 minutes, 10 to 15 minutes, 15 to 35 minutes, 15 to 30 minutes, 15 to 25 minutes, 15 to 20 minutes, 20 to 35 minutes, or 25 minutes. It can be from minutes to 30 minutes.

In the method of the present invention, step (b-1) may take 6 to 13 minutes per 96 samples. That is, step (b-1) per round of the method of the present invention takes 6 to 13 minutes. The time refers to the time required to dispense the sample into each well after directly dispensing the lysis buffer into each well of the container.

The total time required for step (b-1) is determined by the performance of the sample preparation device in the automated molecular diagnostic system, the performance of the liquid dispensing module in the sample preparation device, the number of pipetting channels included in the liquid dispensing module, and the number of pipetting channels included in the liquid dispensing module. Depending on whether or not it is pre-divided, the time may vary from 6 to 13 minutes.

The total time required for step (b-1) is 6 minutes to 13 minutes, 7 minutes to 13 minutes, 8 minutes to 13 minutes, 9 minutes to 13 minutes, 10 minutes to 13 minutes, 11 minutes to 13 minutes, and 12 minutes. to 13 minutes, 6 minutes to 12 minutes, 6 minutes to 11 minutes, 6 minutes to 10 minutes, 6 minutes to 9 minutes, 6 minutes to 8 minutes, 6 minutes to 7 minutes, 7 minutes to 12 minutes, 7 minutes to 11 minutes, 7 minutes to 10 minutes, 7 minutes to 9 minutes, 7 minutes to 8 minutes, 8 minutes to 12 minutes, 8 minutes to 11 minutes, 8 minutes to 10 minutes, 8 minutes to 9 minutes, 9 minutes to 12 minutes, It may be 9 minutes to 11 minutes, 9 minutes to 10 minutes, 10 minutes to 12 minutes, or 10 minutes to 11 minutes.

In the method of the present invention, step (b-2) may take 3 to 5 minutes per 96 samples. That is, step (b-2) per round of the method of the present invention takes 3 to 5 minutes. The time refers to the time during which the mixture of sample and direct lysis buffer is incubated.

The total time required for step (b-2) may overlap with other steps. For example, 3 to 5 minutes of step (b-2) may be performed simultaneously while mixing the lysate and the isothermal amplification reagent in step (b-3).

The total time required for step (b-2) may be 3 minutes to 5 minutes, 3 minutes to 4 minutes, or 4 minutes to 5 minutes.

In the method of the present invention, step (b-3) may take from 1 minute to 18 minutes per 96 samples. That is, step (b-3) per round of the method of the present invention takes 1 to 18 minutes. The time refers to the time required to dispense the lysate into the reaction vessel and then dispense the isothermal amplification reagent.

The total time required for step (b-3) is determined by the performance of the sample preparation device in the automated molecular diagnosis system, the performance of the liquid dispensing module in the sample preparation device, the number of pipetting channels included in the liquid dispensing module, and the isothermal amplification reagent in the container. It may vary from 1 minute to 18 minutes depending on whether it is pre-divided or not.

The total time of step (b-3) is 1 minute to 18 minutes, 1 minute to 15 minutes, 1 minute to 10 minutes, 1 minute to 5 minutes, 5 minutes to 18 minutes, 5 minutes to 15 minutes, 5 minutes. It may be from 10 minutes to 10 minutes, from 10 minutes to 18 minutes, from 10 minutes to 15 minutes, or from 15 minutes to 18 minutes.

In the method of the present invention, step (c) may take 1 to 4 minutes per 96 samples. That is, step (c) takes 1 to 4 minutes per round of the method of the present invention. The time refers to the time required to move the reaction vessel from the sample preparation device to the sample analysis device and, optionally, seal the reaction vessel using an automatic vessel sealer.

The total time required for step (c) may vary in the range of 1 to 4 minutes depending on the performance of the sample transfer device in the automated molecular diagnosis system and, optionally, the sealing speed of the automatic container sealer.

The total time required for step (c) may be 1 minute to 4 minutes, 1 minute to 3 minutes, 1 minute to 2 minutes, 2 minutes to 4 minutes, 2 minutes to 3 minutes, or 3 minutes to 4 minutes.

In the method of the present invention, step (d) may take 10 to 20 minutes per 96 samples. That is, step (d) takes 10 to 20 minutes per round of the method of the present invention. The time refers to the time required to perform isothermal amplification in the sample analysis device.

The total time required for step (d) may vary in the range of 10 to 20 minutes depending on the performance of the sample analysis device in the automated molecular diagnosis system.

The total time required for step (c) is 10 minutes to 20 minutes, 10 minutes to 17 minutes, 10 minutes to 15 minutes, 10 minutes to 13 minutes, 13 minutes to 20 minutes, 13 minutes to 17 minutes, and 15 minutes to 20 minutes. minutes, or 15 to 17 minutes.

Forms for practicing the invention

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention in more detail, and it is clear to those skilled in the art that the scope of the present invention as set forth in the appended claims is not limited by these examples. something to do.

Example

Example 1: High-throughput isothermal amplification using an automated molecular diagnostic system

According to the present disclosure, it was confirmed whether a high-throughput isothermal amplification reaction can be performed using an automated molecular diagnostic system.

For this purpose, RT-LAMP was performed using an automated molecular diagnostic system configured as follows:

(i) a sample preparation device comprising a liquid dispensing module,

(ii) reaction vessel transfer device,

(iii) automatic container sealer, and

(iv) A sample analysis device including a nucleic acid amplification device and a nucleic acid detection device.

The liquid dispensing module in the automated molecular diagnosis system includes eight pipetting channels.

The sample prepared in Example 1-1 below, the direct lysis buffer (Swab: Direct ^TM Extraction Kit, Cat. No. 9799140107), and the RT-LAMP reagent for target nucleic acid detection prepared in Example 1-2 were previously added to the sample preparation device. It was installed.

1-1. Preparation of samples

As a target nucleic acid, the gene of the bacteriophage MS2 (ATCC: 15597-B1) genome, which consists of RNA, was used, and bacteriophage MS2 prepared at a concentration of 10 ⁴ PFU/μL was transferred to the specimen transport medium (Transport media, Copan Universal Transport medium, Serially diluted 10-fold using Cat. No. 3C047N), a total of 3 concentrations (concentration 1: 4×10 ³ PFU/μL, concentration 2: 4×10 ² PFU/μL, concentration 3: 4×10 ¹ PFU). /μL) of bacteriophage MS2 samples were prepared three times each. Nine bacteriophage MS2 samples of the three concentrations prepared above and three negative control samples containing distilled water were directly loaded into the sample preparation device in the automated molecular diagnosis system along with lysis buffer and RT-LAMP reagent.

1-2. Preparation of RT-LAMP reagents

(1) Preparation of RT-LAMP reagent containing fluorescent dye

To amplify the bacteriophage MS2 target nucleic acid through RT-LAMP reaction, a total of six primer sets (F3, B3, FIP, BIP, LF and LB) were prepared as shown in Table 1 below.

Subsequently, F3 (SEQ ID NO: 1) 4 pmole, B3 (SEQ ID NO: 2) 4 pmole, FIP (SEQ ID NO: 3) 32 pmole, BIP (SEQ ID NO: 4) 32 pmole, LF (SEQ ID NO: 5) 8 pmole, LB (SEQ ID NO: 6) 8 pmole, WarmStart ^® RTx Reverse Transcriptase (NEB, Cat. No. M0380L) 4 μL, Bst 2.0 WarmStart ^® DNA polymerase (NEB, Cat. No. M0538L) 8 μL, 10 mM A final volume of 150 μL of RT-LAMP reagent (1) was prepared, including 28 μL of dNTP Mix, 12 μL of 100 mM MgSO ₄ , 15 μL of 10X Isothermal Amplification Buffer (NEB, B0537S), and 25 μL of 8X Evagreen.

(2) Preparation of RT-LAMP reagent containing assimilated probe

To amplify the bacteriophage MS2 target nucleic acid through RT-LAMP reaction, the primer sets (F3, B3, FIP, BIP, LF and LB) and assimilation probes (LB-assimilation probe and quenching oligo) shown in Table 1 are as shown in Table 2 below. Prepared as follows.

Subsequently, F3 (SEQ ID NO: 1) 4 pmole, B3 (SEQ ID NO: 2) 4 pmole, FIP (SEQ ID NO: 3) 32 pmole, BIP (SEQ ID NO: 4) 32 pmole, LF (SEQ ID NO: 5) 10 pmole, LB (SEQ ID NO: 6) 4 pmole, LB-assimilation probe (SEQ ID NO: 7) 6 pmole, quenching oligo (SEQ ID NO: 8) 8 pmole, WarmStart ^® RTx Reverse Transcriptase (NEB, Cat. No. M0380L ) Contains 4 μL, Bst 2.0 WarmStart ^® DNA polymerase (NEB, Cat. No. M0538L) 8 μL, 10 mM dNTP Mix 28 μL, 100 mM MgSO ₄ 12 μL, and 10X Isothermal Amplification Buffer (NEB, B0537S) 15 μL A final volume of 150 μL of RT-LAMP reagent (2) was prepared.

Afterwards, RT-LAMP was performed using an automated molecular diagnosis system according to the following protocol.

<Protocol>

1. In the sample preparation device,

1-1. Mixing 50 μL of the bacteriophage MS2 sample diluted in the sample transport medium at the concentration 1 to 3 above and 50 μL of the direct lysis buffer.

1-2. Incubate for 5 minutes at room temperature

1-3. Mix 15 μL of RT-LAMP reagent (1) or (2) with 5 μL of the mixture of 1-2 above in a 96-well plate reaction vessel.

2. Move the reaction vessel containing the RT-LAMP reaction mixture of 1-3 above to the automatic vessel sealer with the help of a reaction vessel transfer device.

3. Sealing the upper surface of the reaction vessel containing the mixture of 2 above using an automatic vessel sealer.

4. Move the sealed reaction vessel to the sample analysis device with the help of a reaction vessel transfer device.

5. In the sample analysis device,

5-1. The mixture was subjected to RT-LAMP reaction at 62°C for 20 minutes.

5-2. Detect amplified product every 60 seconds

As a result, as shown in Figures 13 and 14, it was confirmed that the target nucleic acid sequence could be specifically isothermally amplified and detected in 42 minutes by the high-throughput isothermal amplification method using the automated molecular diagnostic system according to the present disclosure. .

Claims (34)

High throughput isothermal amplification method using an automated molecular diagnostic system comprising the following steps:
(a) mounting a sample collected from a subject into a sample preparation device within an automated molecular diagnosis system; The automated molecular diagnosis system includes a sample preparation device, a reaction vessel transfer device, and a sample analysis device, and the sample preparation device includes a direct lysis buffer installed therein and an isothermal amplification for detecting target nucleic acid. Contains reagents;
(b) in the sample preparation device,
(b-1) mixing the sample directly with the lysis buffer;
(b-2) incubating the mixture at 15°C to 35°C for 3 to 5 minutes to obtain a lysate; and
(b-3) mixing the lysate with an isothermal amplification reagent in a reaction vessel to complete sample preparation;
The sample preparation device includes a liquid handling module for receiving the sample, direct lysis buffer, and isothermal amplification reagent into a reaction vessel, wherein the liquid handling module has 8 to 96 pipetting channels. ) and;
(c) moving the reaction vessel containing the mixture to a sample analysis device with the help of a reaction vessel transfer device;
(d) in the sample analysis device,
(d-1) reacting the mixture in the reaction vessel at a temperature selected from 50°C to 75°C for 10 to 20 minutes to amplify the target nucleic acid in the sample;
(d-2) Detecting the amplified product.
Steps (b) to (d) are automatically performed by a control module within the automated molecular diagnosis system. The method of claim 1, wherein the isothermal amplification method is LAMP (Loop-mediated isothermal amplification). The method according to claim 1, wherein the isothermal amplification reagent is a LAMP reagent. The method of claim 1, wherein the isothermal amplification reagent is RT-LMAP (Reverse-transcription Loop-mediated isothermal amplification) reagent. The method of claim 1, wherein the sample derived from the subject is a swab, saliva, or a mixture thereof. The method of claim 5, wherein the swab is nasopharyngeal swab, nostril swab, oropharyngeal swab, oral swab, or saliva swab. ), a genital swab, a rectal swab, or a mixture of two or more of these. The method of claim 1, wherein each of the sample preparation device and the sample analysis device is a stand-alone device that is individually operated. The method of claim 1, wherein at least one of the sample analysis device and/or the reaction vessel transport device is located inside a spatially enclosed enclosure. The method of claim 1, wherein the sample preparation device and the closed structure form a defined passage through which the reaction vessel is transported, and the reaction vessel transfer device transports the reaction vessel using the defined passage. How to do it. The method of claim 1, wherein the sample preparation device is located above or below the sample analysis device. The method of claim 1, wherein the reaction vessel is a 96-well plate. The method of claim 1, wherein the automated molecular diagnosis system further includes an automated container sealer for sealing the upper surface of the reaction vessel. 13. The method of claim 12, wherein the automatic vessel sealer is located in the sample preparation device. 13. The method of claim 12, wherein the automatic container sealer is located in the closure structure. The method of claim 1, wherein the target nucleic acid is derived from a virus. The method of claim 15, wherein the virus is an RNA virus. The method of claim 16, wherein the RNA virus is SARS-CoV-2. The method of claim 1, wherein the target nucleic acid is selected from the group consisting of the E gene, N gene, RdRP gene, S gene in SARS-CoV-2, and combinations thereof. The method of claim 1, wherein the isothermal amplification reagent for detecting the target nucleic acid includes an oligonucleotide containing a fluorescent molecule and an oligonucleotide containing a quencher molecule. The method of claim 1, wherein the isothermal amplification reagent additionally includes oligonucleotides for amplifying and detecting an internal control. The method of claim 20, wherein the internal control group is RNase P. The method of claim 1, wherein the isothermal amplification reagent for detecting target nucleic acids is an isothermal amplification reagent for detecting a plurality of target nucleic acids. The method of claim 22, wherein the plurality of target nucleic acids are 2 to 5 target nucleic acids. 2. The method of claim 1, further comprising the step of moving the reaction vessel to an automatic vessel sealer with the aid of a reaction vessel transfer device and sealing the upper surface of the reaction vessel prior to step (c). 25. The method of claim 24, wherein sealing the upper surface of the reaction vessel is performed by heat or adhesive. The method according to claim 1, wherein the temperature of step (d-1) is any temperature selected from 60°C to 65°C. The method according to claim 1, wherein step (d-2) is performed at regular time intervals while performing step (d-1). The method of claim 1, wherein the method takes 18 to 60 minutes per 96 samples. The method of claim 1, wherein step (b) takes 7 to 36 minutes per 96 samples. The method of claim 1, wherein step (b-1) takes 6 to 13 minutes per 96 samples. The method of claim 1, wherein step (b-2) takes 3 to 5 minutes per 96 samples. The method of claim 1, wherein step (b-3) takes 1 to 18 minutes per 96 samples. The method of claim 1, wherein step (c) takes 1 to 4 minutes per 96 samples. The method of claim 1, wherein step (d) takes 10 to 20 minutes per 96 samples.

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