KR20250090414A - Thermo-responsive multiple emulsion and method for amplifying nucleic acid using same - Google Patents

Abstract

The present disclosure relates to a W/O/W (water-oil-water) type thermoreactive emulsion composition including an inner layer including water-in-oil droplets containing PCR primers and probes; and an outer layer including a water-in-oil droplet; a nucleic acid amplification device including the same; and a nucleic acid amplification method including the same. The present disclosure delivers primers and/or probes required for nucleic acid detection or diagnosis through a thermoreactive multiple emulsion composition, thereby stably storing the primers and/or probes until a PCR reaction and releasing them when the PCR reaction starts. This prevents nonspecific reactions that may occur before a PCR reaction, allows each composition to be manufactured target-specifically, and enables multiplex diagnosis to be implemented simply by adding nucleic acids extracted from a specimen to a reaction reagent including the composition and performing PCR.

Description

{Thermo-responsive multiple emulsion and method for amplifying nucleic acid using same}

Disclosed herein are a nucleic acid amplification device and method using an emulsion.

In order to increase the reliability of diagnosis and achieve precise diagnosis in multiplex diagnosis, the development of technology that can simultaneously analyze many target nucleic acids in one channel using polymerase chain reaction (PCR) is required. However, the current technology only allows multiplex diagnosis of up to 10 genes in one reaction by changing the fluorescence wavelength or nucleic acid melting temperature. Even this is limited in use because a high level of calculation is required for primer design to reduce non-specific signals when performing multiple gene target PCR in one reaction. As a result, in the case of newly emerging infectious diseases or mutations, they are being released as independent reagents rather than being added to existing multiplex diagnosis sets. In this case, since multiple reactions must be performed from one sample for multiplex diagnosis, the cost of diagnosis is high and the diagnosis time is long, which is a disadvantage. For example, in the case of diagnostic technologies using microwells in which primers, probes, and other reagents are pre-loaded into each well, the unit cost for manufacturing the microwell array is expensive, and the process of pre-loading reagents into each well is also complicated. In addition, since the well configuration must all be changed when a new infectious disease variant or mutation occurs, it takes a long time to respond. In the case of diagnostic technologies using microchambers in which different primers react in each chamber, there is a disadvantage in that the operating cost is expensive because a microfluidic chip with complex valve control is required.

Therefore, there is a need to develop a diagnostic technology that enables multiplexed diagnosis without the design of complex primers, including multiple reactors within a single channel in a simpler form.

Republic of Korea Publication Patent No. 10-2015-0048964 (May 11, 2015)

The problem to be solved in the present disclosure is to provide a composition for nucleic acid diagnosis with improved storage stability and accuracy of PCR primers and probes, and a nucleic acid amplification device including the same.

The problem to be solved in the present disclosure is to provide a composition for nucleic acid amplification capable of multiplex diagnosis in a simpler form, and a nucleic acid amplification device including the same.

To solve the above problems, one embodiment of the present disclosure comprises an inner layer including a water-in-oil type droplet; and

Including the award-winning trauma

It is an emulsion composition of the W1/O/W2 (water1-oil-water2) type.

The above water-in-oil (W1/O) droplet contains at least one of a PCR (polymerase chain reaction) primer and probe in the inner phase, the water phase (W1), and

The above emulsion composition is heat-reactive,

A multiple emulsion composition is provided.

In addition, one embodiment of the present disclosure comprises the multi-emulsion emulsion composition; and

A reaction chamber in which the above multi-emulsion emulsion composition is arranged;

A nucleic acid amplification device including a .

In addition, one embodiment of the present disclosure is a method for amplifying a nucleic acid, comprising the steps of: injecting the multi-emulsion emulsion composition into a reaction chamber; and

A step of amplifying the target nucleic acid by performing polymerase chain reaction (PCR) on the target nucleic acid;

A method for amplifying a nucleic acid comprising:

In the case of conventional solution PCR, if several types of target nucleic acids are to be detected, multiple PCRs must be performed to detect the targets through primer and probe handling for each, which has the disadvantages of taking a long time and being complicated, but according to the present disclosure, each composition can be manufactured target-specifically, and several types of target nucleic acids can be detected by adding nucleic acids extracted from a specimen to a reaction reagent containing the composition and performing only one PCR, thereby resolving the primer and probe handling issue, thereby enabling simultaneous multiplex diagnosis. In addition, according to the present disclosure, primers and/or probes necessary for nucleic acid detection or diagnosis are delivered through a thermoreactive multiple emulsion composition, so that the primers and/or probes can be stably stored until the PCR reaction and released when the PCR reaction begins, thereby preventing nonspecific reactions that may occur before the PCR reaction. Therefore, the present disclosure can quickly respond to and diagnose a newly mutated virus or new mutation simply by adding target nucleic acids.

FIG. 1 is a schematic diagram illustrating the structure of an emulsion composition of a multiple emulsion formulation comprising water-in-oil droplets (W1/O) including water 1 droplets (W1) in oil globules (O) in a water 2 continuous phase (W2) according to one embodiment of the present disclosure.
FIG. 2a is an image showing a multi-emulsion formulation of a composition according to one embodiment of the present disclosure, taken using a transmission electron microscope (TEM).
FIG. 2b is an image showing a multi-emulsion formulation of a composition according to one embodiment of the present disclosure, taken using a transmission electron microscope (TEM).
FIG. 3 is a schematic diagram illustrating the effect of a composition according to one embodiment of the present disclosure on suppressing the generation of non-specific amplification products in PCR.
FIG. 4 is a schematic diagram showing the structure of a composition in which the trauma is a hydrogel and a method of arranging a plurality of hydrogels (A, B, C, D) in a single PCR chamber to simultaneously detect target nucleic acids (target genes) as an embodiment of the present disclosure.
FIG. 5 is an image of a multi-emulsion formulation containing water-in-oil droplets inside a hydrated gel state as an example of the present disclosure, confirmed by scanning electron microscope (SEM).
FIG. 6 is a diagram comparing the fluorescence levels of each hydrogel particle to confirm the particle uniformity of a multi-emulsion emulsion composition in the form of a hydrogel formulation as an example of the present disclosure.
FIG. 7 is a diagram showing the results of confirming the formulation stability according to the content of a lipophilic surfactant included in a composition according to one embodiment of the present disclosure.
FIG. 8 is a diagram showing the results of analyzing the particle size of water-in-oil droplets according to the content of a lipophilic surfactant included in a composition according to one embodiment of the present disclosure.
FIG. 9 is a diagram showing the results of confirming the emulsion particle size distribution and the uniformity of emulsion particles according to the surfactant concentration of a multi-emulsion emulsion composition (W1/O/W2) according to one embodiment of the present disclosure.
FIG. 10 is a diagram confirming the formulation stability according to the types of oil and surfactant included in the oil phase in a multi-emulsion emulsion composition (W1/O/W2) according to one embodiment of the present disclosure.
FIG. 11a is a diagram showing the melting point (Tm) of a water-in-oil emulsion (W1/O) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 11b is a diagram showing the melting point (Tm) of a water-in-oil emulsion (W1/O) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 11c is a diagram showing the melting point (Tm) of a water-in-oil emulsion (W1/O) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 11d is a diagram showing the melting point (Tm) of a water-in-oil emulsion (W1/O) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 11e is a diagram showing the melting point (Tm) of a water-in-oil emulsion (W1/O) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 11f is a diagram illustrating the melting point (Tm) of a water-in-oil emulsion (W1/O) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 11g is a diagram showing the melting point (Tm) of a water-in-oil emulsion (W1/O) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 12a is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 12b is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 12c is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 12d is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 12e is a diagram illustrating the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 12f is a diagram illustrating the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of lipophilic surfactant as an example of the present disclosure.
FIG. 13a is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of hydrophilic surfactant as an example of the present disclosure.
FIG. 13b is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of hydrophilic surfactant as an example of the present disclosure.
FIG. 13c is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of hydrophilic surfactant as an example of the present disclosure.
FIG. 13d is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of hydrophilic surfactant as an example of the present disclosure.
FIG. 13e is a diagram illustrating the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of hydrophilic surfactant as an example of the present disclosure.
FIG. 13f is a diagram illustrating the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of hydrophilic surfactant as an example of the present disclosure.
FIG. 13g is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of hydrophilic surfactant as an example of the present disclosure.
FIG. 13h is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of hydrophilic surfactant as an example of the present disclosure.
FIG. 13i is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of hydrophilic surfactant as an example of the present disclosure.
FIG. 13j is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of hydrophilic surfactant as an example of the present disclosure.
FIG. 13k is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of hydrophilic surfactant as an example of the present disclosure.
FIG. 13l is a diagram showing the melting point (Tm) of an oil-in-water emulsion (O/W2) according to the type of hydrophilic surfactant as an example of the present disclosure.
FIG. 14a is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when Tween 20 (0.25 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
FIG. 14b is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when Tween 20 (0.25 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
FIG. 14c is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when Tween 20 (0.25 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
FIG. 14d is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when Tween 20 (0.25 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
FIG. 14e is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when Tween 20 (0.25 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
FIG. 14f is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when Tween 20 (0.25 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
FIG. 15a is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when Tween 85 (0.09 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
FIG. 15b is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when Tween 85 (0.09 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
FIG. 15c is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when Tween 85 (0.09 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
FIG. 16a is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when TritonX-100 (0.1275 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
FIG. 16b is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when TritonX-100 (0.1275 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
FIG. 16c is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when TritonX-100 (0.1275 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
FIG. 17 is a diagram showing the thermal reactivity of a multiple emulsion composition according to the type of lipophilic surfactant when Tween80 (0.25 w/w%) is used as a hydrophilic surfactant as an example of the present disclosure.
Figure 18 is a diagram showing the results (right) of confirming the effect of reducing non-specific reactions when a liquid multi-emulsion emulsion composition of one embodiment of the present disclosure is supplied to a liquid phase and liquid RT-qPCR is performed, compared to the control group (left).
Figure 19 is a diagram showing the results of confirming the RT-qPCR efficiency of Comparative Example 1 according to the prior art.
Figure 20 is a diagram showing the results of confirming the RT-qPCR efficiency of Example 3 according to one embodiment of the present disclosure.
Figure 21 is a diagram showing the results of confirming the RT-qPCR efficiency of Example 4 according to one embodiment of the present disclosure.
FIG. 22 is a diagram showing the results of confirming the qPCR efficiency of Example 2, which is a multi-emulsion composition in the form of a hydrating gel, as an example of the present disclosure.
FIG. 23 is a diagram showing the results of confirming the qPCR efficiency of Example 2, which is a multi-emulsion composition in the form of a hydrating gel formulation, as an example of the present disclosure.
FIG. 24 is a diagram showing the results of confirming the RT-qPCR efficiency of Example 2, which is a multi-emulsion composition in the form of a hydrating gel, as an example of the present disclosure.
Figure 25 is a diagram showing the RT-qPCR results of the control group NTC (No Template Control) in Test Example 9.
FIG. 26 is a diagram showing the results of performing multiple diagnosis according to one embodiment of the present disclosure.
FIG. 27 is a diagram showing the results of performing multiple diagnosis according to one embodiment of the present disclosure.

Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the attached drawings.

The embodiments of the present disclosure disclosed in the text are merely illustrative for the purpose of explanation, and the embodiments of the present disclosure may be implemented in various forms and should not be construed as limited to the embodiments described in the text. The present disclosure may have various modifications and may have various forms, and the embodiments are not intended to limit the present disclosure to a specific disclosure form, but should be understood to include all modifications, equivalents, or substitutes included in the spirit and technical scope of the present disclosure.

Singular expressions include plural expressions unless the context clearly indicates otherwise. In this application, the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, part or combination thereof described in the specification, but do not exclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts or combinations thereof.

One embodiment of the present disclosure provides an emulsion composition having a water1-oil-water2 (W1/O/W2) type, which includes an inner phase comprising water-in-oil type droplets; and an outer phase comprising an aqueous phase. In this case, the water-in-oil (W1/O) type droplets include at least one of a PCR (polymerase chain reaction) primer and probe in an inner phase, an aqueous phase (W1), and the emulsion composition may be thermoreactive.

In the present disclosure, 'thermal reactivity' means a characteristic in which, as the temperature rises, the instability of a surfactant increases, so that the oil phases are separated, the innermost phase (W1) of the composition is released outside the oil phase (O), and the water phase (W1) and the outer phase (W2) are united so that the PCR primers and/or probes included in the water phase (W1) are released outside the water-in-oil (W1/O) droplets. The critical temperature at which such thermal reactivity appears, i.e., the release temperature of the included PCR primers and/or probes, can be controlled according to the type and concentration of the surfactant included in the composition and/or the type of oil. For example, the critical temperature can be higher than 55°C, higher than 56°C, higher than 57°C, higher than 58°C, higher than 59°C, or higher than 60°C.

The attached Fig. 1 illustrates an exemplary form of the present disclosure. Referring to Fig. 1, a composition (Emulsion) according to the present disclosure may be an emulsion composition of a multiple emulsion formulation including water-in-oil droplets (W1/O) including water 1 droplets (W1) in oil globules (O) in an aqueous phase (Water 2 continuous phase, W2). In one embodiment, the water-in-oil droplets (W1/O) included in the composition may be one or multiple, as exemplarily illustrated in Fig. 1. Specifically, the number of the droplets may be 10 ¹¹ /μl or less, 10 ¹⁰ /μl or less, 10 ⁹ /μl or less, 10 ⁸ /μl or less, 10 ⁷ /μl or less, 10 ⁶ /μl or less, 10 ⁵ /μl or less, 10 ⁴ /μl or less, 10 ³ /μl or less, 10 ² /μl or less, or 10 ¹¹ /μl or less, but is not limited thereto. In one embodiment, the number of water phases (W1) included in one of the water-in-oil type droplets (W1/O) may be 1, or may be 2 or more as exemplarily illustrated in FIG. 1. Specifically, the number of the water phases (W1) may be 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, and may be 50 or less, 40 or less, 30 or less, 20 or less, or 10 or less. If the number of the water phases (W1) included in the composition is too small, the amount of the reagent participating in the reaction may be too low, so that the reaction may not occur well, and the amount of the oil phase may be relatively too large, so that a decrease in the fluorescence signal according to the reaction may occur. If the number of the water phases (W1) is too large, the reaction efficiency may be reduced by changing the composition of the reaction buffer during the process in which the water phase (W1) is released out of the oil phase (O), and the amount of the oil phase may be relatively too small, so that the stability of the formulation may be reduced.

FIG. 2a and FIG. 2b are images of a multi-emulsion formulation of a composition according to one embodiment of the present disclosure, which are confirmed by a transmission electron microscope (TEM), and it can be confirmed that the innermost phase, the water phase (W1), and the outer phase, the water phase (W2), are separated by an oil film of the oil phase (O) and are stably dispersed in each other.

In addition, as exemplarily illustrated in FIG. 1, in one embodiment, the wound (W2) may include components necessary for a PCR reaction other than the PCR primer and/or probe included in the innermost wound (W1). For example, as illustrated in FIG. 1, the wound may include a PCR primer, an RT primer used for reverse transcription reaction converting RNA into DNA, a probe, etc. Alternatively, the wound (W2) may include a PCR reagent mixture including components necessary for the PCR reaction described above.

FIG. 3 is a diagram illustrating an action of a composition according to an embodiment of the present disclosure in PCR. Specifically, according to the present disclosure, the water-in-oil type droplets (W1/O) (emulsion in FIG. 3) of the composition have thermoreactivity, so that in the reverse transcription (RT) step performed at about 55°C or lower, the water phase of the water-in-oil type droplets can be contained in an oil film to maintain the droplet shape, and in the qPCR step performed at a temperature of about 95°C, as the instability of the droplets increases, phase separation between the oil phase (O) and the water phase (W1) occurs, and as the water phase (W1) and the external water phase (W2) unite, the PCR primers contained in the droplets can be released out of the W1/O droplets. That is, since the composition according to one embodiment of the present disclosure safely stores the PCR primer inside the droplet and releases the PCR primer at a stage where the PCR primer is needed, not only does a non-specific reaction by the PCR primer not occur in the reverse transcription stage, but also the generation of non-specific amplification products in the PCR stage can be suppressed, so that the RNA detection limit (LoD) is improved, thereby enabling quantitative analysis of even a small amount of RNA. In addition, as an example, when a probe is included in the water-in-oil type droplet (W1/O), the probe can be stored in the reverse transcription stage and damage to the probe can be prevented, thereby maintaining its function in the subsequent PCR process, thereby solving the problem that, when a probe is included in a solid phase particle having conventional thermosensitivity, the reporter and target are damaged by the radical chain reaction, making it impossible to apply the cross-linking reaction. When a probe is included, as an example, there is an advantage in that damage caused by the radical chain reaction that occurs when curing as a hydrogel formulation can be prevented, thereby maintaining the function in the subsequent PCR process.

As an example, the PCR primer and the RT primer may each be one or more nucleic acids selected from DNA, RNA, LNA, and PNA, but are not limited thereto. In this case, the PCR primer and the RT primer may each be 10 to 100 bases, more specifically 20 to 50 bases, but the sequence type and sequence length of each primer may be modified without limitation depending on the target nucleic acid.

In one embodiment, the probe may be a selective fluorescent probe. The fluorescent probe binds to a target nucleic acid and provides a fluorescent signal, thereby enabling the detection of the target nucleic acid in real time. In one embodiment, since the target nucleic acid is amplified by a PCR reaction and the fluorescent intensity also increases, the amplified target nucleic acid can be quantified by detecting the fluorescent intensity. The fluorescent probe may be used without limitation in type as long as it complementarily binds to the target nucleic acid and exhibits fluorescent properties. For example, the selective fluorescent probe may include a TaqMan probe, etc. As an example, the TaqMan probe is a nucleic acid modified with a fluorescent substance (such as FAM) at the 5' end and a quencher substance (such as BHQ) at the 3' end. It specifically hybridizes and binds to template DNA in the annealing step, but fluorescence generation is suppressed by the quencher on the probe, and during the extension reaction, the Taq DNA polymerase has 5'→ exonuclease activity decomposes the TaqMan probe that hybridizes and binds to the template, releasing the fluorescent dye from the probe, thereby releasing the inhibition by the quencher and exhibiting fluorescence. As an example, the probe may include a method in which the quencher is not a universal quencher in a FRET pair (fluorescence resonance energy transfer pair) but shifts the emission wavelength so that fluorescence detection is not performed. An example of this is the FAM-TAMRA pair. In one embodiment, the water phase (W1) of the water-in-oil type droplet (W1/O) can include a material having an upper critical solution temperature (UCST) of 20 to 90°C, and various materials can be selected depending on room temperature stability and PCR reactivity. Specifically, the water phase (W1) can include at least one selected from the group consisting of agarose, gelatin, collagen, low melting point agarose (LMPA), and PEG-aCD (a mixture of polyethylene glycol and alpha-cyclodextrin). More specifically, the water phase (W1) can include, but is not limited to, agarose having a critical melting temperature of 80 to 90°C, gelatin having a critical melting temperature of 40 to 50°C, collagen having a critical melting temperature of 30 to 40°C, LMPA having a critical melting temperature of 60 to 80°C, PEG-aCD having a critical melting temperature of 20 to 90°C.

In one embodiment, the type of oil included in the oil phase (O) of the water-in-oil type droplet (W1/O) is not limited, but specifically, at least one selected from the group consisting of hydrofluoroether (HFE) oil, mineral oil, silicone oil, and isopropyl palmitate oil may be included. For example, the silicone oil may be at least one selected from the group consisting of dimethicone, cyclomethicone, polydimethylsiloxane, methylphenylpolysiloxane, methylcyclopolysiloxane, octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, dodecamethylcyclohexasiloxane, tetradecamethylhexasiloxane, and octamethyltrisiloxane.

In one embodiment, the average diameter of the water-in-oil type droplets (W1/O) can be from 100 nm to 10 μm. Specifically, the average diameter can be 100 nm or more, 150 nm or more, 200 nm or more, 250 nm or more, 300 nm or more, 450 nm or more, 500 nm or more, and 10 μm or less, 1 μm or less, 950 nm or less, 900 nm or less, 850 nm or less, 800 nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, or 200 nm or less. Here, the average diameter may be calculated as the average of the maximum diameters of each droplet particle among water-in-oil droplets comprising 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the water-in-oil droplets contained in the composition. If the average diameter of the water-in-oil droplets (W1/O) is too small, the trapping of the droplets in the formulation may not be performed well when manufactured as a hydrogel formulation, and if it is too large, it may cause instability of the hydrogel structure.

In one embodiment, the oil phase (O) of the water-in-oil type droplet (W1/O) may include a lipophilic surfactant, and the water phase (W2) of the outer layer may include a hydrophilic surfactant, and the release temperature and release efficiency of PCR primers and/or probes included in the water-in-oil type droplet may be controlled depending on the type and concentration of these surfactants.

Specifically, the lipophilic surfactant may include a fluoro-based surfactant, and more specifically, may include a fluoro-based anionic surfactant or a fluoro-based nonionic surfactant. Examples of the fluoro-based anionic surfactant include, but are not limited to, poly(hexafluoropropylene oxide) (poly(hexafluoropropylene oxide)), perfluorodecanoic acid (PFDA), perfluorododecanoic acid (PFDoA), and the like. More specifically, the polyhexafluoropropylene oxide may be Krytox 157FS-L, Krytox 157FS-M, and Krytox 157FS-H. Examples of the fluoro-based nonionic surfactant include, but are not limited to, Ran surfactant, Picosurf, and Fluosurf. In one embodiment, the water-in-oil type droplets may include hydrofluoroether oil in the oil phase and a fluorosurfactant as the lipophilic surfactant.

Alternatively, in one embodiment, the lipophilic surfactant can include a silicone-based nonionic surfactant. The silicone-based nonionic surfactant can include, but is not limited to, Abil EM90, for example. In one embodiment, the water-in-oil droplet can include at least one of mineral oil, silicone oil, and isopropyl palmitate oil in the oil phase, and the lipophilic surfactant can include a silicone-based nonionic surfactant.

Specifically, the hydrophilic surfactant may be a surfactant having an HLB (Hydrophile-Lipophile Balance) of 8 to 20. More specifically, the hydrophilic surfactant may be a polyoxyethylene sorbitan-based surfactant, and may be, for example, at least one of Tween 20, Tween 80, Tween 85, Triton X 100, Brij S10, PEG 400 monolaurate, and Pluronic F127.

As an example, the aqueous phase (W2) of the trauma may be a water or water-based salt solution containing the hydrophilic surfactant. Specifically, the aqueous phase (W2) may contain phosphate-buffered saline (PBS).

In another embodiment, the outer surface (W2) may be a hydrogel. More specifically, the hydrogel may be a porous structure including pores, and the water-in-oil type droplets may be positioned within the pores of the hydrogel. In one embodiment, the porosity of the porous structure may be 10% to 80% by volume, more specifically 20% to 70% by volume, based on the total volume of the hydrogel particles. If it is out of the above range, the porosity may be reduced or the structural stability of the hydrogel particles may be unstable, which may be detrimental to the PCR reaction. FIG. 4 is a diagram schematically showing the structure of a composition in which the outer surface is a hydrogel, and FIG. 5 is a diagram showing an image confirmed by a scanning electron microscope (SEM) of a composition in which the outer surface is a hydrogel according to one embodiment. In one embodiment, when the outer surface is in the form of a hydrogel as described above, the water-in-oil type droplets (W1/O) may be physically fixed inside the gel.

As an example, when the trauma (W2) is a hydrated gel as described above, the average diameter of the W1/O/W2 particles may be 10 μm to 10 mm. Specifically, the average diameter may be 10 μm or more, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, 500 μm or more, 600 μm or more, 700 μm or more, 800 μm or more, 900 μm or more, 1000 μm or more, 2000 μm or more, 3000 μm or more, 4000 μm or more, 5000 μm or more, 6000 μm or more, 7000 μm or more, 8000 μm or more or 9000 μm or more, and 10000 μm or less, 9000 μm or less, 8000 μm or less, 7000 μm or less, 6000 μm or less, 5000 μm or less, 4000 μm or less, 3000 μm or less, It may be 2000 μm or less, 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, or 100 μm or less. Here, the average diameter may be the average value of the maximum diameter value of each particle calculated from 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the total W1/O/W2 particles.

As an example, the material of the hydrogel may be any polymer (pre-polymer) capable of solidification, and specifically may include at least one hydrophilic polymer selected from the group consisting of polyethylene glycol-diacrylate (PEG-DA), polyacrylamide (PAAm), agarose, gelatin, and collagen.

As an example, the above-described trauma may further include at least one of an encoder that provides target nucleic acid primer information and a fluorescent marker that provides quantitative information on the amplified nucleic acid. The encoder refers to a material that distinguishes what the PCR primer is in each porous structure by color or shape, and for example, a dye that fluoresces in various colors, a quantum dot, or a metal, plastic, glass, silicon, etc. having a specific shape can be used. Alternatively, when the trauma is a hydrogel, the PCR primer in each hydrogel particle can be distinguished by changing the size or shape of the hydrogel particle itself or by forming a specific mark on the surface of the hydrogel particle without using an encoder. Alternatively, the PCR primer in each hydrogel particle can be distinguished by specifying the position of each hydrogel particle within the array.

In the case of conventional solution PCR, if several types of target nucleic acids are to be detected, multiple PCRs must be performed to detect the targets through primer and probe handling for each, which has the disadvantage of being time-consuming and complicated. However, according to the present disclosure, since several types of target nucleic acids can be detected through a single PCR, the primer and probe handling issue is resolved, enabling simultaneous multiplex diagnosis.

One embodiment of the present disclosure can provide a nucleic acid amplification device including the multi-emulsion emulsion composition; and a reaction chamber in which the multi-emulsion emulsion composition is arranged. In one embodiment, the nucleic acid amplification device can include a plurality of multi-emulsion emulsion compositions, each of which includes a primer for a different target nucleic acid.

As an example, the device may further include a reaction chamber, and the reaction chamber may include an array or tube in which the composition is arranged. The material of the array according to an example is not limited to a type of material, such as glass, plastic, polymer, or silicon, as long as it can apply the temperature conditions of the nucleic acid amplification reaction.

In addition, one embodiment of the present disclosure can provide a nucleic acid amplification method including a step of injecting one or more of the above multi-emulsion emulsion compositions into a reaction chamber; and a step of amplifying the target nucleic acid by polymerase chain reaction (PCR).

As one example, the step of amplifying the target nucleic acid may include destabilizing a surfactant forming the interface of a multiple emulsion (W1/O/W2) composition during a denaturation step in PCR, thereby releasing the inner phase (W1) into the outer phase (W2), thereby releasing the PCR primer contained therein out of the droplet.

As one embodiment, the one or more multi-emulsion emulsion compositions may each contain PCR primers for different target nucleic acids.

As one embodiment, the method may further comprise a step of injecting a solution containing one or more target nucleic acids into the chamber prior to the PCR step of the multi-emulsion emulsion composition. In one embodiment, the solution containing the target nucleic acids may further comprise a primer containing a locked nucleic acid (LNA), such as a 3'-locked nucleic acid primer, Taq polymerase, and the like.

As an example, the reaction chamber may include an array or tube in which the composition is arranged.

As one embodiment, the method may further include a step of analyzing a nucleic acid polymerized within the multi-emulsion emulsion composition. As another embodiment, the method may further include a step of quantitatively analyzing in real time a nucleic acid polymerized within the one or more compositions polymerized simultaneously with the step of performing the polymerase chain reaction, so that different types of target nucleic acids can be simultaneously amplified and detected and quantitatively analyzed in real time.

Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are intended only to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

[Example 1] Preparation of a liquid multi-emulsion emulsion composition

First, to prepare the water-in-oil type droplet phase, 4 μl of 1 mM PCR primer (Forward primer) was added to a 1.7 ml tube, then 16 μl of 2.5% LMPA (low melting point agarose) (Manufacturer: Promega, Product name: Agarose, LMP, Preparative Grade for Small Fragments (10 to 1,000 bp)) (at least 400 μl in volume) heat-treated at 100°C for 10 minutes was added, mixed, and centrifuged until the speed reached approximately 400 rpm. Next, 200 μl of a solution in which a fluoro-based nonionic surfactant (product name: 008-FluoroSurfactant, manufacturer: RAN Biotechnologies) as a lipophilic surfactant was melted into hydrofluoroether oil (product name: HFE7500, manufacturer: Kemis Co., Ltd.) as an oil phase to a concentration of 0.4% (w/w) and heat-treated at a temperature of 100°C was added to the tube, and then emulsified through ultrasonic treatment to produce a water-in-oil type emulsion composition (W1/O).

At this time, the sequence of the PCR primer is as follows.

-PCR primer (Forward primer): 5'-GTTCTTACCTTTCTTTTCCAATGTTAC-3' (SEQ ID NO: 1)

The above-mentioned manufactured water-in-oil emulsion composition (W1/O) was placed on ice and stabilized for 10 minutes, then centrifuged at 13,000 rpm for 40 seconds to separate the water-in-oil emulsion composition (W1/O) into an upper layer and a lower layer containing hydrofluoroether oil, thereby completely removing the hydrofluoroether oil in the lower layer.

As a contrast agent, 1 mL of a solution (PBST 0.25% (w/w)) containing Tween20 (product name: Tween®20, manufacturer: Sigma-Aldrich) as a hydrophilic surfactant dissolved in PBS was added to the tube, and the W1/O/W2 emulsion composition was well dispersed using a pipette. Then, the dispersed W1/O/W2 emulsion composition was sonicated to stabilize the particle size of the W1/O/W2 emulsion composition.

[Example 2] Preparation of a multi-emulsion emulsion composition in the form of a hydrogel formulation

As one embodiment of the present disclosure, a water-in-oil type emulsion composition (W1/O/W2) 35% (v/v) prepared in the same manner as Example 1 except that both the PCR primer (Forward primer) and the probe were added to the composition, and 20% (v/v) of poly(ethylene glycol) diacrylate (PEG-DA, Sigma-Aldrich, MW700) as a hydrating gel monomer added to the external surface, 40% (v/v) of poly(ethylene glycol) (PEG, Sigma-Aldrich, MW600) as a pore-inducing polymer, 5% (v/v) of 20% diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (product name: Water-soluble TPO based nanoparticle photoinitiator, manufacturer: Sigma-Aldrich), and 5% (v/v) of Acrydite Reverse primer were mixed to obtain a total A 100 μL prepolymer hydrogel solution was prepared. At this time, unlike the PCR primer (forward primer) and probe included in the water-in-oil type emulsion composition, the reverse primer is included in the form of an acrydite reverse primer in which an acrydite functional group is attached as a linker to the reverse primer, and is directly fixed to the hydrogel through the linker.

The sequences of each primer and probe used in the above manufacturing are as follows.

-PCR primer (Forward primer): 5'-GTTCTTACCTTTCTTTTCCAATGTTAC-3' (SEQ ID NO: 1)

-RT Primer (Reverse primer): 5'- CCATCATTAAATGGTAGGACAGGG-3' (SEQ ID NO: 2)

-Probe: 5'-FAM/TGGTTCCATGCTATCTCTGGGACC/BHQ1-3' (SEQ ID NO: 3; Product name: DNA oligo, Manufacturer: IDT)

The solution prepared above was prepared in the form of droplets using a plasma-treated PDMS mold, and cured by exposing it to UV (2.5 mW) for 30 seconds.

Figure 6 shows the comparison of the fluorescence levels of the hydrogel particles of the multiple emulsion emulsion composition in order to confirm the particle uniformity of the manufactured hydrogel formulation. The fluorescence values of eight particles among the particles shown in Figure 6 were measured, and the average brightness was 7365, the standard deviation was 596, and the deviation between particles was uniformly less than 10%.

[Example 1]

As an example of the present disclosure, the following experiment was performed to confirm the formulation stability according to the size of water-in-oil droplets (W1/O) and the content of lipophilic surfactant.

First, a water-in-oil emulsion composition (W1/O) was prepared by emulsifying in the same manner as in Example 1, except that Picosurf® (product name: Pico-Surf®, manufacturer: Sphere Fluidics) was used as a lipophilic surfactant. At this time, the content of the lipophilic surfactant was adjusted to 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 wt% based on the total weight of the water-in-oil emulsion composition (W1/O), and the formulation stability according to the content of the lipophilic surfactant was visually confirmed immediately after preparation. In addition, the size of the water-in-oil droplets (W1/O) according to each content was confirmed using a particle size analyzer (Dynamic light scattering system, DLS; product name: Zetasizer Nano ZSP, manufacturer: Malvern Instruments).

As a result, when the content of the lipophilic surfactant was 0.2% (w/w) or less, a creaming phenomenon occurred and the two-phase formulation of water-in-oil was not maintained, and when it was 0.3% (w/w) or more, it was confirmed that a stable formulation of water-in-oil droplets (W1/O) was maintained (see Fig. 7). In addition, at this time, the average diameter of the water-in-oil droplets (W1/O) was 252 ± 8 nm (see Fig. 8).

Next, in order to confirm the formulation stability according to the particle size of the multiple emulsion composition (W1/O/W2) and the content of hydrophilic surfactant, the following experiment was performed.

First, a multiple emulsion emulsion composition (W1/O/W2) was prepared in the same manner as in Example 1, but the content of the hydrophilic surfactant was adjusted to 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 wt% based on the total weight of the multiple emulsion emulsion composition. Then, the size of the W1/O/W2 particles according to the content of the hydrophilic surfactant was confirmed using a particle size analyzer (Dynamic light scattering system, DLS; Manufacturer: Malvern Instruments, Product name: Zetasizer Nano ZSP).

As a result, as shown in Fig. 9, the particle size of the multi-emulsion emulsion composition (W1/O/W2) was found to be approximately 200 to 400 nm, and it was confirmed that the content of the hydrophilic surfactant should be 0.05% (w/w) or more to maintain a stable emulsion formulation.

[Example 2]

As one example of the present disclosure, the formulation stability was compared according to the type of oil and the type of surfactant included in the water-in-oil type droplet.

Specifically, a multiple emulsion emulsion composition (W1/O/W2) was prepared in the same manner as in Example 1, but the types of oil and surfactant included in the oil phase were different. A total of four types of oils, two types of lipophilic surfactants and two types of hydrophilic surfactants were used, and whether they were mixed was visually confirmed, which is shown in Fig. 10 and Table 1 below. At this time, the oil was hydrofluoroether (HFE) oil (3M, HFE-7500), mineral oil (Sigma-aldrich, mineral oil), silicone oil (Sigma-aldrich, silicone oil AR20), isopropyl palmitate (IPP) oil (Sigma-aldrich, Isopropyl palmitate), the lipophilic surfactant was fluorosurfactant (RAN Biotechnologies, 008-FluoroSurfactant-2wtH-50g) 0.4% (w/w), cetyl PEG/PPG-10/1 dimethicone (Abil EM90; KCC, SeraSol SC 83-A) 3% (w/w), and the hydrophilic surfactant was Triton-X (Sigma-aldrich, Triton X-100) 0.1275% (w/w), Tween20 (Sigma-aldrich, Tween 20) It was 0.25%(w/w).

lipophilic surfactant Hydrophilic surfactant 0.4% fluorosurfactant Abil EM90 3% Triton-X 0.1275% Tween20 0.25% HFE Oil mix separation separation separation Mineral oil separation mix separation separation Silicone oil separation mix separation separation IPP Oil separation mix separation separation

As a result, it was confirmed that all four types of oils did not mix with the hydrophilic surfactants Triton-X and tween20, while HFE oil mixed well with the fluorosurfactant, and mineral oil, silicone oil, and IPP oil mixed well with the silicone nonionic surfactant Abil EM90.

[Example 3]

In order to determine the melting point (Tm) of the type of lipophilic surfactant included in one embodiment of the present disclosure, that is, the temperature at which a lipophilic surfactant maintaining a W1/O interface becomes unstable depending on temperature correspondence and a morphological change occurs in a water-in-oil emulsion (W1/O), the following experiment was performed.

First, a water-in-oil emulsion (W1/O) was prepared, and the Tm of the water-in-oil emulsion (W1/O) was confirmed. At this time, the water phase was fixed as PBS and the oil phase was fixed as HFE, and the surfactants in Table 2 below were used as lipophilic surfactants, and their properties are shown in the table.

Poly(hexafluoropropylene oxide) PFDA
(Perfluorodecanoic acid) PFDoA
(Perfluorododecanoic acid) Ran Picosurf Fluosurf Krytox 157FS-L Krytox 157FS-M Krytox 157FS-H Ionic Anionic Nonionic Mw 2500 5000 7000-7500 514.08 614.1 1000~20000 Da 414 7000-13000 Da viscosity 99.4-149 - 703-1055 - - - 1.24 (Absolute) - density 1.91 - 1.88 1.8 1.8 1.8 1.5 - Boiling point ( ^o C) 170-200 - 170-200 218 249 - >100 -

A 1.7 mL tube was filled with 20 μL of PBS containing 200 μM FAM-DNA (5'-GGTCGAGGGTGGCTACGGCTGAACT[FAM]-3'; SEQ ID NO: 4) and 200 μL of HFE oil containing a lipophilic surfactant, and then a water-in-oil emulsion (W1/O) was prepared by tip sonication (Amplitude 40%, 45 sec). 16 μL of the water-in-oil emulsion (W1/O) solution was injected into a microfluidic chip, and the chip was placed on a hot plate. The temperature was raised from 30 °C to 120 °C at a rate of 0.2 °C/sec, and fluorescence images of the water-in-oil emulsion (W1/O) were obtained once every 5 s. After plotting the fluorescence value graph of the water-in-oil emulsion (W1/O) according to temperature, the melting point (T _m ) value was analyzed based on the inflection point.

As a result, as shown in Figs. 11a to 11g, it was confirmed that both the water-in-oil emulsion (W1/O) had a Tm value at a high temperature of 90°C or higher when Krytox, an anionic surfactant, and Ran, a nonionic surfactant, were used. The Tm value of Krytox decreased as the molecular weight increased. Regardless of the amount used (volume percentage), the water-in-oil emulsion (W1/O) showed a Tm value of 94±1°C.

Next, an oil-in-water emulsion (O/W2) was prepared in the same manner as above using 0.25% (w/w) of the hydrophilic surfactant Tween20 included in the outer layer (W2) of the multiple emulsion (W1/O/W2), and the Tm according to the lipophilic surfactant in the oil-in-water emulsion (O/W2) was confirmed again. In actual PCR, no primers or probes are added to the W2 phase, but in this experiment, 200 μM FAM-DNA (5'-GGTCGAGGGTGGCTACGGCTGAACT[FAM]-3'; SEQ ID NO: 4) was added to the W2 phase together with the hydrophilic surfactant in order to observe the state change of the emulsion through the fluorescence change.

At this time, the traumatic injury (W2) was fixed with PBS containing 0.25% (w/w) of Tween 20, and the internal oil phase was each made of HFE and the surfactants in Table 2 were used as lipophilic surfactants.

As a result, as shown in Figs. 12a to 12f, it was confirmed that both the oil-in-water emulsion (O/W2) and the nonionic surfactant had a Tm value at a high temperature of 90°C or higher when anionic surfactants and nonionic surfactants were used. Among these, the Tm value of Krytox decreased as the molecular weight increased.

[Example 4]

In order to determine the melting point (Tm), that is, the temperature at which a morphological change occurs in an oil-in-water emulsion (O/W2), depending on the type of hydrophilic surfactant included in one embodiment of the present disclosure, the following experiment was performed.

First, an oil-in-water emulsion (O/W2) was prepared, and the Tm in the oil-in-water emulsion (O/W2) was confirmed. At this time, the water phase was fixed as PBS and the oil phase was fixed as HFE, and the surfactants in Table 3 below were used as hydrophilic surfactants, and their properties are shown in the table.

Tween 20 Tween 80 Tween85 Triton X 100 Span 20 Span 80 Brij S10 PEG 400
Monolaurate Pluronic F127 SDS
(Sodium dodecyl surfactant) Ionic Nonionic Nonionic Nonionic Nonionic Nonionic Nonionic Nonionic Nonionic Nonionic Nonionic Mw(g/mol) 604.8 833.9 1839 625 346.47 428 711 400 12600 288.38 CMC 0.06 mM 0.012 mM 0.06 mM 0.24 mM (0.04 mM) 0.43 mM 90 uM (0.1 mM) 129.97 μg/mL 8.2 mM Boiling point (℃) 695 695.8 100 270 516.1 579.3 - > 260 > 149 204-207 HLB 17.0 15.0 11.0 13.5 8.6 4.3 12 13 18-23 40.0

200 μL of PBS containing 200 μM FAM-DNA (5'-GGTCGAGGGTGGCTACGGCTGAACT[FAM]-3'; SEQ ID NO: 4) and a hydrophilic surfactant, and 200 μL of HFE oil were added to a 1.7 mL tube, and then an oil-in-water emulsion (O/W2) was prepared through tip sonication (Amplitude 40%, 45 sec). At this time, the molar concentrations of other hydrophilic surfactants except Tween 20 were adjusted to be the same as Tween 20 0.25%. (2.2 mM)

16 μL of the oil-in-water emulsion (O/W2) solution was injected into a microfluidic chip, the chip was placed on a hot plate, and the temperature was raised from 30°C to 120°C at a rate of 0.2°C/sec, and a fluorescence image of the oil-in-water emulsion (O/W2) was obtained every 5 seconds. A graph of the fluorescence value of the oil-in-water emulsion (O/W2) according to temperature was drawn, and the melting point (T _m ) value was analyzed based on the inflection point.

As a result, as shown in Figs. 13a to 13l, among the hydrophilic surfactants used, tween20 showed the highest Tm value, and it was confirmed that the Tm value decreased as the amount of tween20 decreased. Span20 showed poor formulation stability because the emulsion was largely clumped together even at room temperature.

[Example 5]

Based on the results of Test Examples 3 and 4 included in one embodiment of the present disclosure, the following experiment was performed to compare the DNA release efficiency according to temperature of a multiple emulsion composition (W1/O/W2) depending on the type of hydrophilic surfactant and lipophilic surfactant.

Preparation of W1/O/W2 multi-emulsion emulsion composition

A 1.7 mL tube was filled with 20 μL of PBS containing 200 μM FAM-DNA (5'-GGTCGAGGGTGGCTACGGCTGAACT[FAM]-3'; SEQ ID NO: 4) and 200 μL of HFE oil containing a lipophilic surfactant, and a water-in-oil emulsion (W1/O) was prepared by tip sonication (Amplitude 40%, 45 sec).

After stabilizing the above-mentioned water-in-oil emulsion (W1/O) on ice for 10 minutes, the water-in-oil emulsion (W1/O) (upper layer) and HFE oil (lower layer) were separated by centrifugation (13,000 rpm, 40 sec), and the HFE oil in the lower layer was removed using a pipette. After injecting 1 mL of PBS containing a hydrophilic surfactant (0.25%), the W1/O/W2 multiple emulsion emulsion composition was dispersed using a pipette.

The above dispersed W1/O/W2 multiple emulsion composition solution was tip sonicated (Amplitude 40%, 8 sec) to prepare a second W1/O/W2 multiple emulsion emulsion composition, which was then centrifuged again (10,000 rpm, 10 sec) to remove the PBS in the upper layer.

Standard Curve for DNA Quantification

FAM-DNA solutions at various concentrations (2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.1 μM) were prepared, 80 μL of each FAM-DNA solution was loaded onto a 96-well black plate, and the fluorescence amount was measured using a fluorescence spectrometer. A standard curve was obtained based on the measured fluorescence amounts at each FAM-DNA concentration.

Determination of DNA concentration in W1/O/W2 multiple emulsion compositions

After diluting the W1/O/W2 multiple emulsion composition prepared above 10-fold, 80 μL was added to a 96-well black plate, and the fluorescence amount was measured using a fluorescence spectrometer (product name: FLUOstar Omega, manufacturer: BMG LABTECH). By substituting the fluorescence value of the W1/O/W2 multiple emulsion emulsion composition into the above standard curve, the encapsulated DNA concentration was obtained.

Confirmation of DNA release efficiency according to temperature change of W1/O/W2 multi-emulsion emulsion composition

20 μL of the W1/O/W2 multiple emulsion composition prepared above was heated in a heating block for 10 minutes. At this time, each experiment was conducted under temperature conditions of 10°C intervals from 30°C to 120°C.

The heated W1/O/W2 multiple emulsion composition was centrifuged (10,000 rpm, 30 sec) to separate the supernatant, diluted 10-fold, and 80 μL of the diluted supernatant was loaded onto a 96-well black plate. The fluorescence amount was measured using a fluorescence spectrometer (product name: FLUOstar Omega, manufacturer: BMG LABTECH), and the released DNA concentration was obtained by substituting each fluorescence value. This was substituted into the mathematical equation below to calculate the DNA release efficiency for each temperature, and this was represented graphically.

[Mathematical Formula 1]

DNA release efficiency = (DNA concentration released at each temperature * 100) / (encapsulated DNA concentration)

As a result, as shown in FIGS. 14a to 17, both of the multiple emulsion compositions containing a hydrophilic surfactant in the outer layer and a lipophilic surfactant in the inner layer showed a tendency toward thermal reactivity. In the case of the hydrophilic surfactant, the highest DNA release efficiency was observed when tween85 was used, and in the case of the lipophilic surfactant, the release efficiency was confirmed to be high in the order of krytox > Ran > picosurf > fluosurf. In addition, in the case of the lipophilic surfactant, the DNA release efficiency increased in most cases as the surfactant content decreased.

[Example 6]

In order to confirm the RT-qPCR non-specific reaction inhibition effect of the multi-emulsion emulsion composition (W1/O/W2) according to one embodiment of the present disclosure, the following experiment was performed.

First, as an example, a multiple emulsion emulsion composition (W1/O/W2) was prepared using the same method as described in Test Example 5, but the inner phase (W1) used PBS, the oil phase (O) used HFE oil containing 0.4% of ran as a lipophilic surfactant, and the outer phase (W2) used PBS containing 0.25% of Tween20.

At this time, the primer used in the composition is a SARS-COV-2 N gene primer set, and the base sequences of the two primers are as follows.

-Forward primer: 5'-GACCCCAAAATCAGCGAAAT-3' (SEQ ID NO: 5)

-Reverse primer: 5'-TCTGGTTACTGCCAGTTGAATCTG-3' (SEQ ID NO: 6)

As a control, a PCR solution in which the two primers were freely present in the solution was prepared, containing both bidirectional primers in a PBS solution containing 0.25% Tween20.

The experiment was conducted by using RT-qPCR in the above example and control group in a human total RNA (complex sample) environment containing 2*10 ² copies of the SARS-COV-2 target gene, and gel electrophoresis was performed on the PCR-completed solution using a 3% agarose gel.

As a result, as shown in Fig. 18, compared to the control group (left), the example of the present disclosure (right) showed a decrease in the band of non-specific amplification products in a complex sample environment. This is because, in the example of the present disclosure, when RT-qPCR is performed, the primers are encapsulated in the inner phase of the multi-emulsion emulsion composition, physically separated from the reaction environment, and released under high temperature conditions of 95°C to participate in the PCR reaction, thereby suppressing non-specific reactions.

[Example 7]

To verify the RT-qPCR performance according to one embodiment of the present disclosure, the following experiments were performed. As the target gene, SARS-CoV-2 viral RNA (NCCP No.: 43326) gene sample (Template) 2x10 ⁵ , 2x10 ⁴ , 2x10 ³ , 2x10 ² , 2x10 ¹ copies/μl and NTC (No Template Control) were used.

The primers and probes used in this experiment are as follows.

-Forward primer: 5'- GGGAGCCTTGAATACACCAAAAG-3' (SEQ ID NO: 7)

-Reverse primer: 5'- TGTAGCACGATTGCAGCATTG-3' (SEQ ID NO: 8)

-Probe: 5'-(FAM)-TCACATTGGCACCCGCAATCCTGC-(BHQ1)-3' (SEQ ID NO: 9)

The PCR equipment used was a CFX opus 96 real-time PCR equipment from Bio-Rad. The compositions of each PCR mixture for the examples and comparative examples are as follows (in μl).

Comparative Example 1 (Prior Art)

_-H2O : 4.1

-2x PCR reaction mixture (SuperScript ^TM III Platinum ^TM One-Step qRT-PCR Kit, Thermo Fisher): 12.5

-MgSO ₄ (50 mM) (SuperScript ^TM III Platinum ^TM One-Step qRT-PCR Kit, Thermo Fisher): 0.4

-Human Total RNA (50 ng/μl) (Total RNA Control (Human), Thermo Fisher): 1

-Forward primer (10 μM): 1.5

-Reverse primer (10 μM): 2

-Probe (10 μM): 0.5

-Enzyme mixture (SuperScript ^TM III Platinum ^TM One-Step qRT-PCR Kit, Thermo Fisher): 1

-Template: 2

Example 3 (multi-emulsion emulsion composition)

_-H2O : 4.1

-2x PCR reaction mix (SuperScript ^TM III Platinum ^TM One-Step qRT-PCR Kit, Thermo Fisher): 12.5

-MgSO ₄ (50 mM) (SuperScript ^TM III Platinum ^TM One-Step qRT-PCR Kit, Thermo Fisher): 0.4

-Human Total RNA (50 ng/μl) (Total RNA Control (Human), Thermo Fisher): 1

-Emulsion -Forward primer (10 μM): 1.5

-Reverse primer (10 μM): 2

-Probe (10 μM): 0.5

-Enzyme mixture (SuperScript ^TM III Platinum ^TM One-Step qRT-PCR Kit, Thermo Fisher): 1

- Template: 2

Example 4 (multi-emulsion emulsion composition)

_-H2O : 4.1

-2x PCR reaction mixture (SuperScript ^TM III Platinum ^TM One-Step qRT-PCR Kit, Thermo Fisher): 12.5

-MgSO ₄ (50 mM) (SuperScript ^TM III Platinum ^TM One-Step qRT-PCR Kit, Thermo Fisher): 0.4

-Total human RNA (50 ng/μl) (Total RNA Control (Human), Thermo Fisher): 1

-Emulsion -Forward primer (10 μM): 1.5

-Reverse primer (10 μM): 2

-Probe (10 μM): 0.5

-Enzyme mixture (SuperScript ^TM III Platinum ^TM One-Step qRT-PCR Kit, Thermo Fisher): 1

- Template: 2

The PCR equipment temperature conditions were reverse transcription (RT) (55℃, 10 minutes)-PCR (pre-denaturation 94℃, 3 minutes/denaturation 94℃, 15 seconds/annealing 58℃, 30 seconds), repeated for 45 cycles. The result of amplifying the target nucleic acid through PCR was confirmed with a real-time curve, and after PCR, whether the amplified product matched the target to be detected was confirmed through gel electrophoresis (mupid 2plus equipment; manufacturer: TaKaRa, product name: mupid 2plus).

As a result, as shown in FIG. 19, Comparative Example 1 according to the prior art was capable of detecting up to a concentration of 2x10 ³ copies/μl, and as shown in FIG. 20 (Example 3) and FIG. 21 (Example 4), the example of the present disclosure was capable of detecting up to a concentration of 2x10 ² copies/μl. That is, when the emulsion according to the present disclosure is used, quantitative analysis is possible up to an RNA concentration of 200 copies/μl, and the limit of detection (LoD) for RNA was improved by 1 /10 times. This is because the forward primer stored in the emulsion according to the present disclosure did not cause nonspecific reactions with other targets during the RT process, and a sufficient amount of the forward primer was used only to amplify the detection target during the PCR process.

[Example 9]

As one embodiment of the present disclosure, the following experiments were performed to confirm the performance of the multi-emulsion composition of the hydrating gel formulation of Example 2 in qPCR and RT-qPCR.

qPCR

The target gene used was 2x107, ^2x106 , ^2x105 , ^2x104 copies/μl of COVID19 alpha mutant synthetic DNA (Template, IDT) and NTC (No Template Control), and the PCR equipment used was Mykobiomed's G2-4. A PCR mixture containing 7 μl of _H2O , 8 μl of 2x Mykobiomed Master Mix, and 1 μl of template was used. The PCR equipment temperature conditions were pre-denaturation 95°C, 8 sec - denaturation 95°C, 4 sec - annealing 51°C, 30 sec, repeated for 40 cycles. As shown in Figures 22 and 23, the qPCR efficiency of Example 2 was 97.24%.

The COVID19 alpha variant synthetic DNA used above and the primers and probes used are as follows.

-COVID19 alpha variant synthetic DNA: 5' TCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCA -3' (SEQ ID NO: 10)

-Forward primer: 5'- TCAACTCAGGACTTGTTCTTACCT -3' (SEQ ID NO: 11)

-Reverse primer: 5'- TGGTAGGACAGGGTTATCAAAC -3' (SEQ ID NO: 12)

-Probe: 5'- TCCATGCTATCTCTGGGACC -3' (SEQ ID NO: 13)

RT-qPCR

Same as the above qPCR, COVID19 alpha mutant viral RNA (Template) NCCP.43381_ 86.2 ng/μl was used as the target gene, and the same primers and probes were used. The PCR equipment used was Genechecker UF-350 from Genesystem. A PCR mixture containing 7 μl of _H2O , 8 μl of 2x MycoBioMed Master Mix, and 1 μl of template was used. The PCR equipment temperature conditions were reverse transcription (RT) 55°C, 10 minutes - PCR pre-denaturation 95°C, 30 seconds - denaturation 95°C, 4 seconds - annealing 51°C, 30 seconds, repeated for 40 cycles. Figure 24 shows the results of Example 2, and Figure 25 shows the results of NTC (No Template Control).

From the above two results, it can be confirmed that the composition according to the present disclosure normally amplifies the target in both qPCR using synthetic DNA and RT-qPCR using viral RNA, and that the amplification efficiency is about 97%, and the target is amplified about 1.97 times per cycle.

[Example 10]

Multiplex diagnosis was performed using a composition according to one embodiment of the present disclosure as follows.

To distinguish the mutations of the COVID-19 virus, microparticles targeting the mutant virus were produced and placed all together in one reaction, and the sample was added and the reaction was performed in the same manner as RT-qPCR in Test Example 9 above.

The mutant virus target primers and probe sequences used in this experiment are as follows.

As a result, as shown in FIGS. 26 and 27, the fluorescence of the microparticles brightened in line with the inserted target, and it can be confirmed that the multiple diagnostic efficiency is improved when the present disclosure is used.

Attach electronic file of sequence list

Claims (18)

an inner layer comprising water-in-oil droplets; and
Including the award-winning trauma
It is an emulsion composition of the W1/O/W2 (water1-oil-water2) type.
The above water-in-oil (W1/O) droplet contains at least one of a PCR (polymerase chain reaction) primer and probe in the inner phase, the water phase (W1), and
The above emulsion composition is heat-reactive,
A multiple emulsion composition. In the first paragraph, the oil phase (O) of the water-in-oil (W1/O) droplet contains a lipophilic surfactant,
The above award (W2) contains a hydrophilic surfactant,
A multiple emulsion composition. A multi-emulsion emulsion composition, wherein the number of droplets included in the composition in the first paragraph is two or more. A multi-emulsion emulsion composition, wherein the number of water droplets (W1) contained in each droplet is two or more in the first paragraph. A multi-emulsion emulsion composition in claim 1, wherein the average diameter of the droplets is 100 nm to 10 μm. In the second paragraph, the water-in-oil (W1/O) droplet contains hydrofluoroether oil in the oil phase (O),
The lipophilic surfactant comprises a fluorosurfactant,
A multiple emulsion composition. A multi-emulsion emulsion composition in claim 6, wherein the fluoro-based surfactant is a fluoro-based anionic surfactant or a fluoro-based nonionic surfactant. In the second paragraph, the water-in-oil (W1/O) droplet contains at least one of mineral oil, silicone oil and isopropyl palmitate oil in the oil phase,
A multi-emulsion emulsion composition comprising a silicone-based nonionic surfactant as the above lipophilic surfactant. A multiple emulsion composition in claim 2, wherein the hydrophilic surfactant is a surfactant having a hydrophilic-lipophile balance (HLB) of 8 to 20. A multiple emulsion composition in claim 9, wherein the hydrophilic surfactant is a polyoxyethylene sorbitan-based surfactant. A multi-emulsification emulsion composition in claim 1, wherein the water phase (W1) of the water-in-oil (W1/O) droplets comprises at least one selected from the group consisting of agarose, gelatin, collagen, LMPA (low melting point agarose), and PEG-aCD (a mixture of polyethylene glycol and alpha-cyclodextrin). A multi-emulsion emulsion composition in the first aspect, wherein the trauma (W2) is a hydrogel. In claim 12, the hydrating gel has a porous structure including pores,
A multi-emulsion emulsion composition wherein the water-in-oil (W1/O) droplets are positioned within the pores of the hydrogel. In claim 12, the hydrating gel is a multi-emulsion emulsion composition comprising at least one selected from the group consisting of polyethylene glycol-diacrylate (PEG-DA), polyacrylamide (PAAm), agarose, gelatin, and collagen. A multi-emulsion emulsion composition according to any one of claims 1 to 14; and
A reaction chamber in which the above multi-emulsion emulsion composition is arranged;
A nucleic acid amplification device comprising: In claim 15, the nucleic acid amplification device comprises a plurality of multi-emulsion emulsion compositions, each of which contains primers for different target nucleic acids. As a method for amplifying nucleic acids,
A step of injecting a multi-emulsion emulsion composition according to any one of claims 1 to 14 into a reaction chamber; and
A step of amplifying a target nucleic acid by polymerase chain reaction (PCR);
A nucleic acid amplification method comprising: A nucleic acid amplification method further comprising a step of analyzing a nucleic acid polymerized in the multiple emulsion composition in claim 17.