WO2022177367A1 - Composition for multiplex detection of target nucleic acid, based on isothermal amplification and fluorescent ring pattern, and detection method using same - Google Patents

Abstract

The present invention relates to a platform technology for target nucleic acid detection, based on isothermal amplification and a fluorescent ring pattern, and, more particularly, to: a composition for target nucleic acid detection, comprising a probe for rolling circle amplification and a fluorescent substance bound to the end of capture DNA; and a detection kit, a sensor and a detection method comprising same. A detection method according to the present invention integrates rolling-circle-amplification-based isothermal amplification and a difference in driving power according to substance size, and enables only a target nucleic acid to be specifically detected with high sensitivity even for a very small amount of a target nucleic acid in a picomole unit, uses capture DNA-fluorescent substances to use fluorescent substances of various wavelengths, and thus enable the multiplex detection of one or more target genes, and enables immediate identification of the presence of the target nucleic acid by observing the shape of formed fluorescence ring patterns without extra equipment and drying time after dropping an amplification product-capture DNA aggregate onto filter paper, and thus the effects of a dry environment do not need to be taken into consideration. Therefore, the detection method of the present invention can be easily used in onsite diagnosis at low cost, so as to be ultimately usable as a rapid gene test platform in the diagnosis of infectious diseases and improvement of patient disease control through early pathogen detection and efficient monitoring.

Description

Composition for multiple detection of target nucleic acid based on isothermal amplification and fluorescence ring pattern and detection method using same

The present invention relates to a platform technology for detecting a target nucleic acid based on isothermal amplification and a fluorescence ring pattern, and more particularly, a composition for detecting a target nucleic acid comprising a probe for rolling-ring amplification and a fluorescent material bound to the end of a capture DNA; It relates to a kit for detection, a sensor, and a detection method comprising the composition.

Infectious diseases account for 25% of global mortality and pose a serious threat. As of January 2022, the novel coronavirus pandemic has infected more than 300 million people worldwide, and the death toll has exceeded 5.5 million. The risk of infectious diseases is increasing.

The RT-PCR molecular diagnostic method, which is used as the gold standard method for diagnosing novel coronavirus, requires expert skill, is labor-intensive, requires expensive equipment from amplification to detection, and is difficult to apply to on-site diagnosis due to its high cost. There are limits. In addition, there is an antibody test method that can be used simply in the field, but the accuracy is significantly lower than that of molecular diagnosis, and there are limitations in that the diagnosis can be made after a certain amount of time has passed after the onset of symptoms.

In vitro diagnostic methods that can be used simply in the field have been developed, but most of them are known as methods of detecting two or more of the Orf1ab gene (RdRp gene), S gene, E gene, and N gene of the corona virus by RT-PCR, a molecular diagnostic technology. . On the other hand, in order to overcome the limitations of RT-PCR, in vitro diagnostic technology using isothermal amplification methods, Cas12, 13-based methods, etc. have been developed and used. Recently, one of the isothermal amplification methods, loop-mediated isothermal amplification -Mediated amplification (LAMP)-based self-diagnosis all-in-one kit 'Lucira COVID-19 All-In-One Test Kit' has received emergency FDA approval for the first time.

As such, no clear treatment has been developed so far, the production of antibodies decreases over time, and the limitations of vaccines that are difficult to respond to mutated viruses. Since this method is the most efficient, a molecular diagnostic technology that detects a target genetic material quickly and accurately is required. In addition, in the case of the novel coronavirus, the rate of asymptomatic infection is high, and even though it is asymptomatic, it has excellent virus transmission ability, so it is of utmost importance to develop a simple and inexpensive diagnostic kit that allows self-diagnosis to reduce contact between people as much as possible.

On the other hand, isothermal genetic testing has a great advantage in that it is simpler and can specifically identify a target at a low cost compared to the existing RT-PCR platform. Previously, isothermal amplification methods such as rolling circle amplification (RCA), loop mediated isothermal amplification (LAMP) and nucleic acid sequence-based amplification (NASBA) have been used to reduce colorimetric and fluorescence It is integrated with various sensing platforms based on

When a solution is dropped on a paper with pores, the particles in the solution spread radially from the center of the droplet to the edge by capillary action to form a ring pattern. If the moving particle is larger than the size of the pore, it is blocked by the pore, and movement is restricted, and is located close to the center of the circle. By making the pore size of the paper smaller and uniform and controlling the size of the particles, it is possible to suppress the ring pattern formed by the particle material in the droplet.

Accordingly, the present inventors amplified the target genetic material into a long single-stranded DNA repeating a predetermined sequence using RCA, one of the isothermal amplification methods, and at the same time hybridized the capture DNA-fluorescent material having a complementary sequence to the product. Then, an analysis method was established to determine the presence or absence of a target nucleic acid by observing the shape of a fluorescent ring pattern formed by droplets containing aggregates on paper.

The present invention was devised to overcome the limitations of conventional molecular detection and diagnosis methods for target nucleic acids, and the present inventors have developed an analysis method combining the RCA-based isothermal amplification reaction and the difference in driving force according to the size of the material, And it was confirmed that the simple and excellent detection effect without the influence of environmental factors was confirmed, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for detecting a target nucleic acid and a target nucleic acid detection sensor or kit comprising the composition.

Another object of the present invention is to provide a method for detecting a target nucleic acid using the composition for detection.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a probe comprising a nucleic acid sequence complementary to a target nucleic acid; a primer for rolling circle amplification (RCA); And it provides a composition for detecting a target nucleic acid comprising a fluorescent material bound to the end of the capture DNA.

In one embodiment of the present invention, the composition may further include a ligase (ligase) and a DNA polymerase.

In another embodiment of the present invention, the probe may include a nick.

In another embodiment of the present invention, the capture DNA may be complementary to a rolling-circle amplification product.

In another embodiment of the present invention, the fluorescent material is fluorescein (FAM), Texas Red (TexasRed), rhodamine, Alexa, cyanine (Cy), BODIPY (BODIPY) ), acetoxymethyl ester (Acetoxymethyl ester), coumarin (coumarin), and may be at least one selected from the group consisting of quantum dots (quantum dot).

In another embodiment of the present invention, the fluorescent material may have an emission wavelength of 300 to 800 nm.

In another embodiment of the present invention, the target may be a bacterium, a virus or a fungus.

In another embodiment of the present invention, the bacteria may be methicillin-resistant Staphylococcus aureus (MRSA).

In another embodiment of the present invention, the virus may be SARS-CoV-2.

In another embodiment of the present invention, the detection of the target nucleic acid may be made by observing the shape of the fluorescent ring pattern.

In another embodiment of the present invention, the target nucleic acid may be plural.

In addition, the present invention provides a kit for detecting a target nucleic acid comprising the composition for detection.

In addition, the present invention provides a target nucleic acid detection sensor comprising the composition for detection.

In addition, the present invention comprises the steps of adding and reacting a biological sample derived from a subject to the composition for detection; Comprising the step of observing the shape of the fluorescent ring pattern after dropping the reaction solution on the filter paper,

The reaction solution provides a method for detecting a target nucleic acid, characterized in that the isothermal amplification reaction and the binding reaction of the capture DNA-fluorescent material and the target nucleic acid occur simultaneously.

In one embodiment of the present invention, the biological sample may be selected from the group consisting of tissues, organs, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine.

In another embodiment of the present invention, the reaction may be carried out for 1 minute or more.

In another embodiment of the present invention, the reaction may be carried out at a temperature of 20 to 50 ℃.

In another embodiment of the present invention, the size of the pores of the filter paper may be 0.01 to 100 μm.

The detection method according to the present invention integrates the isothermal amplification based on the rolling ring amplification and the difference in driving force according to the size of the material. - By using a fluorescent material, multiple detection of one or more target genes is possible using fluorescent materials in various wavelength bands, and by observing the shape of the fluorescent ring pattern, the amplification product-captured DNA aggregate is dropped on filter paper, The presence or absence of the target nucleic acid can be checked immediately without drying time, so there is no need to consider the effect of the drying environment. Therefore, since the detection method of the present invention can be conveniently used for on-site diagnosis at a low cost, it can be used as a rapid genetic test platform to ultimately diagnose infectious diseases and improve patient's disease management through early pathogen detection and efficient monitoring.

1 shows a schematic diagram of the isothermal amplification gene multiplex detection method of the present invention based on the formation of a fluorescent ring pattern.

2 shows the fluorescence ring pattern pattern and image analysis results formed by the amplification product-captured DNA aggregate observed in paper filters of various pore sizes.

3 shows the fluorescence ring pattern and image analysis results of amplification products-captured DNA aggregates obtained under various conditions. Specifically, FIG. 3a is an image result obtained by varying the fluorescence excitation wavelength, and FIG. The obtained image and the result of quantitative analysis thereof are shown, and FIG. 3C is the image obtained by varying the amplification culture temperature and the result of quantitative analysis thereof.

4 shows the detection result by the detection method according to the present invention according to the concentration of the target nucleic acid. Specifically, FIG. 4a is an image result of a fluorescence ring pattern by isothermal amplification and capture DNA-fluorescent material, and FIG. 4b is the above figure. It is the result of quantitative analysis of the image of 4a.

5 shows the selectivity results of a fluorescent ring pattern-based detection method by adding a target gene material. Specifically, FIG. 5a is a target gene (RBD) alone or a non-target gene (RdRp and/or mecA) in a mixed treatment condition Isothermal amplification and capture DNA-image results of the fluorescence ring pattern by the fluorescent material, Figure 5b is a result of quantitative analysis of the image of Figure 5a.

Figure 6 is for a fluorescence ring pattern-based multiple detection method. Specifically, Figure 6a shows a schematic diagram for this, and Figure 6b is an image of a fluorescence ring pattern that appears when two target genes (RBD, RdRp) are present alone or together. is shown.

7 shows the detection results when the composition of the capture DNA-fluorescent material and the Cellulose Nitrate filter paper is applied to the normal filter paper and the capture DNA-quantum dot, respectively, in the detection method according to the present invention. Specifically, FIG. 7a is isothermal on the general filter paper. The image of the fluorescence ring pattern of the aggregate of the amplified and captured DNA-fluorescent material and the result of an orthogonal analysis thereof. It is the result of an objective analysis.

The present invention relates to a genetic testing platform technology capable of rapid and simple multiple detection of pathogens with excellent sensitivity and specificity. Specifically, the present invention provides a method for detecting a target nucleic acid through a rolling ring amplification reaction and a binding reaction between a target nucleic acid sequence and a capture DNA-fluorescent material in the amplification product, and whether a fluorescent ring pattern is formed in the resulting reaction solution. it's about how

Hereinafter, the present invention will be described in detail.

The present invention provides a probe comprising a nucleic acid sequence complementary to a target nucleic acid;

a primer for rolling circle amplification (RCA); and

Provided is a composition for detecting a target nucleic acid comprising a fluorescent material bound to the end of the capture DNA.

In addition, the composition for detection may further include a ligase and a DNA polymerase.

As used herein, the term “target nucleic acid” refers to all kinds of nucleic acids to be detected, and gene sequences derived from different species, subspecies, or variants or genes within the same species. may contain mutations. The nucleic acid may be any type of DNA including genomic DNA, mitochondrial DNA, viral DNA, and any type of RNA including mRNA, ribosomal RNA, non-coding RNA, tRNA, viral RNA, and the like, but is not limited thereto. .

In the present invention, the target may be preferably a bacterium, a virus, or a fungus, and more preferably, the bacterium may be a methicillin-resistant Staphylococcus aureus (MRSA). And, the virus may be SARS-CoV-2, but is not limited thereto.

As used herein, the term “probe” refers to a nucleic acid capable of specifically binding to mRNA having a length of several bases to several hundreds of bases produced through enzymatic chemical separation and purification or synthesis. Probes can be labeled with radioactive isotopes or enzymes to check the presence or absence of mRNA, and can be designed and modified by a known method.

In the present invention, the probe can specifically bind to the target nucleic acid including a nucleic acid sequence complementary to the target nucleic acid to be detected, and includes a nick at the site complementary to the target nucleic acid . When the target nucleic acid is complementary to the probe, the 5' end and the 3' end of the nick, which is an open part of the probe, are adjacent to each other, and are linked by a ligase to form a ring.

As used herein, the term "complementary" refers to the ability for correct pairing between two nucleotides. That is, two nucleic acids are considered complementary to each other at that position if a nucleotide at a given position in a nucleic acid can form a hydrogen bond with a nucleotide in another nucleic acid. The degree of complementarity between nucleic acid strands can significantly affect the efficiency and strength of hybridization between nucleic acid strands.

In the present invention, the ligase may preferably be a T4 ligase, but is not limited thereto, and as long as it has a function of linking the nick portion of the probe like the T4 ligase, a person skilled in the art may appropriately select and use it. .

As used herein, the term “primer” refers to an oligonucleotide synthesized for use in diagnosis, DNA sequencing, etc. as a short gene sequence serving as a starting point of DNA synthesis. The primers can be synthesized and used with a length of typically 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation, capping, etc. by a known method.

In the present invention, when the target nucleic acid binds to the probe, the primer is connected to the probe to form a circular template, and the DNA polymerase can induce rolling ring amplification using the primer as a starting point.

As used herein, the term "rolling circle amplification (RCA)" refers to a nucleic acid amplification reaction for amplifying a circular nucleic acid template through a rolling circle mechanism. The rolling circle amplification reaction is initiated by hybridization of primers to a circular, often single-stranded, nucleic acid template. Next, the nucleic acid polymerase repeats the sequence of the nucleic acid template by extending the primers hybridized to the original nucleic acid template by proceeding around the original nucleic acid template. Rolling circle amplification typically produces concatemers comprising tandem repeat units of a circular nucleic acid template sequence. The rolling ring amplification can be linear RCA (LRCA), which exhibits linear amplification kinetics (eg, RCA with a single specific primer), or can be exponential RCA (ERCA), which exhibits exponential amplification kinetics. Rolling circle amplification may also be performed using multiple primers to generate hyperbranched concatemers (multiple primed rolling circle amplification or MPRCA). For example, in double primed RCA, one primer may be complementary to the original nucleic acid template, as in linear RCA, while the other primer may be complementary to the tandem repeat unit nucleic acid sequence of the RCA product. The rolling ring amplification can be carried out under isothermal conditions using a DNA polymerase suitable therefor. In the present invention, the product produced through rolling-circle amplification may be a long single-stranded DNA comprising one or more target nucleic acid sequences and a repeating sequence with spatial and structural margins.

In the present invention, the DNA polymerase may be a phi29 DNA polymerase suitable for rolling ring amplification, but is not limited thereto, and if it has a function of performing rolling ring amplification using the nucleic acid sequence of the probe as a template A person skilled in the art can use it by selecting it appropriately.

In the present invention, the fluorescent material is bound to the end of one capture DNA, and the capture DNA can complementarily bind to a target nucleic acid sequence in the long single-stranded DNA generated as a result of the rolling circle amplification.

One or more of the fluorescent materials may be used simultaneously as long as they have different wavelength bands, and preferably, fluorescein (FAM), Texas Red (TexasRed), rhodamine, Alexa, cyanine; Cy), body P (BODIPY), acetoxymethyl ester (Acetoxymethyl ester), coumarin (coumarin) and may be at least one selected from the group consisting of quantum dots (quantum dot), but is not limited thereto, the capture DNA nucleic acid strand Any known fluorescence emitting material that can be attached to the fluorescence can be used.

In the present invention, the fluorescent material is 300 to 800 nm, 300 to 700 nm, 300 to 650 nm, 400 to 800 nm, 400 to 700 nm, 400 to 650 nm, 450 to 800 nm, 450 to 700 nm or 450 It may have an emission wavelength of to 650 nm.

When the fluorescent material has an emission wavelength of less than 300 nm or greater than 800 nm, the fluorescent ring pattern may not be distinguished.

In the present invention, in a specific example, it was confirmed that the detection method according to the present invention can detect one or more target nucleic acids simply and with high accuracy by observing the shape of the fluorescent ring pattern formed by the droplet (see Example 1).

In one embodiment of the present invention, as a result of dropping an aggregate of an isothermal amplification product and a capture DNA-fluorescent material on a paper filter having various pore sizes, the stronger the fluorescence signal at the center of the droplet is as it has smaller and more uniform pores. It was confirmed that it appeared (see Example 2).

In another embodiment of the present invention, as a result of confirming the pattern of droplets formed by the amplification product-captured DNA fluorescent material by varying the fluorescence excitation wavelength, amplification incubation time, and temperature conditions of the detection method, the ring more clearly at 254 nm wavelength The pattern was suppressed to obtain a dense fluorescence signal in the center of the droplet, and it was observed that the fluorescence ring pattern was changed even with an amplification incubation time of 5 minutes or more, and it was confirmed that the amplification culture temperature of 37 ° C is most suitable ( see Example 3).

In another embodiment of the present invention, the aggregate formed by the isothermal amplification reaction and the binding reaction of the target nucleic acid of the amplification product and the capture DNA-fluorescent material under each treatment condition with different concentrations of the target gene nucleic acid is coated on Cellulose Nitrate filter paper. As a result of dropping, the formation of a fluorescence ring pattern was suppressed, and it was confirmed that the fluorescence signal located in the center of the circle became stronger as the concentration of the target gene nucleic acid increased (see Example 4).

In another embodiment of the present invention, one to three SARS-CoV-2 derived RBD and RdRp genes and one to three mecA genes derived from methicillin-resistant Staphylococcus aureus in order to confirm that the target genetic material is selectively detected even when the target genetic material is not present alone As a result of treatment with a dog combination and the RBD gene as a target nucleic acid and analysis using a capture DNA-fluorescent material, it was possible to observe a dense fluorescence signal in the center of the circle only in the condition in which the RBD gene was treated. Under the condition, a fluorescence signal was shown at the edge of the circle to form a fluorescence ring pattern, confirming the selectivity of the fluorescence ring pattern inhibition by the addition of the target nucleic acid (see Example 5).

In the present invention, the target nucleic acid may be plural. Specifically, when there are a plurality of target nucleic acids, that is, two or more, the detection composition comprises probes comprising a nucleic acid sequence complementary to each target nucleic acid, primers for each probe, and each target nucleic acid. Capture DNA complementary to the rolling-ring amplification product of - may include fluorescent materials, and the fluorescent material may be bound to a different fluorescent material for each type of capture DNA.

In another embodiment of the present invention, as a result of performing the analysis method of the present invention by targeting the RBD and RdRp genes derived from SARS-CoV-2 in order to confirm whether multiple analysis of simultaneously detecting one or more target substances is possible, one target It was confirmed that when there is a nucleic acid, only one fluorescence signal corresponding thereto appears in the center of the droplet, and when two or more targets exist, the fluorescence signals are mixed and appear in the center (see Example 6).

In another embodiment of the present invention, a normal filter paper rather than a Cellulose Nitrate filter paper is used, and as an example of a capture DNA-fluorescent material, a capture prepared using quantum dots known to generate much stronger fluorescence than a general fluorescent material in a narrow wavelength band As a result of performing the analysis method of the present invention using DNA-quantum dots, it was confirmed that the fluorescence ring pattern was suppressed by confirming a strong fluorescence signal at the center of the circle when the target nucleic acid was present (see Example 7).

The above results prove that the method for detecting a target nucleic acid according to the present invention is a platform technology capable of sensitively and rapidly detecting and diagnosing a target nucleic acid from various samples.

Accordingly, as another aspect of the present invention, the present invention provides a target nucleic acid detection sensor comprising the composition for detection. Any sensor technology capable of detecting a target nucleic acid using the composition for detection may be used, and is not particularly limited to the type or characteristic of the sensor technology.

The sensor system according to the present invention may be provided in the form of a kit. In the present invention, the kit is a target nucleic acid amplification reaction such as a buffer, a DNA polymerase cofactor, and deoxyribonucleotide-5-triphosphate (dNTP) together with the detection composition (e.g. , polymerase chain reaction) may include all reagents necessary for carrying out the reaction. In addition, the optimal amount of reagents to be used in a particular reaction of the kit can be readily determined by one of ordinary skill in the art having the disclosure herein.

As another aspect of the present invention, the present invention comprises the steps of adding and reacting a biological sample derived from a subject to the composition for detection;

Comprising the step of observing the shape of the fluorescent ring pattern formed after dropping the reaction solution on filter paper,

The reaction solution provides a method for detecting a target nucleic acid, characterized in that the isothermal amplification reaction and the binding reaction of the capture DNA-fluorescent material and the target nucleic acid occur simultaneously.

As used herein, the term "fluorescent ring pattern" refers to a ring pattern formed when a solution containing a fluorescent material moves radially by capillary action on paper and the fluorescent material is positioned at the edge of a circle do.

When a target nucleic acid is present in the biological sample, the nick portion of the probe is linked by a ligase enzyme by complementary binding between the target nucleic acid and the probe specific for the target nucleic acid of the present invention to form a circular template. rolling circle amplification occurs, leading to repetitive and long single-stranded DNA amplification. Amplification of repetitive sequences is induced indefinitely as long as sufficient dNTPs are supplied, and the amplification product consisting of a sequence complementary to the probe is pushed away by the newly formed product and is separated from the probe as a template, resulting in a fluorescent material bound to it. It forms a single strand of long repeating sequence capable of forming complementary bonds with the capture DNA. Aggregation is induced by the complementary binding of the target nucleic acid sequence and the capture DNA repeatedly present in the long single-stranded DNA. When the reaction solution containing the aggregate is dropped on filter paper, the pattern of droplets formed by the fluorescent material bound to the capture DNA can be immediately confirmed by fluorescence, and preferably observed under a UV lamp.

In the present invention, the biological sample may be selected from the group consisting of extracorporeally isolated tissues, organs, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine, and the tissue is, for example, connective, skin, muscle or nerve. Tissues may be included, wherein said organs are, for example, eye, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testis, ovary, uterus, Rectal, nervous system, glandular and internal blood vessels may be included, but the scope of the sample is not limited thereto.

In the present invention, the reaction may be carried out for 1 minute or more, specifically for 3 minutes or more, and more specifically for 5 minutes or more. When the reaction is performed for less than 1 minute, the reaction does not occur sufficiently so that the fluorescence ring pattern may not be clearly distinguished or observed, and when the reaction is performed for more than 1 minute, the fluorescence ring pattern is clearly distinguished or observed can be

In addition, in the present invention, the reaction is 20 to 50 ℃, 20 to 45 ℃, 20 to 40 ℃, 25 to 50 ℃, 25 to 45 ℃, 25 to 40 ℃, 30 to 50 ℃, 30 to 45 ℃ or It may be carried out at a temperature of 30 to 40 ℃.

When the reaction is performed at a temperature of less than 20° C. or more than 50° C., the reaction may not occur properly, and the fluorescence ring pattern may not be clearly distinguished or observed.

In the present invention, the filter paper may preferably be Cellulose Nitrate, but it is not limited thereto because it is possible even when a general filter paper is used.

In addition, the size of the pores of the filter paper is ok as long as the isothermal amplification product-capture DNA-fluorescent material can move slower than the capture DNA-fluorescent material due to steric hindrance, but it is small enough that the capture DNA-fluorescent material cannot pass through. It shouldn't be the size. For example, the size of the pores of the filter paper is 0.01 to 100 μm, 0.01 to 50 μm, 0.01 to 10 μm, 0.05 to 100 μm, 0.05 to 50 μm, 0.05 to 10 μm, 0.1 to 100 μm, 0.1 to 50 μm or 0.1 to 10 μm.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Detection of a target nucleic acid through observation of a fluorescent ring pattern shape (FIG. 1)

Developing a method to detect genetic material quickly and simply is very important for the diagnosis and subsequent treatment of diseases. Among these, gene-based diagnosis can analyze a small amount of target with high specificity through nucleic acid amplification technology, and the isothermal amplification method is more suitable for a point-of-care diagnostic platform because it can be performed through simple and miniaturized equipment.

Accordingly, the present inventors amplify the target genetic material into a long single-stranded DNA repeating a predetermined sequence using one of the isothermal amplification methods, rolling circle amplification (RCA), and at the same time, a sequence complementary to the product A simple diagnostic method was developed to determine the presence or absence of a target by hybridizing a capture DNA-fluorescent material with

More specifically, when the nick portion of the nucleic acid probe (Padlock probe) including a sequence complementary to the nucleic acid of the target gene is close by binding to the target gene, it is ligated by the T4 ligase enzyme. A circular DNA template is created. Based on this template, starting from the RCA primer under isothermal conditions (37°C), the phi29 polymerase generates a long single-stranded DNA containing multiple copies of the target sequence through unidirectional replication. As described above, the amplification product, long single-stranded DNA, has a structure in which a specific target sequence is repeated, and at the same time amplified, it is complementarily combined with the capture DNA-fluorescent material to form an amplification product-capture DNA aggregate. -Fluorescent labeling process through binding to fluorescent material is sufficiently completed within 30 minutes.

The required volume of the solution containing the aggregates used to observe the fluorescent ring pattern for diagnosis is a small volume of 1-5 μl, and when this drop is dropped on Cellulose Nitrate filter paper, the solution is transferred from the center of the circle to the edge by capillary action. spread out in a circle If the particles in the solution are larger than the pore size of the filter, they are concentrated in the center of the droplet due to the limitation of the driving force. The amplification product-capture DNA aggregate material having a size of several microns is located in the center of the droplet because it cannot move because it is blocked by the pores, and the capture DNA-fluorescent material moves to the edge of the droplet to form a ring pattern. According to the present invention, the shape of the fluorescence ring pattern can be immediately confirmed by the drop dropped on the paper without a separate drying time, so there is an advantage that there is no need to consider the effect of the drying environment.

Example 2. Identification of fluorescence ring pattern according to pore size of filter paper

The present inventors predicted that the change in the fluorescence ring pattern would be greater as the pores of uniform and small size became relatively difficult to move, and to confirm this, alpha-cellulose (AC) having pores of 11 μm or 2.5 μm And after performing the experiment in the same manner as in Example 1 on Cellulose Nitrate (CN) filter paper having pores of 0.45 μm or 0.2 μm in size, the shape of the fluorescence ring pattern was observed and the image was analyzed.

As a result, as shown in FIG. 2 , it was confirmed that, as expected, the more uniform and smaller the pores, the more the amplification product-captured DNA aggregates gathered in the center to show a strong fluorescence signal.

Example 3. Confirmation of Optimization Conditions for Fluorescence Ring Pattern-based Detection Method

The present inventors tried to determine under what conditions the detection method based on the fluorescence ring pattern is most optimally performed. To this end, the change in the fluorescence ring pattern was observed by varying the excitation wavelength of the fluorescence, the amplification incubation time, and the temperature.

As a result of confirming the fluorescence ring pattern by changing the excitation wavelength of the fluorescence, as shown in FIG. 3a , the fluorescence signal exhibited by the amplification product-captured DNA aggregate concentrated in the center of the droplet at 254 nm was more clearly obtained. It was confirmed that the fluorescence ring pattern image appeared more clearly because the signal-to-noise ratio increased because the 254 nm wavelength emitted a stronger fluorescence signal than the 365 nm wavelength.

In addition, when the fluorescence ring pattern was confirmed while changing the amplification incubation time to 5 minutes, 10 minutes, 20 minutes and 30 minutes, the fluorescence ring pattern was suppressed even after the incubation time was 5 minutes or more, as shown in FIG. 3b. could see The droplet of the solution with amplification process for 5 min had a fluorescence signal throughout the circle, whereas no target showed a signal only at the edge of the circle. The longer the incubation time, the more dense the signal was observed in the central region of the droplet, and the solution incubated for more than 20 minutes showed a higher signal in the central region than the outside.

Finally, as a result of performing the amplification process under various temperature conditions (25°C, 28°C, 32°C, 34°C and 37°C) to confirm a suitable amplification culture temperature, as shown in FIG. 3C, 34°C and 37°C It was confirmed that the fluorescence ring pattern was most efficiently suppressed under the conditions.

Example 4. Fluorescence ring pattern observation and image analysis according to target gene nucleic acid concentration

The present inventors tried to determine whether the efficiency of the fluorescent ring suppression pattern of the aggregate varies depending on the concentration of the target, based on the results of the above examples. To this end, the target nucleic acid was treated with various concentrations (0, 0.1, 0.3, 0.5, 0.8, 1, 5 and 10 pmol) and the shape of the fluorescence ring pattern was observed after performing the experiment in the same manner as in Example 1, and Images were analyzed.

As a result, as can be seen from the fluorescent ring pattern images of FIGS. 4A and 4B , it was observed that the higher the concentration of the target nucleic acid, the stronger the fluorescence signal located at the center of the circle.

Example 5. Confirmation of selective detection of target gene

Next, the present inventors tried to find out whether the target genetic material can be selectively detected even if it does not exist alone. To this end, a gene encoding a receptor binding domain (RBD) in the spike protein of SARS-CoV-2, an RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2, and The mecA gene of methicillin-resistant Staphylococcus aureus (MRSA) was treated with the RBD gene alone, two genes, or three genes, and the RBD gene was used as a target nucleic acid for analysis using a capture DNA-fluorescent material.

As a result, as shown in FIGS. 5A and 5B , it was confirmed that the fluorescence signal was concentrated in the center of the droplet under the conditions in which RBD, the target gene material, was treated. It was confirmed that the phenomenon that the fluorescence ring pattern was suppressed with

Example 6. Confirmation of detection of multiple target genes

In addition, the present inventors believe that, since the detection method of the present invention uses a method of measuring a pattern of a fluorescence signal, multiplex analysis targeting multiple genes is possible by using a capture DNA-fluorescent material using a fluorescent material in various wavelength bands. expected.

To confirm this, as shown in Fig. 6a, a specific target material is amplified and labeled by one nucleic acid probe (Padlock probe) and a capture DNA-fluorescent material set corresponding thereto, so that different target gene materials (RBD, RdRP and mecA) Multiple analysis was performed using a nucleic acid probe having a different sequence to bind to a gene) and a capture DNA-fluorescent material of emission (520 nm and 610 nm) of a different wavelength band.

As a result, as can be seen in FIG. 6b, when there is one target nucleic acid, only one fluorescence signal appears in the center of the droplet, and when two or more targets exist, the fluorescence signals are mixed and appear in the center. did. It was found that the present technology can reduce the possibility of false-positive or false-negative results by detecting two or more target genetic materials.

Example 7. Detection of a target nucleic acid through observation of a fluorescence ring pattern under conditions using quantum dots or AC filter paper

The present inventors determined the shape of the fluorescent ring pattern when using AC filter paper instead of Cellulose Nitrate (CN) filter paper in the method for detecting a target nucleic acid according to the present invention, and when using quantum dots instead of general fluorescent material as capture-DNA fluorescent material We tried to find out whether it was possible to check whether the target nucleic acid was detected by observation. To this end, the RCA isothermal amplification reaction was performed in the same manner as in Example 1 by treating the target nucleic acid at various concentrations (0, 0.01, 0.05, 0.1, 0.5, 1, 3, 5, 10, 50 and 100 pmol). After hybridization was carried out using a capture DNA-fluorescent material or a capture DNA-quantum dot, a drop of the aggregate was dropped on general filter paper and the shape of the fluorescent ring pattern was observed.

As a result, as shown in FIG. 7a , even if a cheaper general filter paper was used instead of CN filter paper, if the target nucleic acid was present, it was possible to confirm the fluorescence signal concentrated in the center of the circle, as shown in FIG. 7b , the capture DNA - Even when quantum dots were used, the same phenomenon was observed in that the fluorescent ring pattern located in the center of the circle was suppressed as the concentration of the target nucleic acid increased.

The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (18)

1. a probe comprising a nucleic acid sequence complementary to a target nucleic acid;

   a primer for rolling circle amplification (RCA); and

   A composition for detecting a target nucleic acid comprising a fluorescent material bound to the end of the capture DNA.
2. According to claim 1,

   The composition is characterized in that it further comprises a ligase (ligase) and a DNA polymerase, the composition for detection.
3. According to claim 1,

   The probe is characterized in that it comprises a nick (nick), the composition for detection.
4. According to claim 1,

   The capture DNA is characterized in that complementary binding to the rolling-circle amplification product, the composition for detection.
5. According to claim 1,

   The fluorescent material is fluorescein (FAM), Texas Red (TexasRed), rhodamine (rhodamine), Alexa (alexa), cyanine (cyanine; Cy), body P (BODIPY), acetoxymethyl ester (Acetoxymethyl ester) ), coumarin (coumarin) and quantum dots (quantum dot), characterized in that at least one selected from the group consisting of, the composition for detection.
6. According to claim 1,

   The fluorescent material is characterized in that it has an emission wavelength of 300 to 800 nm, the composition for detection.
7. According to claim 1,

   The target is a composition for detection, characterized in that the bacteria, virus or fungus.
8. 8. The method of claim 7,

   The bacterium is methicillin-resistant Staphylococcus aureus (MRSA), characterized in that, the composition for detection.
9. 8. The method of claim 7,

   The virus is characterized in that SARS-CoV-2, the composition for detection.
10. According to claim 1,

    Detection of the target nucleic acid, characterized in that made by observing the shape of the fluorescent ring pattern, the composition for detection.
11. The method of claim 1,

    The target nucleic acid is a plurality of, characterized in that the detection composition.
12. A kit for detecting a target nucleic acid, comprising the composition of any one of claims 1 to 11.
13. A target nucleic acid detection sensor comprising the composition of any one of claims 1 to 11.
14. adding and reacting the subject-derived biological sample to the composition of claim 1; and

    Comprising the step of observing the shape of the fluorescent ring pattern after dropping the reaction solution on the filter paper,

    The reaction solution is characterized in that the isothermal amplification reaction and the binding reaction of the capture DNA-fluorescent material and the target nucleic acid occur simultaneously.
15. 15. The method of claim 14,

    Wherein the biological sample is selected from the group consisting of tissue, organ, cell, whole blood, blood, saliva, sputum, cerebrospinal fluid and urine, a method for detecting a target nucleic acid.
16. 15. The method of claim 14,

    The method of detecting a target nucleic acid, characterized in that the reaction is carried out for 1 minute or more.
17. 15. The method of claim 14,

    The method of detecting a target nucleic acid, characterized in that the reaction is carried out at a temperature of 20 to 50 ℃.
18. 15. The method of claim 14,

    The method of detecting a target nucleic acid, characterized in that the size of the pores of the filter paper is 0.01 to 100 μm.

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