KR102083396B1 - Target nucleic acids detection method using quantum dot for dispersed light - Google Patents

Abstract

The present invention detects a target nucleic acid based on the fluorescence characteristics of a fluorescent dye which changes state when a detecting probe hybridizes to the target nucleic acid. Excitation does not require bioconjugation of the quantum dots, thereby making it possible to easily prepare and use reagents for detection of target nucleic acids, and to prevent the fluorescence of the fluorescent dye and the fluorescent light of the fluorescence dye from interfering with each other. The present invention relates to a method for detecting a target nucleic acid that can accurately detect a target nucleic acid. A detection probe having a nucleotide sequence complementary to a target nucleic acid is hybridized with a target nucleic acid in an environment in which a quantum dot is interspersed, and thus, fluorescent characteristics are different depending on whether or not hybridization is performed. A hybridization step of inducing a state change of the fluorescent dye to be changed; And a detection step of detecting the target nucleic acid by detecting the fluorescent light of the fluorescent dye which receives the emission light of the quantum dot serving as a scattered light source by exciting the scattered quantum dots. It includes.
The research related to the present invention was performed with the support of the Ministry of Trade, Industry and Energy's core material source technology development project (development of PNA-nanohybrid fusion material and molecular diagnostic system for femtomol level oncogene diagnosis, task number: 10047831).
In addition, the verification experiment applying the target nucleic acid detection method according to the invention to the actual clinical sample was performed by receiving a clinical sample from Incheon St. Mary's Hospital human-derived material resource bank.

Description

Target nucleic acid detection method using quantum dots as a scattered light source {TARGET NUCLEIC ACIDS DETECTION METHOD USING QUANTUM DOT FOR DISPERSED LIGHT}

The present invention detects a target nucleic acid based on the fluorescence characteristics of a fluorescent dye which changes state when a detection probe hybridizes to the target nucleic acid. Excitation does not require bioconjugation of the quantum dots, thereby making it possible to easily prepare and use reagents for detection of target nucleic acids, and to prevent the fluorescence of the fluorescent dye and the fluorescent light of the fluorescence dye from interfering with each other. The present invention relates to a method for detecting a target nucleic acid capable of accurately detecting a target nucleic acid by analysis.

The research related to the present invention was carried out with the support of the Ministry of Trade, Industry and Energy's core material source technology development project (PNA-nanohybrid fusion material and molecular diagnostic system for femtomol level oncogene diagnosis, task number: 10047831). In addition, the verification experiment applying the target nucleic acid detection method according to the invention to the actual clinical sample was performed by receiving a clinical sample from Incheon St. Mary's Hospital human-derived material resource bank.

As the relationship between the presence or mutation of a particular gene and the disease continues to be identified, genetic testing, traditionally performed to confirm or discriminate genetic disease in patients with symptoms of the disease, may be used to prevent disease, diagnose early, Treatment and management are expanding to reduce the morbidity and mortality of the disease.

In particular, gene abnormalities such as oncogenes mutations, tumorsuppressor genes variations, deregulations and chromosomal abnormalities are involved in the development of cancer and the prognosis of drugs. Various studies have been reported with this finding (Fearson ER et al., Cell., 1990; KW Kinzler et al., Cell., 1996; Manuel Serrano et al., Cell., 1997; Amado RG et al., J Clin.Oncol., 2008, Raponi M et al., Curr. Opin.Pharmacol., 2008; Siena S et al., J. Natl. Cancer Inst., 2009). According to this, by analyzing genes specific to cancer, it is possible to indirectly confirm whether the cancer has developed.

As such, the occurrence of mutations in disease-related genes, in particular cancer-related genes, affects early diagnosis, onset of disease, and prognosis, as well as selection and modification of therapeutic agents. Therefore, various mutation detection methods have been developed according to the purpose of analysis or the type of gene (Taylor CF, Taylor GR.Method Mol. Med., 92: 9, 2004). In many cases, however, the number of cancer cells with a specific mutation present in the assay sample is significantly less than normal cells and / or cancer cells that do not have the mutation. et al, Clin Endocrinol, 2006 / Trovisco et al, J Pathol., 2004).

Traditionally, cancer-related gene mutation tests have been performed with invasive tissue biopsy specimens, which not only cause patient pain, but also many patients are unable to biopsy tissue at all, and in addition, heterogeneity of cancer tissue cells (Heterogeneity) Because of the risk of bias due to), the need arises for testing from non-invasive, homogeneous liquid biopsy specimens, such as blood, which results in smaller genes. There is a need for accurate detection of mutations.

Genetic variation testing includes traditional methods such as Sanger sequencing, pyrosequencing, recently developed real-time PCR, and digital PCR.

In addition, allele specific PCR methods (Rhodes et al., Diagnmol pathol., 6:49, 1997), using primers specific for mutations to selectively increase small amounts of mutated genes, Scorpion Real-time allele specific PCR (DxS 'scorpions and ARMS) method (Mark et al., Journal of Thoracic Oncology, 4: 1466, 2009), allele specific primer CAST PCR suppresses amplification of wild-type genes using technology and minor groove binder (MGB) probes, selectively amplifies only mutant genes, and then detects amplification products without using the Taqman probe. Method (Didelot A et al., Exp Mol Pathol., 92: 275, 2012), Cold-PCR method that can increase the sensitivity of mutations using critical denaturation temperature (Tc) (Zuo et al, Modern Pathol, 22:.. 1023, 2009) There are a variety of selective amplification method based on the real-time PCR (polymerase chain reaction, polymerase chain reaction) technology has been introduced and the like. These methods make it possible to provide relatively easy and fast results of diagnosis and analysis of mutations in various cancer-related genes (Bernard et al., Clinical Chemistry, 48: 1178, 2002).

In the selective amplification method based on the real-time PCR technology, it is common to use fluorescent light of a fluorescent substance labeled with a probe as a method for detecting a target gene mutation.

In particular, techniques using quenchers that absorb fluorescent light and reduce the intensity of fluorescent light when in proximity to the fluorescent material are often used. According to Patent No. 10-1496671, when the probe is in a single strand, Adsorbed to graphene oxide, the fluorescent material of the probe is quenched, and when the probe and the target nucleic acid are combined and double stranded, the fluorescent material of the probe is not absorbed by the graphene oxide.

In addition, according to Patent Publication No. 10-2015-0139096, using a molecular beacon capable of complementarily binding a specific gene associated with the disease of interest to exosomes from cells suspected of the disease of interest, one side of the molecular beacon At the end, a detection label (fluorescent material) was bound, and at the other end of the molecular beacon, a detection label and a corresponding quencher were bound. Therefore, when the molecular beacon is not bound to the specific gene, the detection label is quenched by the binding of the detection label and the quencher. When the molecular beacon is bound to the specific gene, the detection label is spaced apart from the quencher to recover the fluorescence. The gene can be detected.

Commonly used fluorescent materials have a narrow light absorption spectrum to excite, and therefore, use a different light source or an optical polarizing filter that transmits or reflects only light of a corresponding wavelength depending on the fluorescent material. B has a problem that the number is limited or the detection apparatus is complicated.

In addition, a fluorescently labeled probe is often used in combination with a real-time PCR technology-based selective amplification method. However, there is a problem in that the probe may inhibit the PCR amplification to decrease the sensitivity.

As a result, efforts are continuing to meet the clinical needs of more accurate and sensitive detection methods.

Recently, a melting point (Tm: melting temperature) analysis method has been proposed as a polymorphism detection method. The polymorphism detection method through melting point analysis uses a probe complementary to the region containing the polymorphism to be detected, forms a hybrid (double stranded nucleic acid) by complementary binding of the nucleic acid and the probe, and then heats the hybrid to heat the temperature. As the temperature rises, the melting point at which the hybrid is separated into single-stranded nucleic acids is obtained by measuring signals such as absorbance, and the polymorphism is determined therefrom. In general, the melting point is derived from the measured melting curve by melting curve analysis, and then the polymorphism is determined from the melting curve.

The melting point is higher as the complementarity of both hybridized single-stranded nucleic acids and lower as the complementarity is low, so that the melting point is detected and selected as an evaluation reference value for the hybrid generated by the complementary coupling of the gene and the probe. By comparing the melting point of the hybrid by the probe with the evaluation reference value, it is possible to determine whether the site of detection of the nucleic acid is the same as the target gene. In addition, when the measured value is lower than the test evaluation reference value, it can be discriminated that the detection target site of the test nucleic acid has a different type. Accordingly, the polymorphism can be detected by only performing signal measurement after the PCR reaction by adding the detection probe.

As a measurement signal for measuring the melting point, it is common to use fluorescent light of a fluorescent material labeled on a probe. Typically, fluorescent materials have a narrow light absorption spectrum for excitation, so that different light sources may be used depending on the fluorescent material. An optical polarizing filter that transmits or reflects only light having a corresponding wavelength must be used, which causes a problem in that the type or number of fluorescent materials to be applied is limited or the device is complicated.

In addition, since the concentration of the target nucleic acid in the sample is very low, the target nucleic acid is amplified or mutated selectively by PCR or real-time PCR, and the melting point measurement is performed. Still exists.

Quantum dots (QDs) are characterized by spectral changes in size (i.e., characteristics that emit light of different wavelengths as the size changes), improved brightness, excellent stability against photo bleaching, simultaneous multiple fluorescence excitation, etc. It is a nano material that has attracted a lot of attention recently in the bio field because of its unique useful optical properties.

In particular, it has a broad absorption spectrum and a narrow emission spectrum, and is used as a donor or acceptor in a biosensor or diagnostic system based on fluorescence resonance energy transfer (FRET) as well as replacing a conventional fluorescent substance. Many attempts have been made.

The FRET phenomenon indicates that when fluorescent materials in two different wavelength regions are adjacent to each other, energy is transferred from one of the excited fluorescent materials to another fluorescent material through non-radiation, thereby representing the intrinsic fluorescence of the other fluorescent material. It is a physical phenomenon. In this case, a substance that gives energy is called a donor, and a substance that receives energy is called an acceptor.

Since the FRET phenomenon generally occurs when the donor and the acceptor are in a range of approximately 10 kV to 100 kV corresponding to the dimensions of most macromolecules in living organisms, the donor and acceptor may be applied by applying a bioconjugation technique. The local distance between them must be guaranteed.

Many of the FRET-based sensors using quantum dots use protein-quantum dot conjugates or streptavidin-quantum dot systems and conjugate biomolecules to quantum dots through nonspecific adsorption, electrostatic interactions and covalent bonds. Cases have also been reported. FRET-based sensor-related techniques using quantum dots include quantum dot-conjugated hybridization probes for screening siRNA sequences, high sensitivity DNA nanosensors, and nuclease tolerant FRET probes based on DNA-quantum dot conjugation. Reported.

As a specific example of the prior art using the quantum dots there is a Patent No. 10-0704011 and Patent No. 10-1510513.

FRET-based techniques using most quantum dots, including the prior art, require not only to label most target materials (nucleic acid, genetic material, for example DNA) with dye prior to conjugation to donor quantum dots, In some cases, the procedure is relatively complicated because a plurality of probes have been previously prepared or involved in biotin treatment and streptavidin treatment, respectively, to the target material and quantum dots prior to conjugation. In addition, bioconjugation of the probe to quantum dots may interfere with hybridization between the probe and the target material.

KR 10-0704011 B1 2007.03.29. KR 10-1510513 B1 2015.04.02. KR 10-1496671 B1 2015.02.23. KR 10-2015-0139096 A 2015.12.11.

The present invention solves the problems of conventional bioconjugation, inaccuracy in detecting fluorescent materials, complexity of optical system, and limitations of fluorescent materials as described above, and thus provides a target nucleic acid detection method that can detect target materials more simply and quickly and accurately. The purpose is to provide.

The present invention provides a hybridization step of hybridizing a detection probe having a nucleotide sequence complementary to a target nucleic acid with a target nucleic acid to induce a state change of a fluorescent dye whose fluorescence properties vary depending on whether or not to hybridize; And a detection step of detecting a target nucleic acid by measuring fluorescent light of a fluorescent dye. The invention provides a method for detecting a target nucleic acid for achieving the above object. The mixture is interspersed so that the detection probe hybridizes with the target nucleic acid in an environment in which the quantum dots are scattered, and excites the quantum dots scattered around the hybrid generated by the hybridization of the detection probe and the target nucleic acid to serve as a scattering light source. The target nucleic acid is detected by measuring the fluorescent light inherent in the fluorescent dye which is emitted by excitation of the quantum dots by absorbing the fluorescent dye.

Accordingly, the quantum dots do not need to be bioconjugated to the detection probes, do not interfere with the hybridization of the detection probes and the target nucleic acids, and the object to be excited and the object to be detected are separated from each other. Can be excited to detect fluorescent light without interference.

Accordingly, the optical system can be simplified, and by using quantum dots targeted to a specific fluorescent dye according to the characteristics of the quantum dots having a narrow band emission spectrum, the target nucleic acid is accurately measured and the target nucleic acid is accurately detected. It provides a target nucleic acid detection method that can be.

In addition, a target nucleic acid detection method capable of multiplex detection is provided by inserting different probes labeled with fluorescent dyes having different wavelengths of absorbing and emitting fluorescent light and quantum dots of different sizes corresponding to the absorbing wavelengths of each fluorescent dye. Can provide.

In addition, a detection probe having a fluorescent dye coupled to one end and a quencher at the other end is used to extinguish fluorescent light when the detection probe is not hybridized to the target nucleic acid and to emit fluorescent light when hybridized to the target nucleic acid. A detection method that utilizes a characteristic that is characterized in that it is used, and a detection probe that combines a fluorescent dye at one end with a quencher adsorbing the detection probe is separated from the quencher when the detection probe hybridizes to the target nucleic acid to emit fluorescent light It provides a target nucleic acid detection method that can be applied to a variety of detection methods, such as detection method utilizing.

The present invention also provides a method for detecting a target nucleic acid using a dispersed light source stably exciting a fluorescent dye in performing a melting curve analysis in a detection step.

According to an embodiment of the present invention, the surface of the quantum dot is modified with a hydroxyl group (-OH), a carboxyl group (-COOH), an amine group (-NH2), or a thiol group (-SH) to show water solubility, Keep it evenly distributed. This provides a method for detecting a target nucleic acid having a dispersed light source capable of uniformly fluorescing a fluorescent dye.

According to one embodiment of the present invention, amplification step of amplifying a polymerase chain reaction (PCR) amplification of the sample containing the target nucleic acid, the hybridization step is a reagent in which the detection probe and the quantum dots are evenly distributed After preparing, the detection reagent is added to the PCR amplified sample by the amplification step. Through this, since the problem of quantum dot degradation in the PCR amplification process does not occur, it is possible to provide a target nucleic acid detection method that guarantees detection accuracy.

According to an embodiment of the present invention, the detecting step may include an excitation light for exciting the quantum dots using an ultraviolet ray or a laser, and a dichroic mirror for reflecting the excitation light and transmitting the fluorescent light of a fluorescent dye. With help, the optical axis of the excitation light and the fluorescent light are matched to the optical axis. This provides a target nucleic acid detection method for accurately detecting a target nucleic acid even by a simple optical system.

According to the present invention as described above, the fluorescent dyes can be excited evenly by using a quantum dot evenly distributed in the hybridization sample reagent reaction solution as a new concept of distributed light source, and have characteristics of a quantum dot having a broad incident spectrum and a narrow band emission spectrum. It is less constrained by the light source to excite the quantum dots, and by emitting a fluorescent light to target a specific fluorescent dye, not only can accurately detect the target nucleic acid to be detected, but also accurate multiple detection, bioconjugation technology It is simple because it is not necessary.

In addition, the present invention can prevent the interference between the irradiated light and the fluorescent light to be detected to excite, thereby increasing the detection accuracy, simplify the optical system, wide selection of fluorescent materials, and various fluorescence The simultaneous use of the materials can facilitate multiple detections that simultaneously detect multiple target nucleic acids at once, and can also be readily applied to various target nucleic acid detection methods using detection probes.

1 is a schematic diagram showing an embodiment in which the present invention is applied to a method for detecting a target nucleic acid using a probe combining a fluorescent dye and a quencher.
Figure 2 is a schematic diagram showing an embodiment in which the present invention is applied to a method for detecting a target nucleic acid in which a probe incorporating a fluorescent dye is adsorbed to a material having a quencher function.
3 is a kit 100 for practicing the present invention. Kit 100 shows the flow chip 110 and cover 120 as a mechanical component, showing a perspective view, a top view and a cross-sectional view of the flow chip 110 and cover 120, the flow chip 110 The top perspective view (a) and the top view (b) are shown, and the cover 120 is shown by the bottom perspective view (d) and the bottom view (e), of the cover 120 which is in contact with the top surface side of the flow chip 110. Show the bottom side in detail.
4 is a block diagram of a target nucleic acid detection apparatus configured by adding an optical system 200 and a heater 300 to the kit 100 of FIG. 3.
5 and 6 are flowcharts of a target nucleic acid detection method performed by a nucleic acid detection apparatus including a kit 100.
7 is a melting curve graph obtained according to a mutation rate using KRAS G12D mutant DNA and wild type DNA extracted from a cell line.
8 is a melting curve graph obtained as a result of diagnosing a clinical sample provided by Incheon St. Mary's Hospital Human-Derived Material Resource Bank in order to obtain an experimental result applied to an actual clinical sample.

Referring to the embodiment shown in the schematic diagram of Figures 1 and 2, the method for detecting a target nucleic acid according to the present invention of the fluorescent dye (fluorescent dye) that varies in the fluorescent properties depending on whether the hybridization between the detection probe (detecting probe) and the target nucleic acid In detecting a target nucleic acid by detecting a change in state, a quantum dot (QD) that emits light in an absorption wavelength band of a fluorescent dye in all directions is used as a scattering light source.

Specifically, the hybridization step of hybridizing a detection probe having a complementary base sequence for the target nucleic acid with the target nucleic acid in the environment interspersed with the quantum dots to induce a state change of the fluorescent dye, the fluorescence properties vary depending on whether or not hybridization ; And a detection step of detecting the target nucleic acid by detecting the fluorescent light of the fluorescent dye which receives the emission light of the quantum dot serving as a scattered light source by exciting the scattered quantum dots; It includes.

Target nucleic acid means any kind of nucleic acid to be detected, and there are a variety of DNA, RNA, chromosomes, metabolites, and the like. Target nucleic acids may include mutant genes that are associated with, for example, the likelihood of disease development and the risk of development. A sample containing the target nucleic acid is taken to detect the target nucleic acid.

It is common for a sample to contain a small amount of the target nucleic acid, in the embodiment of the present invention prior to the PCR (polymerase chain reaction, polymerase chain reaction) amplification step to amplify the target nucleic acid in the sample before the hybridization step Then, the hybridization step is performed by adding a mixed solution containing the detection probe and the quantum dots evenly distributed to the sample containing the amplification result as a reagent for detection.

Through this, since the thermal effect in the PCR amplification process does not extend to the quantum dots and the detection probes, there is no problem of deterioration of the quantum dots or a problem due to the thermal characteristics difference between the PCR reagent and the detection probes.

On the other hand, PCR amplification is a well-known technique for amplifying a target nucleic acid by repeating a series of processes including denaturation, annealing, and elongation in a state in which a PCR amplification reagent is added. do.

<Detecting probe>

The detecting probe is any nucleic acid or nucleic acid analog that binds to and hybridizes to a target nucleic acid, and may be selected from the group consisting of oligonucleotides, peptide nucleic acids (PNA), and locked nucleic acids (LNA), respectively. Can be. Since a polymerase for PCR amplification generally has nuclease activity, which may cause damage to the detection probe, it is preferable to use a synthetic nucleic acid such as PNA that is stable against nucleases. In addition, since the detection probe serves to selectively detect the gene of the target nucleic acid, a detection probe for detecting a nucleic acid well known in the art to which the present invention pertains may be used.

In one embodiment of the present invention, a PNA probe is preferably used. Since the PNA probe is a synthetic DNA, specific binding with target DNA or target RNA, and stable to nuclease, is based on a probe melting curve. Melting curve analysis is possible.

The detection probe at this time may be to hybridize only to a specific position gene of the target nucleic acid. For example, it may be a detection probe that hybridizes with a mutant gene except for a wild-type gene.

Hybridization means that complementary single stranded nucleic acids, as is well known, complementarily bind to form a double stranded nucleic acid, ie a hybrid. If the target nucleic acid is double stranded, the target nucleic acid must be separated into single strands to cause a hybridization reaction with the detection probe, such as heat, alkali, formamide, urea or glycoxal treatment, enzymatic methods, binding Proteins and the like are known, but in the description of the embodiments of the present invention, it may be understood that they are achieved by heat treatment by the heater 300.

Fluorescent dyes are materials that fluoresce by absorbing and emitting light of a particular wavelength, and are used in the target nucleic acid detection arts to determine whether hybridization has occurred between target nucleic acids and detection probes.

Such fluorescent dyes are fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC, oregon green (Oregon green), Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, Cyanine-based dyes and thiadicarbocyanine dyes It may be one or more selected from the group consisting of, but is not limited thereto, and fluorescent materials known in the art may be used.

Since the fluorescent dye used herein has the property of absorbing and emitting light of a specific wavelength, the fluorescent dye must be absorbed to emit light of an appropriate wavelength, and according to the present invention, a quantum dot (QD) is formed of the fluorescent dye. It provides light of a suitable wavelength for emitting fluorescent light.

On the other hand, the state of the fluorescent dye changes depending on whether or not the hybridization between the detection probe and the target nucleic acid, that is, the characteristics of emitting fluorescent light or quenching the fluorescent light are different (for example, in FIGS. State change before and after the hybridization) is induced, and an embodiment of the present invention uses a quencher.

Quencher refers to a material that absorbs fluorescent light emitted from a fluorescent dye and reduces the fluorescence intensity. The amount of quenching varies depending on the distance from the fluorescent dye, and varies depending on the type, but within a predetermined distance. When it comes close to the fluorescent dye, it exhibits an extinction characteristic that can be identified by the optical system.

These quenchers are: Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa black FQ, Iowa Black RQ, IRDye QC-1 It may be one or more selected from the group, but is not limited thereto, and the range of reducing the fluorescence intensity varies depending on the type thereof, and may be selected and used in consideration of this.

According to an exemplary embodiment of the present invention illustrated in FIG. 1, by using a detection probe labeled with a fluorescent dye at one end and a quencher at the other end, before the detection probe hybridizes to the target nucleic acid, the fluorescent probe may be fluorescence according to the characteristics of the detection probe. Since the distance between the dye and the quencher is close, the fluorescent light of the fluorescent dye is quenched by the quencher even when the external light (the emission light of the quantum dot in the present invention) is absorbed. However, when the detection probe hybridizes to the target nucleic acid, it is unfolded and bound to the target nucleic acid, so that the distance between the fluorescent dye and the quencher becomes farther, and thus the extinction characteristic by the quencher cannot be exerted and the fluorescence by external light is prevented. Fluorescent light of the dye is emitted. Such a detection probe may be, for example, those disclosed in the prior art document 10-2015-0139096, but is not limited thereto.

According to the exemplary embodiment of the present invention illustrated in FIG. 2, a fluorescent dye is labeled at one end, and a detection probe that is adsorbed to a quencher is used. According to this, the fluorescent light of the fluorescent dye by external light is quenched, and when the detection probe hybridizes to the target nucleic acid, it is separated from the quencher and hybridized, so that the quenching property is not exhibited, and the fluorescent light of the fluorescent dye by external light It is released as it is not quenched. Such detection probes and quencher may be, for example, those disclosed in the prior art document 10-1496671, but is not limited thereto.

On the other hand, although not shown in the drawings and not mentioned in the prior art, as a method of inducing a change in the state of the fluorescent dye, the fluorescence properties vary depending on whether hybridization, as shown in FIG. 3 of Publication No. 10-2017-0058780 As described above, when the detection probe combined with the fluorescent dye and the quencher hybridizes with the target nucleic acid, the site where the fluorescent dye is bound is separated or adsorbed to a double strand hybrid formed by hybridization of the detection probe and the target nucleic acid. In the method of using a fluorescent material, etc., the present invention using the quantum dot as a scattered light source may be applied.

Preferably, the detection probe of FIG. 1 or FIG. 2 is used to determine the emission and quenching of the fluorescent light according to the change of state without any modification of the detection probe, and more preferably in consideration of the fact that the quantum dot is a scattered light source. The detection probe of FIG. 1 using a mixed solution containing a detection probe and a quantum dot will be used.

Hereinafter, for convenience of description, as illustrated in FIG. 1, a detection probe having a fluorescent dye and a quencher is used, but the present invention is not limited thereto, and other methods of inducing a state change of the fluorescent dye may be used. It is obvious that it can be applied.

<Quantum dot (QD)>

Quantum dots (QDs) are nano-sized particles, and light wavelengths vary according to particle size.

In the conventional nucleic acid detection technology, quantum dots have to be conjugated or linked to probes in order to utilize quantum dots based on fluorescence resonance energy transfer (FRET), but in the present invention, they are not based on FRET. It is not necessary.

That is, according to the present invention, the quantum dots are scattered independently and evenly in the detection probe mixture so that they do not bind to the detection probe, and even when the detection probe and the target nucleic acid hybridize, the quantum dots are evenly distributed together with the hybrid generated by the hybridization. do.

Accordingly, in order to independently scatter quantum dots evenly in an aqueous solution containing hybridization detection probes as well as in an aqueous solution in which hybridization is performed, embodiments of the present invention surface-treat the quantum dots so as to be water-soluble. . In this case, the quantum dot surface treatment may be surface modification with hydrophilic functional groups such as a hydroxy group (-OH), a carboxyl group (-COOH), an amine group (-NH 2), a thiol group (-SH), and the like.

Then, the quantum dots are excited to emit quantum dots to serve as a scattered light source, and as the light emitted from the quantum dots is irradiated in all directions, they are absorbed by the surrounding fluorescent dyes to emit fluorescent light unique to the fluorescent dyes.

The particle size of the quantum dot is selected according to the wavelength range of incidence of the fluorescent dye so as to emit light at a specific incident wavelength range of the fluorescent dye when excited. By matching the quantum dots with the fluorescent dyes as described above, the quantum dots can absorb the light of a specific wavelength band and emit light by targeting the fluorescent dyes emitting the unique fluorescent light to emit the fluorescent light of the target fluorescent dye. .

According to an embodiment of the present invention, two or more types of target nucleic acids are present in a sample, and thus they can be applied to a polymorphic target nucleic acid detection method using two or more types of detection probes capable of hybridizing to each type of target nucleic acid. In addition, the wavelength bands of absorbing (absorption or receiving) light as well as the emission wavelengths of the fluorescent probes are different depending on the types of detection probes. Input and use quantum dots. Accordingly, the two or more types of detection probes emit fluorescent light by receiving light from a quantum dot that emits light in the absorption wavelength band of the labeled fluorescent dye when hybridizing with a target nucleic acid complementary to itself, and thus, multiplex detection. Is possible.

Of course, even when quantum dots of different sizes are used, quantum dots of different sizes can be simultaneously excited by a single excitation light by the characteristics of the quantum dots having a broad absorption spectrum.

<Detection>

In this situation where the quantum dots are scattered around, when the detection probe and the target nucleic acid are hybridized, the fluorescent dye is spaced apart from the quencher to emit fluorescent light, and thus the hybridization can be detected by the fluorescent light. Finally, the target nucleic acid can be detected.

According to an embodiment of the present invention, the excitation light for exciting the quantum dots is ultraviolet or laser that does not interfere with the fluorescent light. Thus, the optical system can accurately detect the fluorescent light even though the optical axis of the excitation light and the optical axis of the fluorescent light for detection are matched through one objective lens.

According to the optical system (or optical system) of the exemplary embodiment illustrated in FIGS. 1 and 2, the optical axis of the excitation light irradiated onto the aqueous solution in which the detection probe and the target nucleic acid are hybridized in the interspersed state of quantum dots and the fluorescent light emitted in the aqueous solution The optical axes are matched, but a dichroic mirror is installed on the matched optical axis line at this time to help excitation light irradiation.

That is, the excitation light is reflected on the dichroic mirror and irradiated to the aqueous solution, and the fluorescent light of the fluorescent dye generated in the aqueous solution passes through the dichroic mirror and is detected by a fluorescent light detector (for example, a CCD sensor or PMT). do.

Here, the dichroic mirror passes all the light exceeding a specific wavelength and reflects light having a wavelength less than that, and reflects light having a wavelength band below the excitation light wavelength and exceeds the excitation light wavelength. All the light in the wavelength range is selected to pass.

On the other hand, according to the present invention, since the emitted light of the quantum dots excited by the excitation light and used as the scattered light source can also be detected by passing through the dichroic mirror, the dichroic mirror is additionally provided between the detection unit and the dichroic mirror. It is installed to reflect the emitted light of the quantum dot, and transmit the fluorescent light.

Through this, by detecting the emitted light of the quantum dot, it is possible to verify whether the quantum dot plays a role as a scattered light source, or to compare and analyze the change of the situation before and after the hybridization by comparing the light before and after the hybridization.

According to an embodiment of the present invention, a method for detecting a target nucleic acid by detecting fluorescent light of a fluorescent dye is performed by melting curve analysis.

Melting Curve Analysis is an analysis method that uses the characteristic that hybridization strength between detection probe and target nucleic acid becomes weaker as the temperature is increased, and is separated when the temperature is higher than a predetermined temperature, and the rate of change of the fluorescence light intensity is gradually increased as the temperature is increased. The fusion curve obtained by the pattern analysis (actually, fusion peak analysis) can be used to determine the presence or absence of the target nucleic acid or the variation in the target nucleic acid. In general, the temperature of the portion where the melting peak of the melting curve is formed is determined as the melting point or Tm (Melting Temperature) value.

Of course, when detecting the fluorescent light of the fluorescent dye and the emission light of the quantum dot, it is preferable to filter the light after filtering with a filter that selectively transmits the wavelength band of the light, respectively.

Hereinafter, a reagent sample reaction analysis kit, a target nucleic acid detection apparatus using the same, and a target nucleic acid detection method that are created for a specific embodiment of a target nucleic acid detection method according to the present invention will be described.

<Reagent Sample Response Analysis Kit>

Referring to FIG. 3, the kit 100 includes a flow chip 110 and a cover 120 as mechanical components. In the description of the kit 100, the sample is illustratively a sample containing a target nucleic acid, and the reagent is a sample for detecting a target nucleic acid, which shows that the target nucleic acid detection method can be used.

The flow chip 110 includes a first well 111 to hold a sample containing a target nucleic acid, a second well 112 to hold a reagent, and a first well when the reagent of the second well 112 overflows. It is a component in which a flow path 113 to be introduced into the 111 is formed.

Looking at the embodiment in detail, the first well 111 and the second well 112 to be formed in the mutually spaced apart position is projected downward in the form of a well, the top is open and gradually decreases toward the bottom, After forming a notch (notch) in the direction facing each other in the opening was formed a channel 113 in the form of a long groove to connect between the notches.

Here, the flow passage 113 is formed by the inclined surface 113a formed to incline downwardly the inner bottom surface of the portion connected to the first well 111 and the portion connected to the second well 112, and the second well 112. The reagent of) is smoothly introduced into the first well 111.

On the other hand, in the upper surface of the flow chip 110 to form a recessed portion 114 in which the periphery of the first, second well (111, 112), the periphery of the flow path 113, and the surface except the rim is formed below, The fitting portion 124 of the cover 120 to be fitted.

The cover 120 is a component that covers and seals the upper portion of the flow chip 110 so that the reagent sample is not leaked. The cover 120 protrudes to the bottom surface of the depression 114 according to the composition of the depression 114. It is in close contact with the upper periphery of the upper opening of the two wells (111, 112) and the upper opening of the flow path 113, and provided with a fitting portion 124 protruding downward to conform to the shape of the depression 114, The portion 124 may be fixed to the upper surface of the flow chip 110 by inserting the recess 114 into the upper surface of the flow chip 110. Accordingly, the reagent overflowing from the second well 112 may not leak elsewhere. Only through the first well 111.

In addition, the cover 120 includes a light-transmitting window 121 formed by dissecting and removing a portion covering the upper opening of the first well 111, and the second cover 112 so as to be contained in the second well 112 by pressing downward. The immersion member 122 formed in the site | part which covers the upper opening of the well 112 is provided.

Means for closing the light transmission window 121, and according to a specific embodiment, to cover the light transmission window 121 with a transparent sealing tape 125, it is to be adhesive or adhesive to prevent the separation.

According to an embodiment of the present invention, the cover 120 is made of a material that has rubber elastic properties such as, for example, a silicone material, which is elastically shrinkable, and the fitting part 124 is pressed against the recessed part 114. When the upper surface of the flow chip 110 is covered by the fitting method, the upper openings of the first well 111, the second well 112, and the flow path 113 may be covered and sealed.

The immersion member 122 includes a portion of the cover 120 that covers the upper opening of the second well 112, protrudes downward to be contained in the second well 112, and has a lower structure protruding downward. It consists of the shape holding part 122a which can hold | maintain the shape in the shape inside the lower side of the 2nd well 112, About the upper side structure, it extends to the lower part by pressing of the shape holding part 122a, or the shape holding part ( As the length required to move the bottom portion 122a) is corrugated, the shape maintaining portion 122a may be pressed down on the exposed portion of the upper side to be in close contact with the inner bottom of the second well 112. Thus, as much reagent as possible in the second well 112 may be introduced into the first well 111.

In the embodiment of the present invention, the rubber elastic material, said upper structure of the immersion member 122 extends from the lower side shape retaining portion (122a) and protrudes over the cover 120, the flat surface of the cover 120 Since the immersion member 122 has a bent shape so as to be continued, the immersion member 122 is pressed to the inner bottom of the second well 112 while being pushed down and spreading the curved upper side structure.

According to the embodiment, the shape maintaining part 122a of the immersion member 122 may be in close contact with the inner surface of the lower side of the second well 112 and may be filled in to form a small shape, resulting in less change in shape due to pressing.

The cover 120 includes backflow preventing means for preventing backflow from the first well 111 to the second well 112. According to a specific embodiment, the backflow preventing means blocks the flow path 113. It is composed of a backflow prevention protrusion 123 elastically deformed in the direction of the first well 111 by the hydraulic pressure of the reagent introduced by the pressing of the immersion member 122 to open the flow passage 113.

At this time, the backflow prevention protrusion 123 protrudes from the bottom surface of the inclined surface 113a formed in the flow passage 113 so as to block the flow passage 113 at the portion where the inclined surface 113a on the side of the first well 111 is formed. The farther away from the first well 111, the lower the height of the protrusion, the elastically deformed toward the first well 111 when the hydraulic pressure is applied by the reagent to open the flow passage 113. Of course, if the hydraulic pressure is not applied to the elastic restoring to block the flow path 113, even if the reverse pressure in the first well 111 is not opened, the flow path 113 is kept closed.

On the other hand, when the immersion member 122 is pressed by the material property of the cover 120, a portion covering the flow path 113 is pulled toward the immersion member 122, thereby opening the backflow preventing protrusion 123. Since the operating force is applied, the opening operation is smoothed.

The kit 100 configured as described above is easy to handle because the sample and the reagents are separately contained in the first and second wells 111 and 112 so that the setting for the reaction between the sample and the reagent is completed. If the pretreatment process is required, the pretreatment process can be performed independently, and the mixing process for the reaction between the sample and the reagent after the pretreatment process is easy, and the result of the reaction can be observed. Overall useful for kits for reagent sample reaction analysis.

On the other hand, it is preferable to use quantum dots of different sizes as components of the kit for multiplex detection.

In the kit 100 described above, the sample containing the target nucleic acid is placed in the first well 111, the reagent for detecting the target nucleic acid is placed in the second well 112, and the reagent is added to the sample, followed by the light transmission window. It can be used as a kit for detecting the target nucleic acid via (121). Here, the reagent is a reagent in which a detection probe having a nucleotide sequence complementary to a target nucleic acid is interspersed with the quantum dots as described above, and the detection probe and the target nucleic acid are dispersed in the environment where the quantum dots are scattered as the reagent is introduced into the sample. By hybridizing it can be induced to change the state of the fluorescent dye, the fluorescence properties vary depending on whether or not hybridization.

This will be described in detail in the following description of the target nucleic acid detection device.

<Target nucleic acid detection device>

Referring to FIG. 4, an optical system (or an optical system 200) and a heater 300 are added to the kit 100 to configure a target nucleic acid detection apparatus.

The heater 300 is a component capable of controlling the temperature by a controller (not shown), and is mounted on a portion where the first well 111 is inserted and seated in the seating stand 301 on which the kit 100 is placed. .

In this case, the kit 100 placed on the mounting table 301 puts a sample containing a target nucleic acid and a PCR amplification reagent into the first well 111, and the second well 112 contains a reagent for detecting the target nucleic acid. After being put into the state, it is sealed with the cover 120. According to an embodiment, the reagent is labeled with a detection probe having a nucleotide sequence complementary to the target nucleic acid, and a fluorescence dye having a quantum dot to be used as a scattered light source evenly, depending on whether the hybridization is hybridized.

On the other hand, the temperature control by the controller (not shown) is a temperature control for amplifying a polymerase chain reaction (PCR) by giving a temperature change to the sample of the first well 111 before adding the reagent, In the state in which the reagents of the two wells 112 are injected into the first wells 111, the temperature control is performed to give a temperature change to the first wells 111 to perform a melting curve analysis. Since the present invention is well known in the art, detailed description thereof will be omitted.

Here, the pressing means which can press the immersion member 122 of the cover 120 may be constituted by a hydraulic cylinder or the like, and the introduction of the reagent may be made by the controller.

The optical system 200 is disposed on the light transmission window 121 of the kit 100 mounted on the heater 300. Specifically, the objective lens unit 210, the first dichroic mirror 220, the second dichroic mirror 230, the fluorescent light detection filter 241 and the fluorescent light detector 240 on the light transmission window 121 The excitation light source 221 which is disposed in the vertical direction and detects fluorescent light and excites a quantum dot coincides with the optical axis of the fluorescent light with the help of the reflection of the first dichroic mirror 220. To be irradiated to the first well 111 through the objective portion 210.

Since the light emitted from the quantum dot is emitted from the first well 111 together with the fluorescent light of the fluorescent dye, the second dichroic mirror 230 reflects the emission light of the quantum dot and then fills it with a filter 232 to quantum dot It is detected by the light detector 231.

Here, the first and second dichroic mirror 220 is a dichroic mirror having a characteristic of passing all light exceeding a specific wavelength and reflecting light having a wavelength less than the first and second dichroic mirrors. The dichroic mirror 220 transmits the wavelength band including the fluorescent light and the quantum dot emission light, but reflects the wavelength band light of the excitation light, and the second dichroic mirror 230 passes the wavelength band light of the fluorescent light but the quantum dot emission light. Use appropriately selected wavelengths to reflect light. In addition, as in the case of multi-detection, it is to be noted that when a detector for a wavelength band to be detected is added, a dichroic mirror may be additionally installed or may be installed in different directions.

The filters 232 and 241 may be configured as filters that selectively pass only the wavelength band of light to be detected, respectively. In addition, in some cases, filters having different wavelength bands may be disposed on the rotating plate to selectively rotate to detect light of various wavelength bands.

The fluorescent light detector 240 and the quantum dot light detector 232 may be configured as, for example, a CCD sensor or a photo multiplier tube (PMT).

A specific embodiment of the target nucleic acid detection method using the target nucleic acid detection apparatus configured as described above will be described.

Specific Embodiments

An embodiment in which the KRAS G12D mutation is detected by applying the present invention will be described with reference to FIGS. 5 to 8.

First, the preparation step (S10) was performed.

In this step, 20 μl of the PCR amplification reagent for amplifying the KRAS G12D mutation and 5 μl of the sample containing the KRAS G12D mutation are sequentially added to the first well 111 (S11, S12), and the reagent 36.8 containing the detection probe is used. Dilutions of the μL and the quantum dots that are components of the kit were sequentially placed in the second well 112 (S13, S14).

In a specific example, two kinds of PNA detection probes having different base sequences were used to hybridize with a target mutation to simultaneously detect G12D and G12V among the KRAS mutation types.

Among the detection probes, a detection probe for detecting G12D was labeled with a HEX fluorescent dye, a detection probe for detecting G12V was labeled with a Texas red fluorescent dye, and the quencher was made of dacyl. Such a detection probe was supposed to be suitable for the present invention by utilizing the PNA probe shown in Patent Application No. 10-2015-0054633 filed by the applicant of the present invention.

Then, two kinds of quantum dots consisting of 530 QD for exciting the HEX fluorescent dye and 590 QD for exciting the Texas red fluorescent dye are used. Each quantum dot was diluted 1/100 with distilled water, and 6.6 μl was added to the second well 112.

Next, the assembly step (S20) was performed.

In this step, the flow chip 110 was covered with the cover 120 (S21), and the transparent window 121 was blocked with the sealing tape 125 (S22) to seal the flow chip 110. That is, the cover 120 is fixed to the flow chip 111 by inserting the fitting portion 124 into the recess 114 to open the upper portions of the first and second wells 111 and 112 and the flow passage 113. The opening is covered with a cover 120 and sealed. At this time, the flow path 113 is blocked by the backflow preventing protrusion 123, and the immersion member 112 is contained in a part of the second well 112. In addition, you may fix the cover 120 which previously blocked the light transmission window 121 with the sealing tape 125 to the floating chip 110. FIG.

Next, the PCR step (S30) was performed.

In this step, the kit 100 is seated on the seating table 301, and the cycle of heating and cooling the first well 111 with the heater 300 is repeated to amplify the target nucleic acid (here, G12D). Specific PCR amplification cycle, for example, applied to the embodiment applied to the detection of KRAS mutant gene in the patent application No. 10-2015-0054633 filed by the applicant of the present invention.

As described above, since PCR amplifies the detection reagent of the second well 112 containing the quantum dots in the first well 111, the degradation of the quantum dots due to PCR amplification does not occur.

In addition, since the first well 111 is sealed by the cover 120 including the sealing tape 125 and the flow path 113 which is a passage with the second well 112 is also blocked by the backflow preventing protrusion 123. In addition, it prevents the loss of the sample by evaporation during PCR amplification.

Next, the hybridization step (S40) was performed.

In this step, the immersion member 122 provided on the cover 120 was pressed until the shape maintaining part 122a reached the inner bottom of the second well 112. That is, the detection reagent of the second well 112 is pressured to flow toward the first well 111 through the flow passage 113, and elastically deforms the backflow preventing protrusion 123 to open the flow passage 113. Was introduced into the first well 111.

As a result, hybridization proceeded between the detection probe and the target nucleic acid (ie, G12D) in the mixed solution in which the quantum dots were scattered in the first well 111.

Next, the detection step (S50) was performed.

In this step, the heater 300 is controlled to give a temperature change to the first well 111, and detected by the optical system 200 to perform melting curve analysis. Even if the sealing tape 125 is removed, if the problem such as evaporation does not significantly affect the melting curve analysis process, the detection step S50 may be performed while the sealing tape 125 is removed.

At this time, the optical system 200 detects fluorescent light and quantum dot emission light detected while irradiating ultraviolet light or laser into the first well 111. In addition, when obtaining a melting curve, it is preferable to increase the precision of the melting peak by increasing the temperature at 0.5 ° C intervals and measuring fluorescent light.

Although two kinds of quantum dots are used, the characteristics of the quantum dots having a broad incidence spectrum can excite all the quantum dots to serve as distributed light sources. In addition, since the two quantum dots emit light of a narrow band corresponding to their respective sizes, each of the two quantum dots excites fluorescent dyes targeted to the emission light to emit intrinsic fluorescent light.

In a specific embodiment, the quantum dots were excited with ultraviolet rays having a wavelength of 405 nm.

7 is a graph showing the results of the ratio change of KRAS G12D mutant DNA and wild type DNA, wherein the ratio of KRAS G12D mutant DNA is 0%, 0.5%, 1%, 10%, and 100%. In each case, the result obtained by repeatedly performing the target nucleic acid detection method shown in FIGS. 5 and 6 is shown. According to this, since the Tm of the melt peak converges within the range of 45 ° C. to 52 ° C., this temperature range was selected as a criterion for the KRAS G12D mutation.

On the other hand, the fluorescent light detected by the detection probe and 590 QD for the detection of KRAS G12V was insignificant or less.

Application to actual clinical samples verified the target nucleic acid detection method according to the invention.

To this end, receiving a clinical sample from the Incheon St. Mary's Hospital human-derived material resource bank and applying the target nucleic acid detection method according to the present invention, the graph of Figure 8 according to the melting curve analysis (Melting Curve Analysis) was obtained.

Referring to the graph of FIG. 8, it is determined that a KRAS G12D mutation is detected because a curve having a Tm of a melt peak of 48 ° C. exists within the melting curve and falls within the above criterion.

On the other hand, although the melting curve graph detected by the detection probe and 590 QD input for the detection of the KRAS G12V mutation is shown, the KRAS G12V mutation exists because the Tm of the melt peak does not fall within the determination range of G12V. It can be determined not to. In addition, even when the Tm of the melt peak falls within the determination range, it is possible to determine that the KRAS G12V mutation does not exist by designating the minimum height of the melt peak. For example, the noise criterion is obtained by obtaining a melting peak that appears when detecting a sample without a KRAS G12V mutation and determining a threshold value of the melting peak to be used as a criterion for the presence of a mutation according to the amount of 590 QD. Can also be used.

Specific embodiments have been shown and described in order to illustrate the technical idea of the present invention, but the present invention is not limited to the same configuration and operation as the specific embodiments as described above, and various modifications are made without departing from the scope of the present invention. It can be carried out in. Accordingly, such modifications should also be regarded as belonging to the scope of the present invention, and the scope of the present invention shall be determined by the claims below.

100: Kit
110: flow chip 111: first well 112: second well
113: flow path 113a: slope 114: depression
120 cover 121 floodlight 122 immersion member
122a: shape retaining portion 123: backflow preventing protrusion 124: fitting portion
125: sealing tape
200: optical system
210: objective lens unit
220: first dichroic mirror 221: excitation light source
230: second dichroic mirror 231: quantum dot light detector 232: filter
240: fluorescent light detector 241: filter
300: heater 301: seating table

Claims (10)

In an environment in which quantum dots are independently and evenly dispersed, a detection probe having a nucleotide sequence complementary to a target nucleic acid is hybridized with the target nucleic acid to induce a state change of a fluorescent dye whose fluorescence properties vary depending on whether the hybridization is performed. The fluorescent dye is labeled at one end and a quencher is coupled to the other end, and the fluorescent dye absorbs light of a specific wavelength band and emits a unique fluorescent light, and the quantum dot absorbs a specific wavelength band of the fluorescent dye. A hybridization step having a size capable of emitting light;
A detection step of detecting a target nucleic acid by acting as a scattering light source by exciting scattered quantum dots and detecting fluorescent light of a fluorescent dye receiving the emission light of the quantum dots;
Target nucleic acid detection method comprising a. The method of claim 1,
Target nucleic acid detection method for performing the amplification step of amplifying the target nucleic acid polymerase chain reaction (PCR) prior to the hybridization step. The method of claim 2,
In the hybridization step, a target nucleic acid detection method for preparing a mixed solution in which a detection probe and a quantum dot are evenly dispersed, and putting the mixed solution into a sample containing the target nucleic acid amplified by the amplification step. The method of claim 1,
The detecting step excites the quantum dots with ultraviolet rays or lasers, but with the help of a dichroic mirror that reflects the excitation light and transmits the fluorescent light of the fluorescent dye, a target that matches the optical axis of the excitation light and the optical axis of the fluorescent light. Nucleic Acid Detection Method. The method of claim 1,
The quantum dot is a target nucleic acid detection method surface-treated to exhibit water solubility. delete delete delete The method of claim 1,
A target nucleic acid detection method in which a detection probe is labeled with a fluorescent dye having a different wavelength band of absorbing and emitting fluorescent light for detecting a polymorphic target nucleic acid, and a quantum dot having a different size corresponding to the absorption wavelength band of each fluorescent dye. The method of claim 1,
The detecting step is a target nucleic acid detection method consisting of Melting Curve Analysis (Melting Curve Analysis).

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