Rapid detection method for target genes using plasmon photothermal reaction

Abstract

To solve the problem of the related art in which it was not easy to visually detect a target gene, and make it possible to detect the target gene with excellent sensitivity and specificity in a short period of time.SOLUTION: The present invention relates to a plasmon-based target gene detection method. The present invention involves performing an RT-PCR reaction using plasmonic nanoparticles, then adding SYBR Green I and 3,3',5,5'-tetramethylbenzidine, and visually detecting the presence of a target gene by irradiation with an LED.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to real-time polymerase chain reaction of a target gene using plasmon nanoparticles. The present invention applies plasmon nanoparticles to real-time polymerase chain reaction, not only can detect the presence of a target gene by a colorimetric reaction, but also improve the detection specificity and analysis sensitivity of the target gene. When using the polymerase chain reaction according to the present invention, simple and rapid detection of a target gene is possible, and a diagnostic analysis method that can be distributed on-site can be provided.

[0002] The present invention is derived from research conducted as part of the Individual Basic Research Project of the Ministry of Science and ICT, Korea [Project Specific Number: 1711186785, Project Number: NRF-2022R1F1A1070162, Research Project Name: Development of a Point-of-Care Ultra-High-Speed Real-Time PCR Platform Based on Multifunctional Plasmonic Photothermal Magnetic Nanoparticles], the Construction Project of the Ministry of Education Based on Academic Research in Engineering [Project Specific Number: 1345362911, Project Number: NRF-2021R1A6A1A03039503, Research Project Name: Korea Institute for the Utilization of Indigenous Animal Resources], and the Regional Innovation Megaproject of the Ministry of Science and ICT [Project Specific Number: 20230007, Project Number: 2023-DD-UP-0007, Research Project Name: Development of Source Technologies for the Metaplatform of Marine Bio-Strategic Materials].

Background Art

[0003] The PCR (Polymerase Chain Reaction) method is a test method used in almost all processes of manipulating and experimenting with genetic materials, and is a method of amplifying a specific target genetic material desired to be detected. By polymerase chain reaction, a large amount of genetic material with the same base sequence can be amplified from a small amount of genetic material, so it is used to amplify human DNA and diagnose various types of genetic diseases. It is also applied to the DNA of bacteria, viruses, and fungi and used for the diagnosis of infectious diseases.

[0004] The dengue virus is a single-strand positive-sense RNA virus belonging to the genus Flavivirus in the family Flaviviridae. Dengue fever and dengue hemorrhagic fever are acute febrile illnesses that develop when a human is bitten by an Egyptian forest mosquito (Aedes aegypti) or white thread-tooth mosquito (Aedes albopictus) infected with the dengue virus.

[0005] Over the past few decades, dengue fever has spread rapidly within tropical and subtropical countries, endangering nearly one-third of the world's population. In recent years, it has become one of the most critical public health issues in many resource-constrained countries, threatening human lives and placing significant pressure on healthcare systems from various perspectives.

[0006] More importantly, the combined effects of the COVID-19 pandemic and dengue fever outbreaks could have potentially devastating consequences for patients in endemic areas with various serotypes of DENV. Effective vaccines and early diagnosis of dengue virus (DENV) infection are crucial for improving clinical outcomes and preventing disease transmission. Simple, cost-effective, and sensitive diagnostic tests for field use are essential for disease control, especially in remote areas where access to healthcare providers and facilities is restricted. However, diagnostic tools with such capabilities have yet to be developed.

[0007] Based on this, the present inventors have developed a real-time gene detection test method using plasmon nanoparticles, which, unlike conventional methods, can detect target genes by color change and is rapid. We have confirmed that it can be detected with high sensitivity and specificity, and have completed the present invention. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The present invention aims to provide a method for conducting a plasmon photothermal (PPT)-reverse transcription-colorimetric polymerase chain reaction (RTcPCR) for rapid molecular diagnosis of viral infection.

[0009] The present invention also aims to provide a plasmon-based reverse transcription polymerase chain reaction analysis composition and a target gene detection kit using the same. [Means for solving the problem]

[0010] The present invention will be described in detail below. The advantages and features of the present invention will become clear when referred to in the embodiments described below that achieve them. However, the present invention is not limited to the embodiments presented below and can be embodied in a variety of different forms, and these embodiments are provided merely to complete the presentation of the present invention and to fully inform a person ordinary skill in the art to which the present invention pertains, and the present invention is defined only by the scope of the claims. The same reference numerals throughout the specification refer to the same components.

[0011] Unless otherwise specified, all terms used herein (including technical and scientific terms) are to be used in a sense that can be commonly understood by a person of ordinary skill in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries should not be interpreted ideally or excessively unless explicitly defined otherwise. Terms used herein are for illustrative purposes only and are not intended to limit the present invention. In this specification, the singular form includes the plural form unless otherwise specified in the text.

[0012] To achieve the above objective, the present invention provides a method for conducting a plasmon-based reverse transcription polymerase chain reaction for target gene detection, comprising the steps of: i) mixing a target gene, a primer set, and plasmon nanoparticles, and then proceeding with a reverse transcription polymerase chain reaction; and ii) mixing 3,3',5,5'-tetramethylbenzidine with the product obtained in step i). SYBR Green I can be added in step i) or ii) of the plasmon-based reverse transcription polymerase chain reaction according to the present invention. That is, SYBR Green I can be added before or after the polymerase chain reaction.

[0013] The polymerase chain reaction of the present invention can induce a photothermal reaction of nanoparticles by adding plasmon nanoparticles, 3,3',5,5'-tetramethylbenzidine (hereinafter also referred to as TMB), and SYBR Green I (hereinafter also referred to as SGI). In order to induce the photothermal reaction, the polymerase chain reaction of the present invention may further include step iii) irradiation with a blue LED after step ii). If a target gene is present in the reaction product, the reaction product will turn blue when irradiated with the blue LED. Therefore, if the product from step iii) is blue, it can be determined that a target gene is present.

[0014] In the present invention, the plasmon nanoparticles are metal particles and may preferably consist of a core-shell structure comprising a core containing iron oxide (FeO) and a shell containing gold (Au) attached to the surface of the core. Polyethylene glycol may be bonded to the gold particles of the present invention.

[0015] The target gene to be detected by the polymerase chain reaction of the present invention may include any gene that can be amplified by polymerase chain reaction, but is preferably a viral gene.

[0016] The viruses of the present invention include, but are not limited to, dengue virus, piconavirus, flavivirus, Zika virus, Poissant virus, chikungunya virus, enterovirus, respiratory syncytial virus (RSV), Rift Valley fever, influenza virus, Takaribe virus, Mayarovirus, West Nile virus, yellow fever virus, and coronavirus.

[0017] The present invention also provides a plasmon-based reverse transcription polymerase chain reaction analysis composition comprising plasmon nanoparticles, SYBR Green I, and 3,3',5,5'-tetramethylbenzidine.

[0018] In the present invention, it is preferable that SYBR Green I is added before or after the polymerase chain reaction, and that 3,3',5,5'-tetramethylbenzidine is added after the polymerase chain reaction.

[0019] The present invention also provides a target gene detection kit comprising the plasmon-based reverse transcription polymerase chain reaction analysis composition. The detection kit of the present invention produces a blue color when the target gene is present, allowing for the detection of the target gene with the naked eye. [Effects of the Invention]

[0020] When using the polymerase chain reaction (plasmon photothermal reaction-based RTcPCR (PPT-RTcPCR)) of the present invention, target genes can be rapidly detected within one hour.

[0021] Furthermore, the polymerase chain reaction of the present invention allows for the detection of target genes with high specificity and sensitivity, and reduces the rate of false positives or false negatives.

[0022] Furthermore, the present invention has the advantage that a color reaction occurs when the target gene is present, and the results can be confirmed with the naked eye.

Brief Description of the Drawings

[0023] [Figure 1] FIG. 1 is a schematic diagram showing the overall flow of a method for detecting a target gene using a plasmon photothermal reaction-based RTcPCR (PPT-RTcPCR) platform according to the present invention. [Figure 2A] FIG. 2A shows the workflow of a plasmon photothermal reaction-based RTcPCR (PPT-RTcPCR) platform. FIG. 2A is a progress flowchart of a PPT-RTcPCR platform that combines PPT-based RT-PCR and colorimetric sensing. [Figure 2B] FIG. 2B shows the workflow of a plasmon photothermal reaction-based RTcPCR (PPT-RTcPCR) platform. FIG. 2B shows a colorimetric reading method using plasmon photothermal reaction-based RTcPCR (PPT-RTcPCR). Referring to FIG. 2B, in a negative sample where the target gene is absent, amplification does not occur, and if a dsDNA-SGI complex is not formed, TMB cannot be oxidized, and almost no hue change occurs. On the other hand, in a positive sample where the target gene is present, the amplicon increases geometrically. In this case, TMB can be further oxidized by the dsDNA-SGI complex, causing a hue change that is noticeable from colorless to blue. [Figure 3A] FIG. 3A shows the results of characterizing and photothermal performance of plasmonic nanoparticles (PMN) fabricated according to the present invention. FIG. 3A is a TEM image of plasmonic nanoparticles (PMN) having a core-shell structure. [Figure 3B] FIG. 3B shows the results of characterizing and photothermal performance of plasmonic nanoparticles (PMN) fabricated according to the present invention. FIG. 3B is a high-angle annular dark-field (HAADF) image and an elemental mapping image of plasmonic nanoparticles (PMN). [Figure 3C]Figure 3C shows the characterization and photothermal performance of plasmon nanoparticles (PMNs) fabricated according to the present invention. Figure 3C shows the ultraviolet-visible (UV-Vis) spectral values ​​of the plasmon nanoparticles (PMNs). [Figure 3D] Figure 3D shows the characterization and photothermal performance results of plasmon nanoparticles (PMN) fabricated according to the present invention. Figure 3D shows the magnetization curve of the plasmon nanoparticles (PMN). [Figure 3E] Figure 3E shows the characterization and photothermal performance of plasmon nanoparticles (PMN) fabricated according to the present invention. Figure 3E shows the temperature profile of the solution in the presence of plasmon nanoparticles (PMN) during illumination treatment. [Figure 3F] Figure 3F shows the results of characterization and photothermal performance of plasmon nanoparticles (PMNs) fabricated according to the present invention. Figure 3F is a graph showing the effect of plasmon nanoparticle (PMN) concentration on heating and cooling ramp rates. The optimal ramp rate was observed with 16 OD plasmon nanoparticles (PMNs). [Figure 3G] Figure 3G shows the results of characterization and photothermal performance of plasmon nanoparticles (PMNs) fabricated according to the present invention. Figure 3G shows a typical temperature profile for PPT-based RT-PCR. Thermal circulation began at 50°C (5 mins), followed by 40 repetitions of 90°C (0 sec) and 60°C (8 sec). [Figure 3H] Figure 3H shows the results of characterization and photothermal performance of plasmon nanoparticles (PMNs) fabricated according to the present invention. Figure 3H shows the expanded temperature profile for isothermal reverse transfer. [Figure 3I] Figure 3I shows the characterization and photothermal performance of plasmon nanoparticles (PMNs) fabricated according to the present invention. Figure 3I also shows the enlarged temperature profile for PCR amplification. [Figure 4A]Figure 4A shows the results of evaluating the analytical sensitivity and specificity of the PPT-RTcPCR platform according to the present invention. Figure 4A compares quantitative curves between PPT-RTcPCR and RT-qPCR using RNA extracted from IVT DENV RNA. In Figure 4A, the RT-qPCR results are shown as Ct values ​​inversely proportional to the log of DENV2 RNA copy numbers / μL. [Figure 4B] Figure 4B shows the results of evaluating the analytical sensitivity and specificity of the PPT-RTcPCR platform according to the present invention. Figure 4B compares quantitative curves between PPT-RTcPCR and RT-qPCR using RNA extracted from DENV2 spike serum samples. In Figure 4B, the RT-qPCR results are shown as Ct values ​​inversely proportional to the log of DENV2 RNA copy numbers / μL. [Figure 4C] Figure 4C shows the results of the evaluation of the analytical sensitivity and specificity of the PPT-RTcPCR platform according to the present invention. Figure 4C also shows the results of the absorbance analysis for DENV2 and other nonspecific targets. In Figure 4C, the RT-qPCR results are shown as Ct values ​​inversely proportional to the log of DENV2 RNA copy numbers / μL. [Figure 5A] Figure 5A shows the results of clinical validation of the PPT-RTcPCR platform. Figure 5A shows PPT-RTcPCR result heatmaps for 131 positive samples and 27 negative samples. [Figure 5B] Figure 5B shows the results of clinical validation of the PPT-RTcPCR platform. Figure 5B is an image of the absorbance analysis for clinical trial results. In Figure 5B, samples were classified using a predefined critical value of 0.53 and an ROC optimal critical value of 0.51. [Figure 5C] Figure 5C shows the results of the clinical validation of the PPT-RTcPCR platform. Figure 5C also shows the ROC curve analysis results of the clinical outcomes. In Figure 5C, the AUC is 0.99. [Modes for carrying out the invention]

[0024] The following are various embodiments to aid in understanding the invention. These embodiments are provided solely to facilitate understanding of the invention, and the scope of protection of the invention is not limited to these embodiments.

[0025] This invention presents a method for carrying out a polymerase chain reaction using plasmon nanoparticles.

[0026] The polymerase chain reaction of the present invention is a plasmon photothermal reaction-based (reverse transcription, colorimetric) polymerase chain reaction, and will be referred to as PPT-RTcPCR below.

[0027] The PPT-RTcPCR reaction of the present invention may include the steps of: i) mixing the target gene, primer set, and plasmon nanoparticles, followed by proceeding with a reverse transcription polymerase chain reaction; and ii) mixing 3,3',5,5'-tetramethylbenzidine with the product obtained in step i). In this case, SYBR Green I may be added in step i) or ii).

[0028] In this invention, "PPT" has the same meaning as "plasmonic photothermal." In this invention, "PPT-" refers to various reactions based on the photothermal reaction of plasmonic nanoparticles.

[0029] In this invention, "plasmon nanoparticles (PMN)" refers to nano-sized particles that exhibit plasmon resonance.

[0030] The plasmon nanoparticles of the present invention can utilize metals that exhibit a plasmon resonance effect. Specific examples include gold (Au) nanoparticles, silver (Ag) nanoparticles, or a combination of both gold (Au) and silver (Ag) nanoparticles.

[0031] According to a preferred embodiment of the present invention, the plasmon nanoparticles have a core-shell structure comprising a core containing iron oxide (FeO) and a shell containing gold (Au) attached to the surface of the core. The plasmon nanoparticles may also be configured such that polyethylene glycol is bonded to the gold particles, and polyethylene glycol is exposed on the outside of the core-shell structure.

[0032] In this invention, the term "target gene" means the nucleic acid sequence to be detected, which is annealed or hybridized with the probe under hybridization conditions. "Target gene" is used interchangeably with "target nucleic acid." In this invention, the target nucleic acid may be a gene derived from an animal, plant, bacterium, virus, fungus, etc., or a mutant gene associated with a genetic disease. In this invention, the target gene means a nucleic acid fragment such as DNA or RNA, which may be single-stranded or double-stranded, and may be DNA or RNA capable of exhibiting sense or antisense strands. dsDNA, ssDNA, mixed ssDNA, mixed dsDNA, ssDNa, dsDNA, mRNA, rRNA, tRNA, snRNA, or miRNA are available, but the target gene in this invention is most preferably in RNA form.

[0033] In the present invention, the "target gene" is preferably a viral gene. The viral genes to be detected in the present invention are not limited in type, but include dengue virus types 1-4 (DENV1-4), piconavirus (CVB3), flavivirus, Zika virus, Poissant virus, chikungunya virus, enterovirus, respiratory syncytial virus (RSV), Rift Valley fever, influenza virus, Takaribe virus, Mayarovirus, West Nile virus, yellow fever virus, and coronavirus.

[0034] In the present invention, "detection" means detecting a target gene through photothermal-based PCR of the plasmon nanoparticles of the present invention.

[0035] The product of the PPT-RTcPCR reaction according to the present invention can be irradiated with a blue LED. When irradiated with a blue LED, if the target gene is present in the product, a blue color will appear in the reaction product. Through this colorimetric reaction, if a blue color appears in the reactant, the presence of the target gene can be confirmed and the result can be determined to be "positive".

[0036] In this invention, the terms "complementary binding site" or "complementary binding site" refer to sites that can form complementary base pairs between nucleotide sequences.

[0037] In the present invention, "primer" means a short nucleic acid sequence having a short free 3' hydroxyl group that can form a base pair with a complementary nucleic acid template and serve as a starting point for strand copying of the nucleic acid template. The primer can initiate DNA synthesis in the presence of reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphodes at a suitable buffer solution and temperature.

[0038] The design of the aforementioned primers involves various constraints, such as the ratio of A, G, C, and T content in the primers, prevention of primer conjugate (dimer) formation, and prohibition of repeating the same base sequence more than three times. In addition, under single-PCR reaction conditions, the amount of template DNA, primer concentration, dNTP concentration, and Mg are also factors. 2+ The concentration, reaction temperature, reaction time, and other conditions must be appropriate.

[0039] The aforementioned primers can be modified to incorporate further features that do not alter the basic properties. That is, nucleic acid sequences can be modified using many methods known in the field. Examples of such modifications include methylation, capping, substitution of one or more nucleotides with their respective congeners, and modification of nucleotides to uncharged conjugates such as phosphonates, phosphotriesters, phosphoramidates, or carbamates, or charged conjugates such as phosphorothioates or phosphorodithioates. Furthermore, nucleic acids can have one or more further covalently bonded residues, such as nucleases, toxins, antibodies, signal peptides, proteins such as poly-L-lysine, insertors such as acridine or psoralen, chelating agents such as metals, radiometals, or iron oxide metals, and alkylating agents. [Examples]

[0040] The following examples are provided to aid in understanding the invention and do not limit the scope of protection of the invention.

[0041] [Example 1] Materials and experimental methods 1-1. Preparation of RNA transcribed in vitro The analytical performance of dengue virus (DENV)-specific PPT-RTcPCR analysis was first evaluated using in vitro transcription (IVT) RNA. A partial sequence of the E gene from DENV-2 NGC strains (nt 1453 or 1550; GenBank accession AF038403) was synthesized and cloned into a pGEM-3Z vector downstream of the T7 promoter sequence. The resulting plasmid, pGEM-3Z-DENV, was linearized by SalI restriction digestion and then used as a template for in vitro transcription using the mMESSAGE mMACHINE® T7 transcription kit (Invitrogen, USA). After removing residual RNA using TURBO Dnase (Invitrogen, USA), the disposable IVT RNA sample was stored at -80°C. Subsequently, the IVT RNA (1-10) was analyzed. 6The analytical sensitivity of PPT-RTcPCR was determined using 10-fold serial dilutions of (10 copies / μL). IVT RNA concentrations were calculated using known methods.

[0042] 1-2. Nucleic acid extraction For the analysis sensitivity analysis, 1.15 x 10 6 Viral RNA was extracted from TCF containing DENV2 at a focal unit (FFU) / mL and serially diluted 10-fold with nuclease-free water (10 1 ~10 8 ) went through.

[0043] Furthermore, for analytical specificity analysis, viral RNA was extracted from TCF samples containing DENV1, DENV4, ZIKV, CVB3, or FIPV. Stored human plasma samples were used for nucleic acid extraction without further dilution.

[0044] All samples are from the KingFisher Flex System (Thermo Extraction was performed using the MagMAX®-96 Viral RNA Isolation Kit (Applied Biosystems, USA) from Fisher, USA. After extraction, the nucleic acids were eluted in 90 μL of nuclease-free water, and all nucleic acids were stored at -80°C for further use.

[0045] 1-3. DENV-specific PPT-RTcPCR and optimization DENV-specific PPT-RTcPCR was developed with some modifications from previously known methods. The target DENV2 RNA was first amplified via PPT-based RT-PCR in the presence of plasmon magnetic nanoparticles (PMNs).

[0046] The reaction mixture consisted of 5 μL of 2x reaction mix, 0.2 μL of SuperScript® III RT / Platinum® Taq Mix, 0.1 μL of 50 μM forward (5'-CAG GCT ATG GCA CYG TCA CGA T-3') and reverse (5'-CCA TYT GCA GCA RCA CCA TCT C-3') primers, 2 μL of plasmon nanoparticles (PMN) with an optical density (OD) of 80, 1 μL of viral nucleic acid, 1.6 μL of nuclease-free water, and 20 μL of mineral oil. The thermal circulation protocol began at 50°C for 5 minutes, followed by 40 cycles between 90°C (0 sec) and 60°C (8 sec). Following PPT-based RT-PCR, 10 μL of a colorimetric solution containing 2-(N-morpholino)ethanesulfonic acid (MES) buffer (8 μL, 0.1 M), 40 mM TMB (3,3',5,5'-tetramethylbenzidine, 1 μL), and 80x SGI (SYBR Green I, 1 μL) was added to the reaction solution. Particles were collected with a magnet before irradiation with a blue light LED (300 mA, 14 V) for 2 minutes, utilizing the magnetic properties of PMN. Due to the photocatalytic activity of the dsDNA-SGI complex, TMB was oxidized under blue light LED excitation after adjusting the pH of the solution to approximately 5. The absorbance of the resulting mixture was measured using a NanoDrop® OneC Microvolume UV-Vis Spectrophotometer (Thermo Measured using Scientific® (trademark).

[0047] Reference real-time DENV2 RT-qPCR was performed using a modified known method. Briefly, the reaction mixture contained 5 μL of 2x reaction mix, 0.2 μL of SuperScript® III RT / Platinum® Taq Mix, 0.1 μL of 50 μM forward and reverse primers, 0.18 μL of 10 μM probe (5'- FAM-CTC YCC RAG AAC GGG CCT CGA CTT CAA-BHQ1-3'), 1 μL of viral nucleic acid, and 3.42 μL of nuclease-free water. RT-qPCR was performed using a CFX Opus 96 Real-Time PCR system (Bio-Rad) with the following protocol: 30 minutes at 50°C, 2 minutes at 95°C, then 40 cycles at 95°C for 15 seconds each, and 1 minute at 60°C. For reference real-time RT-PCR, samples with a Ct value greater than 37 were considered negative because the amplification results were difficult to confirm.

[0048] [Example 2] result 2-1. PPTRTcPCR Platform Procedure In this invention, the overall procedure for DENV nucleic acid detection using the PPTRTcPCR platform, from sample collection to target detection, is shown in Figure 1.

[0049] First, viral RNA extracted from plasma samples of patients suspected of having dengue fever was applied to a DENV-specific PPT-RTcPCR reaction. In this system, a nano-sized heater was used for uniform heating of the PMN. When an infrared (IR)-LED is turned on, the reaction temperature can be easily and precisely controlled by adjusting the light intensity. The heating protocol begins with an isothermal process of reverse transcription of RNA into complementary DNA (cDNA), followed by a two-step thermal cycle for cDNA denaturation and specific target expansion.

[0050] Next, a colorimetric solution containing MES buffer, SGI, and TMB is added to visually detect the amplicon using a TMB oxidation-based colorimetric strategy. The amplified dsDNA is the reactant for photocatalysis, which is induced only when SGI is inserted into the dsDNA. Upon irradiation with blue LED light, singlet oxygen is produced by energy transfer from SGI to dissolved oxygen in the solution, which subsequently induces TMB oxidation. The reaction product changes from colorless to blue, indicating amplicon formation. In the absence of viral RNA, there is no specific RT-PCR amplification, no dsDNA-SGI complex is formed, and therefore TMB is not oxidized, keeping the solution colorless.

[0051] Through such a simple design, this device can be developed as a POC (Point of Computing) diagnostic system.

[0052] 2-2. Characterization of the PPT-RTcPCR platform The aforementioned PMN was used in nanoscale heaters because it induces efficient PPT-based heat circulation and convenient magnetic separation. The PMN particles have a magnetic iron oxide core surrounded by a plasmon Au shell (skin) (Figure 3A). The iron oxide core, produced by a solvothermal reaction, was first functionalized at the amine-terminated surface with an organosilane. Subsequently, small AuNPs adhered to the core surface via electrostatic assembly, forming the Au shell.

[0053] To enhance the stability of PMN and minimize nonspecific interactions during amplification, PMN was final-modified with methoxy-polyethylene glycol (mPEG)-thiol. Elemental mapping images show that the core was completely encapsulated by the shell (Figure 3B). PMN also exhibited a strong and broad plasmon wavelength matching the peak wavelength of the IR-LED (Figure 3C). Magnetic measurements also confirmed that PMN possessed excellent magnetic properties after shell coating, with a magnetic moment of 4.75 emu / g (Figure 3D). Using PMN in a PPT-based optical system dramatically altered the temperature of the particle-containing solution under IR-LED illumination. In contrast to the solution where PMN was absent, there was no noticeable temperature change (Figure 3E).

[0054] By adjusting the PMN concentration, the optimal heat circulation efficiency was achieved by forming 16 OD of PMN corresponding to heating and cooling rates of 8.22 ± 0.24 °C / s and 5.27 ± 0.14 °C / s, respectively (Figure 3F). Under these optimal conditions, a temperature profile including an isothermal process (50 °C for 5 minutes) and 40 cycles between 90 °C (0 seconds) and 60 °C (8 seconds) was completed within 20 minutes (Figures 3G, H, and I).

[0055] Through testing with DENV2 RNA, the inventors confirmed that the effect of PMN on amplification is negligible with commercially available PCR equipment. This thermal circulation protocol generated sufficient amplicons even at the low target concentrations of our instrument. To improve the signal-to-noise ratio for subsequent colorimetric detection, relevant mediating parameters such as pH value, SGI and TMB concentrations, LED power, and irradiation time were further optimized. As a result, colorimetric detection was completed within 4 minutes, including PMN acquisition time (<1 minute), blue light irradiation time (2 minutes), and signal measurement time (<1 minute). Leveraging a fast PPT-based thermal circulation and a simple colorimetric strategy, the total analysis time was less than 54 minutes (30 minutes for commercial RNA extraction, <20 minutes for thermal circulation, <4 minutes for signal detection).

[0056] In other words, when detecting viruses using the PPTRTcPCR platform of the present invention, it is not only possible to visualize them through a colorimetric reaction, but also to perform rapid detection within one hour.

[0057] 2-3. Evaluation of the analytical sensitivity and specificity of DENV-specific PPT-RTcPCR In this example, the limit of detection (LoD) for PPT-RTcPCR was investigated using serial dilutions of DENV2 IVT RNA and compared with that of reference real-time RT-qPCR. In this example, the mean OD650 of the nine negative samples was 0.39 ± 0.046, and the critical value for positivity was OD650 0.53 (3x standard deviation of the blank sample).

[0058] The results of this experiment estimated the detection limit of PPT-RTcPCR to be approximately 1.9 copies / μL, which was similar to that of the reference real-time RT-qPCR (2.1 copies / μL) (Figure 4A). The amplified signal generated by PPT-RTcPCR increased as the target concentration increased, and the RNA concentration increased to 10 4 When the concentration is higher than copy / μL, a stable phase is reached, which can be limited by the concentrations of SGI and TMB (see Figure 4A). Furthermore, in samples containing 1 copy / μL DENV IVT RNA, negative results were obtained because the amplification signal was lower than the critical value for PPT-RTcPCR and reference real-time RT-qPCR.

[0059] As a result, the relationship equation A = 0.47 + 0.21 log C (where A is the absorbance at 650 nm, C is the target concentration, and R² = 0.99) can be used to determine when the target concentration changes from 1 copy / μL to 10 4 An excellent linear relationship was obtained when the range was in the copy / μL range.

[0060] The analytical sensitivity was further evaluated in a clinically relevant environment using 10-fold serial dilutions of viral RNA extracted from normal human serum samples supplemented with DENV2. As can be seen in Figure 4B, PPT-RTcPCR showed similar analytical sensitivity to reference real-time RT-qPCR. PPT-RTcPCR detected all samples containing DENV particles at 11.5 FFU / mL as positive, while samples containing 1.15 FFU / mL were not detected as positive.

[0061] Therefore, using the relation A = 0.44 + 0.19 log C, we get 1.15 × 10 4 Excellent linearity (R2 = 0.99) was observed between FFU / mL and 11.5 FFU / mL, indicating that PPT-RTcPCR has similar analytical performance to reference real-time RT-qPCR. The LoD calculated based on the DENV2 titer was approximately 3.42 FFU / mL.

[0062] In this invention, the analytical specificity of PPT-RTcPCR was also evaluated by testing viral RNA extracted from TCF containing DENV1, DENV4, ZIKV (flavivirus), CVB3 (piconavirus), and FIPV (coronavirus). All reactions were negative with absorbance signals below the critical value, indicating the high analytical specificity of PPT-RTcPCR (Figure 4C).

[0063] 2-4. Verification of the clinical performance of DENV-specific PPT-RTcPCR In this example, to confirm the diagnostic ability of DENV-specific PPT-RTcPCR, a panel of 158 human plasma samples collected from clinically suspected dengue fever patients was tested and compared to samples from reference real-time RT-qPCR analysis performed under identical conditions.

[0064] Using a predefined absorbance critical value of OD650 0.53, PPT-RTcPCR detected DENV2 positivity in 125 out of 131 confirmed positive samples, while reference RT-qPCR detected all 131 samples as positive. Furthermore, both PPT-RTcPCR and reference RT-qPCR analyses determined all 27 negative samples to be true negatives (Figures 5A and 5B).

[0065] The clinical sensitivity and specificity of PPT-RTcPCR confirmed by this example were 95.4% (95% CI, 90.4%–97.9%) and 100% (95% CI, 87.5%–100%), respectively.

[0066] To eliminate the possibility that the critical value calculated using the 3σ principle could create the sensitivity difference observed between PPT-RTcPCR and reference RT-qPCR, the optimal critical value was further calculated using ROC (Receiver Operating Characteristic) analysis. As a result, referring to Figure 5C, the optimal critical value was 0.51, and the area under the curve (AUC) was 0.99. The ROC optimal critical value showed a high correlation with the sample set, and in this invention, sensitivity was further increased when the critical value was reduced from 0.53 to 0.51. The analytical and clinical performance of PPT-RTcPCR was re-evaluated using the ROC optimal critical value. The calculated LoD for human serum supplemented with viral nucleic acid and DENV2 was approximately 1.6 copies / μL and 2.69 FFU / mL, respectively.

[0067] [Table 1]

[0068] Considering these results, PPT-RTcPCR identified 127 out of 131 positive samples as true positives, compared to real-time RT-qPCR (Table 1 above). All 27 samples were determined to be true negatives, demonstrating clinical sensitivity and specificity of 97.0% (95% CI, 92.4%–98.8%) and 100% (95% CI, 87.5%–100%). There were four samples tested as false negatives by PPT-RTcPCR, which can potentially be attributed to extremely low concentrations of target RNA, as the colorimetric signal was below the detection limit of the spectrophotometer. The overall agreement between PPT-RTcPCR and reference RT-qPCR was 97.5% (95% CI, 83.4%–99.7%; κ=0.92), indicating that PPT-RTcPCR has excellent diagnostic accuracy.

[0069] The present invention has been described above, focusing on its preferred embodiments. Those with ordinary skill in the art to which the present invention pertains will understand that the present invention may be embodied in modified forms that do not depart from its essential characteristics. Therefore, the disclosed embodiments should be considered in an explanatory rather than restrictive manner. The scope of the present invention is defined in the claims, not in the foregoing description, and all differences within an equivalent scope should be understood as being included within the present invention.

Claims

1. i) The step of mixing the target gene, primer set and plasmon nanoparticles, and then proceeding with the reverse transcription polymerase chain reaction; and ii) The step of mixing 3,3',5,5'-tetramethylbenzidine with the product obtained in step i), In step i) or ii) above, N,N-dimethyl-N'-[4-[(E)-(3-methyl-1,3-benzothiazole-2-ylidene)methyl]-1-phenylquinoline-1-ium-2-yl]-N'-propylpropane-1,3-diamine is added. The plasmon nanoparticles have a core-shell structure consisting of a core containing iron oxide (FeO) and a shell containing gold (Au) attached to the surface of the core. Polyethylene glycol is bonded to the gold particles of the plasmon nanoparticles in the form of methoxy-polyethylene glycol (mPEG)-thiol. A method for detecting target genes using a plasmon-based reverse transcription polymerase chain reaction.

2. The method for detecting a target gene according to claim 1, further comprising the step of irradiating with a blue LED after step ii).

3. The method for detecting a target gene according to claim 2, wherein if the product exhibits a blue color in step iii), it is determined that the target gene is present.

4. The method for detecting a target gene according to claim 1, wherein the target gene is a viral gene.

5. The method for detecting a target gene according to claim 4, wherein the virus is at least one virus selected from the group consisting of dengue virus, piconavirus, flavivirus, Zika virus, Poissant virus, chikungunya virus, enterovirus, respiratory syncytial virus (RSV), Rift Valley fever, influenza virus, Takaribe virus, Mayarovirus, West Nile virus, yellow fever virus, and coronavirus.

6. The plasmon nanoparticles contain N,N-dimethyl-N'-[4-[(E)-(3-methyl-1,3-benzothiazole-2-ylidene)methyl]-1-phenylquinoline-1-ium-2-yl]-N'-propylpropane-1,3-diamine and 3,3',5,5'-tetramethylbenzidine. The plasmon nanoparticles have a core-shell structure consisting of a core containing iron oxide (FeO) and a shell containing gold (Au) attached to the surface of the core. Polyethylene glycol is bonded to the gold particles of the plasmon nanoparticles in the form of methoxy-polyethylene glycol (mPEG)-thiol. Plasmon-based composition for reverse transcription polymerase chain reaction analysis.

7. The plasmon-based reverse transcription polymerase chain reaction analysis composition according to claim 6, wherein the N,N-dimethyl-N'-[4-[(E)-(3-methyl-1,3-benzothiazole-2-ylidene)methyl]-1-phenylquinoline-1-ium-2-yl]-N'-propylpropane-1,3-diamine is added before or after the polymerase chain reaction, and the 3,3',5,5'-tetramethylbenzidine is added after the polymerase chain reaction.

8. A kit for detecting a target gene, comprising the composition described in claim 6.

9. The kit for detecting a target gene according to claim 8, wherein the kit develops a blue color when the target gene is present.

10. The target gene detection kit according to claim 8, wherein the target gene is a viral gene.

11. The target gene detection kit according to claim 10, wherein the virus is at least one virus selected from the group consisting of dengue virus, piconavirus, flavivirus, Zika virus, Poissant virus, chikungunya virus, enterovirus, respiratory syncytial virus (RSV), Rift Valley fever, influenza virus, Takaribe virus, Mayarovirus, West Nile virus, yellow fever virus, and coronavirus.

Images