Rapid target gene detection method using plasmonic 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 target genes using plasmonic nanoparticles. The present invention applies plasmonic nanoparticles to real-time polymerase chain reaction, which can not only detect the presence of target genes by colorimetric reaction, but also improve the detection specificity and analysis sensitivity of target genes. When using the polymerase chain reaction according to the present invention, simple and rapid detection of target genes 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 of Korea [Project Specific Number: 1711186785, Project Number: NRF-2022R1F1A1070162, Research Project Name: Development of a Field-Type Ultra-High-Speed Real-Time PCR Platform Based on Multifunctional Plasmonic Photothermal Magnetic Nanoparticles], the Project for Constructing an Academic Research Base in Engineering of the Ministry of Education [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] Note that the dengue virus is a single-stranded positive-sense RNA virus belonging to the genus Flavivirus of the family Flaviviridae. Dengue fever and dengue hemorrhagic fever are acute febrile diseases that occur during the process of Aedes aegypti or Aedes albopictus, which are infected with the dengue virus, biting humans.

[0005] In the past few decades, dengue infections have spread rapidly within tropical and subtropical countries, putting nearly one-third of the world's population at risk. In recent years, this has become one of the most important public health issues in many resource-limited countries, threatening human lives and imposing great pressure on the medical management system from various perspectives.

[0006] Even more importantly, the combined impact of the COVID-19 pandemic and dengue infections can potentially have devastating consequences for patients in endemic areas with various serotypes of DENV. Effective vaccines and the early diagnosis of dengue virus (DENV) infections are important for improving clinical outcomes and preventing the spread of the disease. Simple, cost-effective, and sensitive diagnostic tests for on-site use are essential for disease control, especially in remote areas where access to medical service providers and facilities is restricted. However, diagnostic tools with such functions have not yet been developed.

[0007] Based on this, the inventors have developed a real-time gene detection test method using plasmonic nanoparticles, which, different from the prior art, can detect target genes through color changes and can be detected quickly with high sensitivity and specificity, thus completing the present invention.

Summary of the Invention

Problems to be Solved by the Invention

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

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

Means for Solving the Problems

[0010] The present invention will be described in detail below. Referring to the merits and features of the present invention and the embodiments described later for achieving them will make it clear. However, the present invention is not limited to the embodiments disclosed below and can be embodied in various different forms. Merely, these embodiments are provided to complete the disclosure of the present invention and to fully inform those with ordinary knowledge in the technical field to which the present invention pertains of the scope of the invention. The present invention is only defined by the scope of the claims. The same reference numerals throughout the specification refer to the same components.

[0011] Unless otherwise defined, all terms (including technical and scientific terms) used in this specification can be used in a meaning commonly understood by those with ordinary knowledge in the technical field to which the present invention pertains. Also, terms defined in commonly used dictionaries are not ideally or excessively interpreted unless specifically defined otherwise. The terms used in this specification are for the purpose of explaining the embodiments and are not intended to limit the present invention. In this specification, the singular form includes the plural form unless otherwise specifically mentioned in the context.

[0012] To achieve the above object, the present invention provides a method for conducting a plasmon-based reverse transcription polymerase chain reaction for detecting a target gene, comprising: i) a step of proceeding with reverse transcription polymerase chain reaction after mixing a target gene, a primer set, and plasmon nanoparticles; and ii) a step of mixing 3,3’,5,5’-tetramethylbenzidine with the product obtained in the step i). SYBR Green I can be added in the 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). To induce the photothermal reaction, the polymerase chain reaction of the present invention can further include iii) a step of irradiating with a blue LED after the step ii). When the target gene exists in the reaction product, when irradiating with the blue LED, the reaction product develops a blue color. Therefore, when the product obtained by the step iii) exhibits a blue color, it can be determined that the target gene exists.

[0014] In the present invention, the plasmon nanoparticles may be metal particles, preferably, a core containing iron oxide (FeO); and a shell containing gold (Au) attached to the surface of the core; and may have a core-shell structure. Polyethylene glycol may be bound 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 all genes that can be amplified by the polymerase chain reaction, but is preferably a viral gene.

[0016] The virus of the present invention includes, but is not limited to, dengue virus, picornavirus, flavivirus, Zika virus, Powassan virus, chikungunya virus, enterovirus, respiratory syncytial virus (RSV), Rift Valley fever, influenza virus, Tacaribe virus, Mayaro virus, West Nile virus, yellow fever virus, and coronavirus.

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

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

[0019] The present invention also provides a kit for detecting a target gene, which includes the composition for plasmon-based reverse transcription polymerase chain reaction analysis. When the target gene exists, the kit of the present invention develops a blue color, and the target gene can be detected with the naked eye.

Advantages of the Invention

[0020] When using the polymerase chain reaction (RTcPCR based on plasmon photothermal reaction (PPT-RTcPCR)) of the present invention, the target gene can be rapidly detected within 1 hour.

[0021] In addition, according to the polymerase chain reaction of the present invention, the target gene can be detected with high specificity and sensitivity, and the ratio of false positives or false negatives can be reduced.

[0022] In addition, the present invention has the advantage that when the target gene exists, a color reaction occurs and the result can be confirmed with the naked eye.

Brief Description of the Drawings

[0023]

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Mode for Carrying Out the Invention

[0024] Hereinafter, various examples are presented to assist in understanding the invention. The following examples are provided solely for the purpose of more easily understanding the invention, and the scope of protection of the invention is not limited to the following examples.

[0025] The present invention presents a method for the progress of polymerase chain reaction using plasmonic nanoparticles.

[0026] The polymerase chain reaction of the present invention is a (reverse transcription, colorimetric) polymerase chain reaction based on plasmonic photothermal reaction, hereinafter referred to as PPT-RTcPCR.

[0027] The PPT-RTcPCR reaction of the present invention can include: i) a step of proceeding with reverse transcription polymerase chain reaction after mixing a target gene, a primer set, and plasmonic nanoparticles; and ii) a step of mixing 3,3’,5,5’-tetramethylbenzidine with the product obtained in the step i). At this time, SYBR Green I can be added in the step i) or ii).

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

[0029] In the present invention, "plasmonic nanoparticles (PMN)" means nano-sized particles showing plasmon resonance.

[0030] The plasmonic nanoparticles of the present invention can use a metal showing a plasmonic resonance effect. Specific examples include gold (Au) nanoparticles, silver (Ag) nanoparticles, or both gold (Au) nanoparticles and silver (Ag) nanoparticles can be used.

[0031] According to a preferred embodiment of the present invention, the plasmonic nanoparticles have a core-shell structure composed of a core containing iron oxide (FeO); and a shell containing gold (Au) attached to the surface of the core. Polyethylene glycol may be bound to the gold particles of the plasmonic nanoparticles, and may be configured in a form in which polyethylene glycol is exposed outside the core-shell structure.

[0032] In the present invention, the term "target gene" means a nucleic acid sequence to be detected, and anneals or hybridizes with a probe under hybridization conditions. The "target gene" is used interchangeably with "target nucleic acid" or "target nucleic acid". In the present invention, the target nucleic acid can be a gene derived from an animal, a plant, a bacterium, a virus, a fungus, etc., or a mutant gene associated with a genetic disease. In the present 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 indicating a sense or antisense strand. dsDNA, ssDNA, hybrid ssDNA, hybrid dsDNA, ssDNa, dsDNA, mRNA, rRNA, tRNA, snRNA or miRNA can be used, but the target gene of the present invention is most preferably in the form of RNA.

[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), picornavirus (CVB3), flavivirus, Zika virus, Powassan virus, chikungunya virus, enterovirus, respiratory syncytial virus (RSV), Rift Valley fever, influenza virus, Takaribe virus, Mayaro virus, West Nile virus, yellow fever virus and coronavirus.

[0034] In the present invention, "detection" means detecting a target gene through the photothermal reaction-based PCR of the plasmonic 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, blue color appears in the reaction product. Through such a colorimetric reaction, when blue color appears in the reactant, it can be confirmed that the target gene is present and determined as "positive".

[0036] In the present invention, the term "binding site that binds complementarily" or "complementary binding site" means a site capable of forming complementary base pairs between nucleotide sequences.

[0037] In the present invention, a "primer" means a short nucleic acid sequence having a short free 3'-hydroxyl group that can form base pairs with a template of a complementary nucleic acid and functions as a starting point for chain copying of a nucleic acid template. A primer can initiate DNA synthesis in the presence of appropriate buffer solutions and reagents for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) and different four types of nucleoside triphosphates.

[0038] When designing the primer, there are various restrictions such as the A, G, C, T content ratio of the primer, prevention of primer dimer formation, and prohibition of repetition of the same base sequence three or more times. In addition, in the single PCR reaction conditions, the amount of template DNA, the concentration of the primer, the concentration of dNTP, Mg 2+ concentration, reaction temperature, reaction time, etc. must be appropriate.

[0039] The foregoing primers can incorporate additional features that do not change their basic properties. That is, the nucleic acid sequences can be modified using many means known in the art. Examples of such modifications include methylation, capping, substitution of one or more homologs of nucleotides, and modification of nucleotides to uncharged linkers such as phosphonates, phosphotriesters, phosphoramidates or carbamates or carbamates, or charged linkers such as phosphorothioates or phosphorodithioates. In addition, the nucleic acid can have one or more additional covalently linked residues such as proteins such as nucleases, toxins, antibodies, signal peptides, poly-L-lysine, intercalating agents such as acridines or psoralens, metals, radioactive metals, chelating agents such as iron-oxidizing metals, and alkylating agents.

Example

[0040] The following examples are presented to assist 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 in vitro transcribed RNA The analytical performance of dengue virus (DENV)-specific PPT-RTcPCR analysis was first evaluated using in vitro transcribed (IVT) RNA. A partial sequence of the E gene of the DENV-2 NGC strain (nt 1453 to 1550; GenBank accession AF038403) was synthesized and cloned into the pGEM-3Z vector downstream of the T7 promoter sequence. After linearizing the generated plasmid pGEM-3Z-DENV by SalI restriction digestion, it was 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 aliquots were stored at -80°C. Then, IVT RNA (1-10 6The analytical sensitivity of PPT-RT cPCR was determined using 10-fold serial dilutions (in copies / μL). The concentration of IVT RNA was calculated by a known method.

[0042] 1-2. Nucleic acid extraction For analytical sensitivity analysis, viral RNA was extracted from TCF containing 1.15 x 10 6 focus forming units (FFU) / mL of DENV2 and serially diluted 10-fold (10 1 ~10 8 ) in nuclease-free water.

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

[0044] All samples were extracted using the MagMAX®-96 Viral RNA Isolation Kit (Applied Biosystems, USA) on the KingFisher Flex System (Thermo Fisher, USA). After the extraction step, 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-RT cPCR and optimization DENV-specific PPT-RT cPCR was developed with some modifications from previously known content. The target DENV2 RNA was first amplified through PPT-based RT-PCR in the presence of plasmon magnetic nanoparticles (PMN).

[0046] The reaction mixture was made to contain 5 μL of 2x reaction mix, 0.2 μL of SuperScript® III RT / Platinum® Taq Mix, 0.1 μL each 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 plasmonic 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 cycling protocol started at 50 °C for 5 minutes, followed by 40 cycles between 90 °C (0 seconds) and 60 °C (8 seconds). After 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. Taking advantage of the magnetic properties of PMN, the particles were collected with a magnet before irradiating with a blue light LED (300 mA, 14 V) for 2 minutes. Due to the photocatalytic activity of the dsDNA-SGI complex, TMB was oxidized under the excitation of the blue light LED by adjusting the pH value of the solution to about 5. The absorbance of the resulting mixture was measured with a NanoDrop® OneC Microvolume UV-Vis Spectrophotometer (Thermo Scientific®).

[0047] The reference real-time DENV2 RT-qPCR was performed by modifying a 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 each 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, followed by 40 cycles of 15 seconds at 95 °C and 1 minute at 60 °C. For reference real-time RT-PCR, samples with a Ct value greater than 37 were considered negative because it was difficult to confirm the amplification results.

[0048] [Example 2] Results 2-1. PPTRTcPCR Platform Proceeding Procedure In the present 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 dengue fever infection was applied to the DENV-specific PPT-RTcPCR reaction. In this system, a nano-sized heater for uniform heating of PMN was used. When the infrared (IR)-LED was turned on, the reaction temperature could be easily and precisely controlled by adjusting the light intensity. The heating protocol started with an isothermal process of reverse-transcribing RNA into complementary DNA (cDNA), followed by two-step thermal cycling for cDNA denaturation and specific target amplification.

[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 a reactant for forming a photocatalyst and is only induced when SGI is inserted into the dsDNA. Upon irradiation with blue light LEDs, singlet oxygen is generated by energy transfer from SGI to dissolved oxygen in the solution, which subsequently induces TMB oxidation. The reaction product changes from colorless to blue, making the amplicon production visible. In the absence of viral RNA, there is no specific RT-PCR amplification, no dsDNA-SGI complex is formed, and thus TMB is not oxidized, keeping the solution colorless.

[0051] Through such a simple design, this device can be developed as a POC diagnostic system.

[0052] 2-2. Characterization of the PPT-RTcPCR Platform Since the PMN induces efficient PPT-based thermal cycling and convenient magnetic separation, it was used for the nano-sized heater. The PMN particles have a magnetic iron oxide core surrounded by a plasmonic Au shell (skin) (Figure 3A). The iron oxide core produced by the solvothermal reaction was first functionalized with an amine-terminated surface by an organic silane. Subsequently, small AuNPs were attached to the core surface through electrostatic assembly, forming the Au shell.

[0053] To enhance the stability of the PMN and minimize non-specific interactions during amplification, the PMN was finally modified with methoxy-polyethylene glycol (mPEG)-thiol. The elemental mapping image shows that the core was completely encapsulated by the shell (Figure 3B). Also, the PMN exhibited a strong and broad plasmon wavelength that coincides with the peak wavelength of the IR-LED (Figure 3C). Magnetic measurements also confirmed that the PMN has excellent magnetic properties after shell coating with a magnetic moment of 4.75 emu / g (Figure 3D). Using the PMN in a PPT-based optical device, the temperature of the particle-containing solution dramatically In contrast, in the solution without PMN, no noticeable temperature change was observed (Figure 3E).

[0054] By adjusting the PMN concentration, an optimal thermal cycling efficiency corresponding to heating and cooling rates of 8.22 ± 0.24 °C / s and 5.27 ± 0.14 °C / s, respectively, was achieved with 16 OD of PMN formed (Figure 3F). The temperature profile including an isothermal process (5 minutes at 50 °C) and 40 cycles between 90 °C (0 seconds) and 60 °C (8 seconds) was completed within 20 minutes under this optimal condition (Figures 3G, H, and I).

[0055] Through tests using DENV2 RNA, the inventors confirmed that the influence of PMN on amplification can be ignored in commercially available PCR equipment. This thermal cycling protocol generated sufficient amplicons even at the low target concentration of our device. To increase the signal-to-noise ratio for subsequent colorimetric detection, relevant medium variables such as pH value, concentrations of SGI and TMB, LED power, and irradiation time were further optimized. As a result, colorimetric detection was completed within 4 minutes, including PMN collection time (<1 minute), blue light irradiation time (2 minutes), and signal measurement time (<1 minute). Utilizing high-speed PPT-based thermal cycling and a simple colorimetric strategy, the overall analysis time was less than 54 minutes (30 minutes for commercial RNA extraction, <20 minutes for thermal cycling, and <4 minutes for signal detection).

[0056] That is, when detecting the virus using the PPTRTcPCR platform of the present invention, it can be seen that not only can it be visualized through a colorimetric reaction, but rapid detection within 1 hour is also possible.

[0057] 2-3. Evaluation of the analytical sensitivity and specificity of DENV-specific PPT-RTcPCR In this example, serial dilutions of DENV2 IVT RNA were used to examine the limit of detection (LoD) for PPT-RTcPCR and compared with reference real-time RT-qPCR. The average OD650 of 9 negative samples in this example was 0.39 ± 0.046, and the critical value for positivity was OD650 0.53 (3 standard deviations of the blank sample).

[0058] The results of this experiment showed that the detection limit of PPT-RTcPCR was estimated to be approximately 1.9 copies / μL, which appeared similar to that of the reference real-time RT-qPCR (2.1 copies / μL) (Figure 4A). The amplification signal generated by PPT-RTcPCR increased as the target concentration increased and reached a plateau when the RNA concentration was higher than 10 4 copies / μL, which could be limited by the concentrations of SGI and TMB (see Figure 4A). Also, in samples containing 1 copy / μL DENV IVT RNA, negative results were derived because the amplification signal was lower than the cut-off values of PPT-RTcPCR and the reference real-time RT-qPCR.

[0059] As a result, a good linear relationship was obtained when the target concentration ranged from 1 copy / μL to 10 4 copies / μL using the regression 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).

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

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

[0062] In the present invention, viral RNAs extracted from TCFs including DENV1, DENV4, ZIKV (Flavivirus), CVB3 (Picornavirus), and FIPV (Coronavirus) were also tested to evaluate the analytical specificity of PPT-RTcPCR. All reactions were negative with absorbance signals below the threshold 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 with samples of a reference real-time RT-qPCR analysis performed under the same conditions.

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

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

[0066] To exclude the possibility that the critical value calculated by the 3σ principle creates the observed difference in sensitivity 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 FIG. 5C, the optimal critical value was 0.51 and the area under the curve (AUC) was 0.99. The ROC optimal critical value had a high correlation with the sample set. In the present invention, when the critical value was decreased from 0.53 to 0.51, the sensitivity became even higher. The analysis and clinical performance of PPT-RTcPCR were re-evaluated using the ROC optimal critical value. The LoD calculated for human serum spiked with viral nucleic acid and DENV2 was approximately 1.6 copies / μL and 2.69 FFU / mL, respectively.

[0067]

Table 1

[0068] Considering such results, PPT-RTcPCR identified 127 out of 131 positive samples as true positives, respectively, compared to real-time RT-qPCR (Table 1 above). And determined all 27 samples as true negatives, showing clinical sensitivities and specificities of 97.0% (95% CI, 92.4% - 98.8%) and 100% (95% CI, 87.5% - 100%), respectively. There were 4 samples that were tested false negative by PPT-RTcPCR, which could potentially be due to the colorimetric signal being lower than the detection limit of the spectrophotometer and extremely low concentrations of target RNA. The overall agreement value 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] As described above, the present invention has been described mainly with reference to its preferred embodiments. Those having ordinary knowledge in the technical field to which the present invention pertains can understand that the present invention can be embodied in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative perspective rather than a limiting perspective. The scope of the present invention is shown not in the foregoing description but in the claims, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims

1. i) mixing the gene of interest, the primer set and the plasmonic nanoparticles, and then carrying out a reverse transcription polymerase chain reaction; and ii) mixing the product obtained in step i) with 3,3',5,5'-tetramethylbenzidine; plasmon-based reverse transcription polymerase chain reaction for detecting target genes, adding SYBR Green I in step i) or ii).

2. The plasmon-based reverse transcription polymerase chain reaction for detecting a target gene according to claim 1 , further comprising the step of: iii) irradiating with a blue LED after step ii).

3. The plasmon-based reverse transcription polymerase chain reaction for detecting a target gene according to claim 2, wherein if the product in step iii) exhibits a blue color, it is determined that the target gene is present.

4. The plasmon-based reverse transcription polymerase chain reaction for detecting a target gene according to claim 1, wherein the plasmon nanoparticle has a core-shell structure composed of a core containing iron oxide (FeO); and a shell containing gold (Au) attached to the surface of the core.

5. The plasmon-based reverse transcription polymerase chain reaction for detecting a target gene according to claim 4, wherein the gold particles of the plasmon nanoparticles are bound to polyethylene glycol.

6. The plasmon-based reverse transcription polymerase chain reaction of claim 1 , wherein the target gene is a viral gene.

7. 7. The plasmon-based reverse transcription polymerase chain reaction of claim 6, wherein the virus is at least one selected from the group consisting of dengue virus, picornavirus, flavivirus, Zika virus, Powassan virus, chikungunya virus, enterovirus, respiratory syncytial virus (RSV), Rift Valley fever, influenza virus, Tacaribe virus, Mayaro virus, West Nile virus, yellow fever virus, and coronavirus.

8. A composition for a plasmon-based reverse transcription polymerase chain reaction assay comprising plasmon nanoparticles, SYBR Green I and 3,3',5,5'-tetramethylbenzidine.

9. The plasmonic-based reverse transcription polymerase chain reaction analytical composition of claim 8, wherein the plasmonic nanoparticles are a core-shell structure consisting of a core comprising iron oxide (FeO); and a shell comprising gold (Au) attached to a surface of the core.

10. The plasmon-based reverse transcription polymerase chain reaction analytical composition of claim 9, wherein the gold particles of the plasmon nanoparticles are bound to polyethylene glycol.

11. The plasmon-based reverse transcription polymerase chain reaction analysis composition of claim 8, wherein the SYBR Green I is added before or after the polymerase chain reaction, and the 3,3',5,5'-tetramethylbenzidine is added after the polymerase chain reaction.

12. A kit for detecting a target gene, comprising the composition according to claim 8.

13. The kit for detecting a target gene according to claim 12, wherein the kit develops a blue color when the target gene is present.

14. The target gene detection kit according to claim 12 , wherein the target gene is a viral gene.

15. The kit for detecting a target gene according to claim 14, wherein the virus is at least one virus selected from the group consisting of dengue virus, picornavirus, flavivirus, Zika virus, Powassan virus, chikungunya virus, enterovirus, respiratory syncytial virus (RSV), Rift Valley fever, influenza virus, Tacaribe virus, Mayaro virus, West Nile virus, yellow fever virus and coronavirus.

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