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JP-7856688-B2 - Rapid detection method for target genes using plasmon photothermal reaction

JP7856688B2JP 7856688 B2JP7856688 B2JP 7856688B2JP-7856688-B2

Inventors

  • イ，ジョンフン

Assignees

  • スンチュンヒャン ユニバーシティ インダストリー アカデミー コオペレーション ファウンデーション

Dates

Publication Date
20260511
Application Date
20240222
Priority Date
20231117

Claims (11)

  1. i) a step of mixing the target gene, primer set and plasmon nanoparticles and then proceeding with a reverse transcription polymerase chain reaction; and ii) a 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 step iii) 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.

Description

Article 30, Paragraph 2 of the Patent Act applies Publication date: June 26, 2023 https://www. sciencedirect. com/science/article/pii/S0003267023007869? via%3Dihub This invention relates to a real-time polymerase chain reaction of a target gene using plasmon nanoparticles. By applying plasmon nanoparticles to a real-time polymerase chain reaction, this invention not only enables the detection of the target gene by colorimetric reaction, but also improves the detection specificity and analytical sensitivity of the target gene. Using the polymerase chain reaction according to this invention, simple and rapid detection of the target gene is possible, providing a diagnostic and analytical method that can be distributed on-site. This invention is derived from research conducted as part of the following projects: Individual Basic Research Project by the Ministry of Science and ICT of Korea [Project Number: 1711186785, Project Number: NRF-2022R1F1A1070162, Research Project Title: Development of a Field-Type Ultra-High-Speed Real-Time PCR Platform Based on Multifunctional Plasmonic Photothermomagnetic Nanoparticles]; Construction of a Science and Engineering Research Base Project by the Ministry of Education [Project Number: 1345362911, Project Number: NRF-2021R1A6A1A03039503, Research Project Title: Korea Institute for the Utilization and Integration of Native Animal Resources]; and Regional Innovation Mega-Project by the Ministry of Science and ICT [Project Number: 20230007, Project Number: 2023-DD-UP-0007, Research Project Title: Development of Source Technology for Metaplatformization of Marine Bio-Strategic Materials]. The Polymerase Chain Reaction (PCR) method is a testing technique used in almost all processes involving the manipulation of genetic material. It amplifies specific target genetic material that is desired for detection. Because polymerase chain reaction allows for the amplification of large quantities of genetic material with the same base sequence from small amounts of genetic material, it is used to amplify human DNA and diagnose various types of genetic disorders. It is also applied to the DNA of bacteria, viruses, and fungi for the diagnosis of infectious diseases. The dengue virus is a single-strand positive-sense RNA virus belonging to the genus Flavivirus of the family Flaviviridae. Dengue fever and dengue hemorrhagic fever are acute febrile illnesses that develop when humans are bitten by Egyptian forest mosquitoes (Aedes aegypti) or white thread-tooth mosquitoes (Aedes albopictus) infected with the dengue virus. 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. 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. Based on this, the inventors developed a real-time gene detection test method using plasmon nanoparticles. Unlike conventional methods, this method can detect target genes by color change and has been confirmed to be able to detect them rapidly with high sensitivity and specificity, thus completing the present invention. Figure 1 is a schematic diagram showing the overall flow of a target gene detection method using a plasmon photothermal reaction-based RTcPCR (PPT-RTcPCR) platform according to the present invention.Figure 2A shows the workflow of a plasmon photothermal reaction-based RTcPCR (PPT-RTcPCR) platform. Figure 2A is a flowchart of the PPT-RTcPCR platform that combines PPT-based RT-PCR with colorimetric sensing.Figure 2B shows the workflow of a plasmon photothermal reaction-based RTcPCR (PPT-RTcPCR) platform. Figure 2B also shows a colorimetric reading method using plasmon photothermal reaction-based RTcPCR (PPT-RTcPCR). Referring to Figure 2B, in negative samples where the target gene is absent, amplification does not occur, and if the dsDNA-SGI complex is not formed, the TMB cannot be oxidized, and little to no hue change occurs. On the other hand, in positive samples where the target gene is present, the amplicons increase geometrically, and in this case, the TMB can be further oxidized by the dsDNA-SGI complex, causing a noticeable hue change from colorless to blue.Figure 3A s