WO2022173266A1 - Recombinase-polymerase isothermal amplification device for performing isothermal amplification and simultaneously generating plasmon fluorescence amplification signals - Google Patents

Abstract

A recombinase-polymerase isothermal amplification device of the present invention enables isothermal amplification and surface fluorescence amplification to be carried out simultaneously so that the device has excellent sensitivity and specificity and can be miniaturized, and thus can be used in the diagnosis of pathogens in a clinical scene.

Description

A recombinant enzyme-polymerase isothermal amplification device that generates a plasmonic fluorescence amplification signal simultaneously with isothermal amplification

The present invention relates to a recombinant enzyme-polymerase isothermal amplification device that generates a plasmonic fluorescence amplification signal simultaneously with isothermal amplification.

Pneumonia, a lower respiratory infection, was the fourth leading cause of death in all age groups, and in 2017 it was the leading cause of death in children under 5 years of age worldwide. Although lower respiratory tract infections are caused by a variety of bacteria and viruses, it is difficult to differentiate them by symptoms. Therefore, in order to avoid unnecessary use of antibiotics and to treat them early, a technology that can quickly and accurately identify pathogens in the field is required.

Since PCR has excellent sensitivity and specificity, it is mainly used for pathogen identification. However, PCR is difficult to apply to on-site diagnosis because it requires a complex process such as a sophisticated temperature change device, long amplification time, and confirmation by electrophoresis.

Recently, various isothermal amplification techniques have been studied for on-site diagnosis. Isothermal amplification technology is attracting attention for point-of-care diagnostics because it can miniaturize temperature devices, but its amplification efficiency, detection sensitivity, and specificity do not reach that of PCR, so there are limitations in clinical application.

In addition, although the detection efficiency for short-length DNA of about 50 bp or less attached to the surface fluorescence amplification substrate has been reported, it is possible to attach a primer to the surface fluorescence amplification substrate and thereby generate a DNA amplification product having a length of 150 bp or more. , and it is not known what the optimal surface fluorescence amplification conditions for this are.

According to one embodiment, a recombinant enzyme-polymerase isothermal amplification device capable of isothermal amplifying a target gene and simultaneously improving detection sensitivity by surface fluorescence amplification, and a recombinant enzyme-polymerase isothermal amplification kit comprising the same.

One aspect of the present invention is a substrate comprising a nano-pillar; a noble metal layer laminated on the surface of the substrate or nanopillars; an insulating layer laminated on the surface of the substrate, nano-pillars, or noble metal layer; noble metal nanoparticles bonded to the surface of the noble metal layer or the insulating layer; And it provides a recombinant enzyme-polymerase isothermal amplification device comprising a forward primer bound to the substrate, nano-pillars, noble metal layer, insulating layer, or the surface of the noble metal nanoparticles.

In one embodiment of the present invention, the forward primer may include 10 thymine linkers.

In one embodiment of the present invention, the insulating layer is a hydrocarbon thiol (alkanethiol), a hydrocarbon disulfide (alkyldisulfide), a fluorocarbon thiol (fluorocarbon thiol), a fluorocarbon silane (fluorocarbon silane), a chlorocarbon silane (chlorocarbon silane) , it may be at least one selected from fluorocarbon carboxylic acid, fluorocarbon amine, fluorocarbon polymer, and derivatives thereof.

In one embodiment of the present invention, the noble metal may be at least one selected from Au, Ag, Cu, Al, Pt, Pd, Ti, Rd, Ru, and alloys thereof.

In one embodiment of the present invention, the recombinant-enzyme polymerase isothermal amplification device performs plasmonic fluorescence amplification when an amplification product is generated by performing recombinant-enzyme polymerase isothermal amplification with a reverse primer labeled with a target gene and a fluorophore. can

Another aspect of the present invention provides a Recombinase-Polymerase Amplification (RPA) kit comprising the following devices and materials: a) a substrate comprising nanopillars; a noble metal layer laminated on the surface of the substrate or nanopillars; an insulating layer laminated on the surface of the substrate, nano-pillars, or noble metal layer; noble metal nanoparticles bonded to the surface of the noble metal layer or the insulating layer; and a recombinant enzyme-polymerase isothermal amplification device comprising a forward primer bound to the substrate, nanopillar, noble metal layer, insulating layer, or surface of noble metal nanoparticles b) fluorophore-labeled reverse primer c) strand displacement polymerase, recombinases, and single-stranded DNA binding proteins.

In one embodiment of the present invention, the forward primer and the reverse primer may consist of sequences having a size of 162 to 231 bp of amplification products by these primer pairs.

In one embodiment of the present invention, the plasmon coupling effect of the plasmonic nucleic acid amplification apparatus may be maximized at the emission wavelength of the fluorophore.

In one embodiment of the present invention, the forward primer and the reverse primer, Streptococcus pneumoniae consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 A pair of primers for detecting the Ply gene; A pair of primers for detecting the OmpA gene of Chlamydophila pneumoniae consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4; A pair of primers for detecting the P6 gene of Haemophilus influenzae consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6; And it may be one or more selected from the group consisting of a primer pair for detecting the ITS1 gene of Mycoplasma pneumoniae consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8.

In one embodiment of the present invention, the forward primer and the reverse primer are a pair of primers for detecting S gene of human coronavirus 229E (Human Coronavirus 229E) consisting of a forward primer of SEQ ID NO: 9 and a reverse primer of SEQ ID NO: 10; A primer pair for detecting the M gene of human coronavirus OC43 (Human Coronavirus OC43) consisting of a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12; A pair of primers for detecting the S gene of human coronavirus NL63 (Human Coronavirus NL63) consisting of a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 14; And it may be at least one selected from the group consisting of a pair of primers for detecting the F gene of human metapneumovirus consisting of a forward primer of SEQ ID NO: 15 and a reverse primer of SEQ ID NO: 16.

In one embodiment of the present invention, the RPA reaction temperature of the kit may be 37 °C to 42 °C.

In one embodiment of the present invention, the RPA reaction time of the kit may be 20 to 40 minutes.

The device according to an embodiment has excellent sensitivity and specificity because surface fluorescence amplification occurs at the same time as isothermal amplification is performed.

The device according to an embodiment can be miniaturized and has superior sensitivity and specificity than the existing isothermal amplification technology, so that pathogens can be quickly and accurately identified in the field.

1A and B show SEM images of the 3D NPOP substrate surface. 1C and D show TEM images of nanopillars of 3D NPOP substrates.

Fig. 2A shows that the 3D NPOP substrate has very strong Rayleigh scattering intensity within the long red range wavelength (650-750 nm) of visible light, and the excitation spectrum (red dotted line) and emission spectrum of the Cy5 fluorophore ( red shaded area). 2B is a distant optical microscope (OM) image showing strong Rayleigh scattering of the 3D NPOP structure.

3 is a result of binding a short-length (25 bp) double-stranded (ds) DNA to a 3D gold NPOP substrate and confirming the PEF.

4 shows a plot of fluorescence intensity enhancer versus Cy5 labeled dsDNA concentration. The enhancer was determined by dividing the average spot intensity of the 3D gold NPOP substrate by the intensity of the general Au substrate.

5 is a result of confirming PEF after binding a short-length (25 bp) single-stranded (ss) DNA to a 3D gold NPOP substrate and hybridizing it with a complementary strand.

6 is a schematic diagram showing the results of gel electrophoresis of the RPA product for the P6 gene of H. influenzae and the process of binding the RPA product to a 3D gold NPOP substrate.

7 is a PEF result for the RPA product bound to the 3D gold NPOP substrate.

8 is a schematic diagram illustrating a process in which solid-state RPA is performed in a plasmonic chip in which a forward primer is bound to a 3D gold NPOP substrate.

9 shows the results of optimization experiments for temperature and reaction time in performing plasmonic RPA on a 3D gold NPOP substrate. A and C are the results of agarose gel electrophoresis of the liquid RPA product, and B and D are the fluorescence microscopic images and fluorescence intensity of solid-phase plasmonic RPA performed with 3D gold NPOP substrate.

10 shows the results of optimization experiments for reaction time in performing plasmonic RT-RPA on a 3D gold NPOP substrate. A is the result of agarose gel electrophoresis of the liquid RT-RPA product, and B is the fluorescence image and fluorescence intensity of solid-phase plasmonic RT-RPA performed with 3D gold NPOP substrate.

11 shows the results and specificity of four types of bacterial DNA detection using a plasmonic RPA array chip fabricated with a 3D gold NPOP substrate. Abbreviations of bacteria used in the drawings are: S: Streptococcus pneumoniae, H: Haemophilus influenzae, C: Chlamydia pneumoniae, and M: Mycoplasma pneumoniae.

12 shows the results and detection sensitivity of four types of bacterial DNA detection using a plasmonic RPA array chip made of a 3D gold NPOP substrate.

13 is a result of the detection of DNA of 4 types of bacteria by liquid phase multiplex PCR.

14 is a result of the detection of four types of bacterial DNA using liquid phase multiplex RPA.

15 shows the results and specificity of four types of viral RNA detection using a plasmonic RT-RPA array chip made of a 3D gold NPOP substrate. Abbreviations of viruses used in the drawings are: E: Corona virus 229E, O: Corona virus OC43, N: Corona virus NL63, and h: human metapneumovirus.

16 shows the results and sensitivities of four types of viral RNA detection using a plasmonic RT-RPA array chip made of a 3D gold NPOP substrate.

17 shows the results of detection of four types of viral RNA by liquid phase multiplex RT-PCR.

18 is a result of the detection of four types of viral RNA by liquid phase multiplex RT-RPA.

19 shows the results of preliminary clinical evaluation of the plasmonic RT-RPA array chip using nasopharyngeal swab samples from patients infected with four types of virus.

20 shows the results of liquid phase multiplex RT-RPA using nasopharyngeal swab samples from patients infected with 4 viruses.

One aspect is a substrate comprising a nanopillar; a noble metal layer laminated on the surface of the substrate or nanopillars; an insulating layer laminated on the surface of the substrate, nano-pillars, or noble metal layer; noble metal nanoparticles bonded to the surface of the noble metal layer or the insulating layer; And it provides a recombinant enzyme-polymerase isothermal amplification device comprising a forward primer bound to the substrate, nano-pillars, noble metal layer, insulating layer, or the surface of the noble metal nanoparticles.

The solid-phase recombinant enzyme-polymerase isothermal amplification (Recombinase Polymerase Amplfication, hereinafter RPA) device performs RPA in a solid-phase by fixing a forward primer to the substrate, a noble metal layer, an insulating layer, or the surface of a noble metal nanoparticle. can do.

The present invention has the following advantages. First, since the forward primer is fixed to a substrate and the like and the reverse primer is prepared separately, non-specific amplification such as primer dimer formation can be reduced. Second, by performing plasmon resonance with the fluorophore labeled in the reverse primer, a strong fluorescence signal can be obtained simultaneously with isothermal amplification. Although plasmon resonance is a known technique, as in the present invention, by performing recombinant enzyme-polymerase isothermal amplification (RPA) with a fixed forward primer and a fluorophore-labeled reverse primer as in the present invention, plasmon fluorescence amplification is performed simultaneously with isothermal amplification. There is no known solid-state isothermal amplification device capable of rapidly detecting a signal by generating a strong signal.

The nanopillar is a columnar nanostructure having a diameter of about 10 nanometers. The nanopillars may be referred to as nanopillars.

The substrate including the nanopillars may have a form in which a plurality of nanopillars are spaced apart from each other on the surface. The substrate may be a polymer, glass, ceramic, metal, paper, resin, silicon, or metal oxide.

The nanopillars may be formed by plasma etching, soft lithography, nanoimprint lithography, photo lithography, or holographic lithography.

According to one embodiment, the forward primer may include 10 thymine linkers.

The noble metal layer may be formed by depositing a Raman active material, for example, may be formed by thermal evaporation, vapor deposition, or a solution process.

According to one embodiment, the noble metal may be a Raman active material, for example, may be at least one selected from Au, Ag, Cu, Al, Pt, Pd, Ti, Rd, Ru, and alloys thereof.

The insulating layer may be formed by depositing on the noble metal layer, for example, may be formed by thermal evaporation, vapor deposition, or a solution process.

According to one embodiment, the insulating layer is hydrocarbon thiol (alkanethiol), hydrocarbon disulfide (alkyldisulfide), fluorocarbon thiol (fluorocarbon thiol), fluorocarbon silane (fluorocarbon silane), chlorocarbon silane (chlorocarbon silane), fluorocarbon carboxylic acid (fluorocarbon) It may be at least one selected from carboxylic acid), fluorocarbon amine, fluorocarbon polymer, and derivatives thereof, and according to an embodiment, it may be PFDT.

According to one embodiment, the recombinant-enzyme polymerase isothermal amplification device performs plasmonic fluorescence amplification when an amplification product is generated by performing recombinant-enzyme polymerase isothermal amplification with a reverse primer labeled with a target gene and a fluorophore. .

The plasmon coupling effect of the recombinant enzyme-polymerase isothermal amplification device may be maximized at the emission wavelength of the fluorophore. According to an embodiment, the fluorophore is Cy5, and the recombinant enzyme-polymerase isothermal amplification device may have a plasmon coupling effect that maximizes at 650 nm.

The recombinant enzyme-polymerase isothermal amplification apparatus may be manufactured in the form of a microarray, a microchip, or the like, or may be manufactured in the form of a multi-microarray capable of simultaneously detecting several target genes.

Another aspect is a Recombinase-Polymerase Amplification (RPA) kit comprising the following device and material, the device and material comprising: a) a substrate including nanopillars; a noble metal layer laminated on the surface of the substrate or nanopillars; an insulating layer laminated on the surface of the substrate, nano-pillars, or noble metal layer; noble metal nanoparticles bonded to the surface of the noble metal layer or the insulating layer; and a recombinase-polymerase isothermal amplification device comprising a forward primer bound to the noble metal layer, the insulating layer, or the surface of the noble metal nanoparticles, b) a fluorophore-labeled reverse primer, c) a strand displacement polymerase, a recombinase, and single-stranded DNA binding proteins.

The contents of the components constituting the kit are the same as those described above.

According to one embodiment, the forward primer and the reverse primer may consist of a sequence having a size of 162 to 231 bp of the amplification product by these primer pairs.

According to one embodiment, the plasmon coupling effect of the recombinant enzyme-polymerase isothermal amplification device may be maximized at the emission wavelength of the fluorophore. According to an embodiment, the fluorophore is Cy5, and the recombinant enzyme-polymerase isothermal amplification device may have a plasmon coupling effect that maximizes at 650 nm. In particular, the present inventors confirmed that the plasmon coupling effect is maximized at 650 nm when the average thickness of the gold layer deposited on the nanopillars is 100 nm, the average thickness of the gold layer deposited after PFDT coating is 80 nm, and the average diameter of the nanoparticles is 70 nm did.

According to one embodiment, the forward primer and the reverse primer are Streptococcus pneumoniae consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 A pair of primers for detecting the Ply gene; A pair of primers for detecting the OmpA gene of Chlamydophila pneumoniae consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4; A pair of primers for detecting the P6 gene of Haemophilus influenzae consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6; And it may be one or more selected from the group consisting of a primer pair for detecting the ITS1 gene of Mycoplasma pneumoniae consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8.

According to one embodiment, the forward primer and the reverse primer are a pair of primers for detecting the S gene of human coronavirus 229E (Human Coronavirus 229E) consisting of the forward primer of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10; A primer pair for detecting the M gene of human coronavirus OC43 (Human Coronavirus OC43) consisting of a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12; A pair of primers for detecting the S gene of human coronavirus NL63 (Human Coronavirus NL63) consisting of a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 14; And it may be at least one selected from the group consisting of a pair of primers for detecting the F gene of human metapneumovirus consisting of a forward primer of SEQ ID NO: 15 and a reverse primer of SEQ ID NO: 16.

According to one embodiment, the RPA reaction temperature of the kit may be 37 ℃ to 42 ℃.

According to one embodiment, the RPA reaction time of the kit may be 20 minutes to 40 minutes.

Another aspect is to prepare an isothermal amplification device comprising a plasmonic substrate having a forward primer fixed to the surface of the nanopillar; Preparing a sample expected to contain the target gene; performing a recombinase-polymerase amplification reaction by contacting the sample and the fluorophore-labeled reverse primer with the forward primer; And it provides a target gene detection method comprising the step of detecting the plasmon fluorescence amplified signal generated in the isothermal amplification device.

Hereinafter, one or more specific embodiments will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1: Fabrication of 3D Au NPOP substrate

A 3D Au NPOP structure having a protrusion-shaped surface on which nano-pillars are formed, a noble metal layer and an insulating layer are stacked, and noble metal nanoparticles bonded thereto was fabricated. (See FIG. 1) A PET polymer substrate (Toray industries Inc.) having a thickness of 188 μm was treated with Ar plasma using a custom-built 13.56 MHz RF etching equipment. The inlet Ar flow rate and reactor operating pressure during the etching process were fixed at 5 sccm and 55 mTorr. Plasma power was maintained at 100 W for an etch time of 2 minutes.

A 100 nm thick Au film was deposited on a PET nanopillar substrate on which nanopillars were formed by etching by thermal evaporation. The thermal deposition conditions were a deposition rate of 2.0 Å/s and a basic pressure of 9.6 μTorr. Gold-coated nanopillars were formed at high density by deposition of the Au film.

Then, 10 μl of a 97% PFDT solution was added to the 3D Au nanopillar substrate over 2 hours. PFDT-coated Au/PET nanopillars were formed by coating PFDT on the gold-coated nanopillars.

Finally, high-density gold nanoparticles (AuNPs) were formed on a 3D curved surface by depositing an 80 nm-thick Au film on a PFDT-coated Au/PET nanopillar substrate at a deposition rate of 0.3 Å/s using a thermal deposition system. . AuNPs, PFDT-coated, and gold-coated PET nanopillar substrates were named 3D Au NPOP substrates.

Example 2: Preparation of plasmonic RPA array chip using 3D Au NPOP substrate

The previously fabricated 3D Au NPOP substrate was cut into 5 mm x 5 mm sections and immersed in streptavidin solution (18 μM in PBS) overnight. It was washed 3 times with nuclease-free water, and washed once with PBS-Tween 1X buffer (Thermofisher). The washed substrate was immersed in 1X Elis Blocker (T & I, Gangwon-do, Korea) at room temperature for 2 hours. After washing three times with nuclease-free water, a biotin-modified forward primer (1 μL, 1 μM) was spotted and fixed on the substrate. After incubation at room temperature for 1 hour, the substrate was washed 3 times with nuclease-free water and dried. The primer-immobilized 3D Au NPOP chip was sealed and refrigerated at 4°C until used for RPA reaction.

Example 3: Method of performing plasmonic RPA

The multiplex RPA and RT-RPA performed on the 3D Au NPOP array chip were performed under most of the same conditions as the liquid phase RPA. 20 μl of the mixture for the RPA reaction without the forward primer was loaded onto the 3D Au NPOP array chip on which the forward primer was fixed. Using ThermoMixer C, the plasmonic RPA reaction was performed at 39°C for 30 minutes, and the plasmonic RT-RPA reaction was performed at 39°C for 40 minutes, respectively. The solution was removed and the array chip was washed 3 times with PBS-Tween 1X and 3 times with Nuclease-free water. After the reaction, the surface fluorescence of the 3D Au plasmonic array chip was imaged with an excitation wavelength of 675 nm and an emission wavelength of 694 nm using a fluorescence microscope. Fluorescence intensity values were analyzed by ImageJ (National Institutes of Health). All experiments were repeated three times and the average value was obtained.

Example 4: Characterization of 3D Au NPOP substrates

A 3D Au NPOP array with high-density spherical AuNPs was fabricated by using slippery perfluorodecanethiol (PFDT) coating on Au nanopillars to improve the surface movement of Au adsorbed atoms (adatom) deposited thereon. Surface migration of gold adsorbed atoms (adatom) due to the large surface free energy difference between the low surface energy of the PFDT layer (γ _PFDT = 0.015 J/m ² ) and the high surface energy of gold atoms (γ _Au = 1.53 J/m ² ) This improved, and spherical gold nanoparticles (AuNPs) were formed. Referring to the scanning electron microscope (SEM) pictures of FIGS. 1A and 1B, it can be seen that the spherical AuNPs completely cover the entire surface of the PFDT-coated gold nanopillars.

The density of the AuNP population was ~350 μm ⁻² . Referring to the transmission electron microscope (TEM) photograph of FIG. 1C, a clear cross-section view of the 3D Au NPOP substrate was confirmed, and the entire curved surface of the Au nano-pillars to which AuNPs were attached was confirmed. Depending on the distance between the nanopillars, AuNPs formed junctions between AuNPs or bridges between the nanopillars. Large AuNPs with a diameter of about 70 nm were formed on the top surface of Au nanopillars by depositing an 80 nm thick Au film.

Multiscale structures in three-dimensional space (i.e., nanoscale junctions and particles of tens of nanometers in diameter in ~100 nm Au pillars) were analyzed using surface-enhanced Raman spectroscopy (SERS) and plasmon-enhanced spectroscopy (Plasmon-enhanced spectroscopy). , PEF). Figure 2 shows the 3D NPOP structure and optical properties of the Cy5 fluorophore. The enhanced light-matter interaction between 3D plasmonic nanostructures causes high Rayleigh scattering values in the long wavelength range of 650-750 nm among the wavelength range of visible light (solid black line). According to the dark-field optical microscopy image of FIG. 2, the 3D NPOP structure exhibited an entire panel of vivid red color corresponding to wavelength-selective Rayleigh scattering under visible light illumination.

Because Rayleigh scattering reflects the plasmon resonance wavelength of plasmonic nanostructures, the 3D NPOP structure has a strong plasmon coupling effect in the long wavelength range of visible light (650–750 nm), which is consistent with the emission wavelength range of Cy5 fluorophores. .

The exact match between the resonance wavelength of the plasmonic nanostructure and the emission wavelength of the fluorophore is a physical mechanism that enhances the fluorescence signal of the plasmon-fluorophore complex for PEF-based biosensing.

Example 5: Plasmon-enhanced fluorescence (PEF) for DNA of 3D Au NPOP substrate

Plasmon-material interactions between plasmonic nanostructures and proximal fluorophores can produce increased quantum yields at optimal distances between 5 and 90 nm.

To evaluate the plasmon-enhanced fluorescence (PEF) of short-length DNA in the previously fabricated 3D Au NPOP nanostructure, 25 bp (about 8.5 nm) double-stranded DNA labeled with Cy5 and biotinylated (about 8.5 nm) was mixed with streptavidin (about 5 nm) to the coated 3D Au NPOP substrate at concentrations of 1 μM, 500 nM, 250 nM, and 100 nM (see FIG. 3A). A blank solution was used as a negative control. To compare the PEF effect, plain Au-coated PET substrates and commercially available plastic substrates for ELISA were also evaluated. The labeled Cy5 was located at a distance of about 13.5 nm from the substrate surface.

FIG. 3B shows fluorescence images after washing for three substrates, and FIG. 3C shows a bar graph showing the loading concentration versus the fluorescence intensity. Compared with normal Au-coated PET substrates and plastic substrates, 3D Au NPOP substrates exhibited highly enhanced fluorescence signals on the entire surface. The fluorescence signal of the 3D Au NPOP substrate was saturated at 500 nM dsDNA and the lower limit of detection was observed to be 100 nM. However, the normal Au-coated PET substrate showed detectable fluorescence signal at a low concentration of 500 nM dsDNA, but the fluorescence signal was in the form of very small dots and the signal intensity was very weak compared to the 3D Au NPOP substrate.

The enhancement factor of the 3D Au NPOP substrate was calculated by comparing the 3D Au NPOP substrate and the general Au coated PET substrate after background subtraction. According to FIG. 4 , the enhancers exhibited 19-fold differences at 1 μM, 15-fold at 500 nM, 35-fold at 250 nM, and 97-fold at 100 nM for each dsDNA concentration. On the other hand, under the same conditions, the plastic substrate showed no detectable fluorescence signal at the dsDNA concentration.

Next, the effect of DNA hybridization on the 3D Au NPOP substrate was evaluated. Biotinylated single-stranded DNA (25 mer) was immobilized on streptavidin-coated 3D Au plasmonic substrate, general Au substrate and plastic substrate. (See FIG. 5A) Then, Cy5-labeled complementary DNA was loaded onto the coated substrate at concentrations of 1 μM, 500 nM, 250 nM and 100 nM. Specific hybridization was confirmed using Cy5-labeled non-complementary DNA (1 μM) as a non-targeting control (NTC). In this case, the labeled Cy5 was located about 13.5 nm away from the substrate surface.

According to B and C of Figure 5, the hybridized ssDNA-binding 3D Au NPOP substrate exhibited a fluorescence pattern similar to that of the previously tested dsDNA-binding substrates. Although the fluorescence intensity was reduced than that of dsDNA (1.5 to 2 times), the 3D Au NPOP substrate showed a significantly improved fluorescence signal compared to the general Au-coated PET substrate and plastic substrate, and even ssDNA at concentrations as low as 100 nM can detect fluorescence signals. did. The decrease in fluorescence of the hybridized ssDNA-bound 3D Au NPOP substrate is most likely due to the decreased number of surface-bound fluorophores, which is due to the lower efficiency of surface solid hybridization compared to solution hybridization. Specific hybridization was confirmed because there was no detectable fluorescence in the non-target control (NTC). On the other hand, normal Au-coated substrates and plastic substrates did not show detectable fluorescence signals even at a concentration of up to 1 μM ssDNA, which was similar to the results confirmed by NTC. According to the above results, it was confirmed that high-efficiency PEF was possible even on the hybridized ssDNA-binding 3D Au NPOP substrate.

To evaluate the PEF of RPA products of long DNA amplicons on 3D Au NPOP substrates, liquid-phase RPA was performed using biotin-labeled forward primers and Cy5-labeled reverse primers and , the RPA product was attached to a 3D Au NPOP substrate coated with streptavidin (about 5 nm). For comparison, a normal Au-coated substrate and a plastic substrate coated with streptavidin were used. The appropriate size of the RPA product is reported to be 100-200 bp (about 34-68 nm) (Lobato et al. 2018). Here, the P6 gene of H. influenzae was used as the target gene. The RPA amplicon for the P6 gene is 201 bp (68.34 nm).

The forward primer immobilized to the substrate was modified with a biotin-10 thymine linker to improve substrate immobilization and hybridization. Therefore, Cy5 indicated in the RPA product was located about 76 nm away from the substrate surface. A liquid phase RPA reaction (10 ¹ copies/reaction (rxn) - 10 ⁷ copies/rxn) to the H.influenzae gene was performed at 39°C for 30 minutes. The S. pneumoniae gene at a concentration of 10 ⁷ copies/rxn was used as the NTC. According to FIG. 6, RPA amplicon of 201 bp was confirmed by agarose gel electrophoresis, which could be detected at a target gene concentration as low as 10 ² copies/rxn.

The liquid phase RPA product was then loaded onto 3D Au NPOP substrates, normal Au coated substrates and plastic substrates. All three substrates were pre-coated with streptavidin. After incubation at room temperature for 10 min and washing with PBS-Tween 20 buffer, the surfaces of the three substrates were imaged with a fluorescence microscope. 7A and 7B show fluorescence images and plots of intensity versus target gene concentration. The 3D Au NPOP substrate to which the RPA product was attached showed a fluorescence pattern similar to that of the short DNA tested previously. 3D Au NPOP substrate showed significantly improved fluorescence signal on the entire surface compared to general Au coated substrate and plastic substrate, and fluorescence detection was possible at a low target gene concentration of 10 ¹ copies/rxn. The normal Au-coated substrate showed a detectable fluorescence signal at a low concentration of 10 ⁵ copies/rxn, but the fluorescence signal was in the form of small dots and the signal intensity was very weak compared to the 3D Au NPOP substrate.

Therefore, 3D Au NPOP substrate showed 10 ⁴ times higher sensitivity than general Au coated substrate and 10 times higher than gel electrophoresis. On the other hand, the plastic substrate showed no detectable fluorescence signal at any concentration. There was no detectable fluorescence for NTC either. Therefore, it was confirmed that the 3D Au NPOP structure exhibited high PEF even for long DNA amplicons. The 3D Au NPOP substrate is expected to overcome the low efficiency of solid-state nucleic acid amplification because the sensitivity is greatly increased.

The primers used in the bacterial DNA detection experiment are shown in Table 1 below.

Bacteria	target gene	Primer sequence	Amplicon length
Streptococcus pneumoniae	Ply	F: Biotin-TTTTTTTTTTGGTCAAGTTTGGTTCTGACTTTGR: Cy5-TCTTGAACACATCTCCTGGATT	162bp
Chlamydophila pneumoniae	OmpA	F: Biotin-TTTTTTTTTTTAACTGCATGGAACCCTTCTTTAR: Cy5-GAGCTTCTGCAGTAAGTGACCAT	183bp
Haemophilus influenzae	P6	F: Biotin-TTTTTTTTTTCTCATTTAGGAGAAATCTAATGAACR: Cy5-GTGATGTCGTATTTATCAAAACC	201bp
Mycoplasma pneumoniae	ITS1	F: Biotin-TTTTTTTTTTGGTAATGGCTAGAGTTTGACTGR: Cy5-TTCACCGCTCCACATGAA	231bp

The primers used in the viral RNA detection experiment are shown in Table 2 below.

Virus	target gene	Primer sequence	Amplicon length
Human Coronavirus 229E	S	F: Biotin-TTTTTTTTTTAGAAAATGTATTTGCTGTTGAGAR: Cy5-AAACATCTGAGGAAAAACCTT	230bp
Human Coronavirus OC43	M	F: Biotin-TTTTTTTTTTTACAATGTATGTTAGGCCGATR: Cy5-ACATAAACAGCAAAACCACTA	215bp
Human Coronavirus NL63	S	F: Biotin-TTTTTTTTTTACTGTGAATGTCACCACACAR: Cy5-ACAGGCCATAAACAAGTTAAACG	218bp
Human Metapneumovirus	F	F: Biotin-TTTTTTTTTTTAAGAATGCCCTCAAAACGR: Cy5-TCTGTTGAATTGACTGAAGC	185bp

Example 6: Plasmonic RPA on 3D Au NPOP Substrate

According to FIG. 8 , a solid-phase RPA reaction (hereinafter referred to as plasmonic RPA) on a 3D Au NPOP substrate may occur simultaneously in solution and on the surface.

According to 1) of FIG. 8, the forward primer is fixed to the surface and is not included in the solution. According to 2) of FIG. 8, the target gene (dsDNA) binds to the reverse primer contained in the solution or the forward primer fixed to the substrate surface by recombinase and single-stranded DNA binding protein (SSB), and strand-displacement polymerase An elongated primer is generated by (strand-displacing polymerase). According to 3 of FIG. 8 , the reverse primer included in the solution may be elongated by binding to the extended forward primer on the surface of the substrate. On the other hand, according to 4) of FIG. 8, the reverse primer extended in solution is released and can bind to the forward primer on the substrate surface. According to 5) of FIG. 8 , the reverse primer extended as a result of the reaction may be present on the substrate surface.

To optimize the RPA reaction conditions on 3D Au NPOP substrate, the H. influenzae P6 gene was used as a target gene. A forward primer modified with biotin and ten thymine linkers was immobilized on a 3D Au NPOP substrate coated with streptavidin. Then, the RPA reaction mixture containing the Cy5-labeled reverse primer, the RPA reagent, and the target gene was loaded onto the 3D Au NPOP substrate on which the forward primer was immobilized. The RPA reaction was carried out at constant temperature and was stopped at the beginning of the washing step.

To optimize the reaction temperature, the RPA reaction was carried out at 25 °C, 37 °C, 39 °C, 42 °C and 45 °C for 30 min. A liquid phase RPA reaction was also performed for comparison. According to A of FIG. 9, as can be seen in gel electrophoresis, the reaction proceeded in the liquid phase RPA at 37°C to 42°C, but the reaction did not proceed at 25°C or 45°C. According to B of FIG. 9 , similarly, in the case of a plasmonic RPA reaction, a fluorescence signal was detected at 37°C to 42°C. This means that the Cy5 labeled reverse primer hybridized by the RPA amplicon but not at 25°C or 45°C.

Referring to the results of the fluorescence signal intensity versus temperature plot, 39 °C was selected as the RPA reaction temperature for the 3D Au NPOP substrate. The signal intensity, standard deviation and non-specific signal of the negative control (blank) were considered for optimization. The RPA reaction time was then tested. According to C of FIG. 9 , the liquid phase RPA amplicon obtained by reacting for 10 minutes was weakly detected in gel electrophoresis, and the signal continued to increase as the reaction time was increased up to 40 minutes. According to D of FIG. 9 , in the plasmonic RPA reaction, a weak fluorescence signal was detected from 10 min after the start of the reaction and a distinguishable fluorescence signal was detected from 20 min. The signal increased with reaction time up to 40 min. Solid-phase amplification can be more retarded than liquid-phase amplification due to the inherent steric hindrance of the solid phase. However, a distinct fluorescence signal was detected after 20 min amplification due to the high PEF of the 3D Au NPOP substrate.

We also optimized the reaction time of reverse transcription RPA (RT-RPA) for viral RNA detection. For the detection of viral RNA, reverse transcriptase was added to the RPA mixture in a manner similar to conventional RT-PCR. Liquid phase RT-RPA of the target gene CoV 229E S was performed at 39°C.

According to Fig. 10A, liquid phase amplification was weakly detected in gel electrophoresis after 10 min, and the signal increased with reaction time for up to 40 min, similar to that observed in liquid phase RPA. According to FIG. 10B , in plasmonic RT-RPA, a weak fluorescence signal was detected after 10 minutes, and a distinguishable fluorescence signal was detected after 20 minutes. The signal increased with reaction time for up to 40 min.

In addition, the fluorescence signal after the reaction at the above-described temperature and time for respiratory infectious bacteria ( S. pneumoniae, C. pneumoniae, M. pneumoniae ) and viruses ( CoV OC43, CoV NL63, hMPV ) was analyzed. In consideration of all reaction efficiencies, the reaction temperature was optimized to 39° C., and the reaction time was optimized to 30 minutes for plasmonic RPA and 40 minutes for plasmonic RT-RPA. Plasmonic RPA of 3D Au NPOP substrate does not differ significantly from conventional liquid-phase RPA in the time required for amplification and has excellent sensitivity, so it can be used as an array chip for high-level multi-detection, and can be used for on-site clinical diagnosis of various respiratory infections. It can be useful.

Example 7: Bacterial DNA Detection by Plasmonic RPA in 3D Au NPOP Array Chip

Using a 3D Au NPOP substrate (plasmonic RPA array chip), a 4-plex RPA array chip for detecting 4 types of bacteria S. pneumoniae, C. pneumoniae, H. influenzae and M. pneumoniae was fabricated and optimized in the above example. Detection was performed under conditions.

Biotinylated forward primers for the Ply gene of S. pneumoniae , the OmpA gene of C. pneumoniae , the P6 gene of H. influenzae and the ITS1 gene of M. pneumoniae were applied to a streptavidin-coated 3D Au NPOP substrate (width 5 mm × 5 mm). ) was immobilized. The immobilization was carried out by dot spotting each primer solution (1 μL, 1 mM) on the substrate.

The RPA reaction mixture containing the Cy5-labeled reverse primer optimized for the Ply, OmpA, P6 and ITS1 genes was loaded onto the array chip. The RPA reaction mixture did not contain a forward primer. Using only the forward primer immobilized on the surface has an advantage in that non-specific reactions between primers can be greatly reduced during highly multiplexed reactions. To evaluate the specificity of the plasmonic RPA array chip for detecting bacteria, DNA at a concentration of 10 ⁵ copies/rxn extracted from S. pneumoniae, C. pneumoniae, H. influenzae or M. pneumoniae was used as the target gene.

According to FIG. 11 , the specific fluorescence signal for the target bacterial DNA was detected at the position where the forward primer for the gene was fixed, and no identifiable fluorescence signal was detected at the position where the forward primer for the other gene was fixed. Therefore, we confirmed that the 4-plex plasmonic RPA array chip is highly specific for the detection of irradiated bacteria.

The sensitivity of the plasmonic RPA array chip for bacterial detection was evaluated and compared with that of conventional liquid-phase multiplex PCR and liquid-phase multiplex RPA. For liquid phase PCR and liquid phase RPA, primers of the same sequence and concentration as those of the plasmonic RPA array chip were used. The concentration of the forward primer was the same as that of the reverse primer. The extracted target genes of S. pneumoniae, C. pneumoniae, H. influenzae and M. pneumoniae were serially diluted from 10 ⁷ copies/rxn to 10 ¹ copies/rxn. The amplification reaction time of liquid phase RPA and plasmonic RPA was 30 minutes, and the amplification reaction time of liquid phase PCR was 80 minutes.

Liquid-phase multiplex RPA and RT-RPA reactions were performed using TwistAmp® Liquid Basic (TwistDX) according to the manufacturer's protocol. Specifically, the liquid phase RPA reaction was performed using 10 μl of TwistAmp rehydration buffer, 2 μl of E-mix buffer, 1.8 mM dNTP, and multiplex primer (250 nM for S. pneumoniae and C. pneumoniae, and 250 nM for H. influenza and M. pneumoniae). 480 nM), 1 μl of 20X Core Reaction Mix, 1 μl of 280 mM magnesium acetate and 1 μl of target gene were used. For the liquid phase RT-RPA reaction, multiplex primers for CoV 229E, CoV OC43, CoV NL63, and hMPV were all added at a concentration of 480 nM, 1 μl of reverse transcriptase (200 U/μl), 1 μl of RNase inhibitor (100 ng/μl) was introduced. Liquid phase RPA and RT-RPA were performed at 39° C. for 40 minutes, and the amplified product was confirmed by agarose gel electrophoresis.

Liquid phase multiplex PCR and RT-PCR were performed using AccuPower® PCR PreMix (Bioneer, Korea) according to the manufacturer's protocol. The primer and target gene concentrations were the same as those used in the liquid phase multiplex RPA reaction. PCR amplification was performed using a 96 well thermal cycler (Applied Biosystems). Amplification conditions were predenaturation at 95°C for 5 minutes, 35 cycle denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, and final elongation at 72°C for 5 minutes. Multiplex RT-PCR was performed using an AccuPower® RT-PCR PreMix. After cDNA synthesis at 42°C for 60 minutes, all amplification conditions were the same as those of PCR. The amplification product was confirmed by 2% agarose gel electrophoresis.

According to FIG. 12 , the fluorescence signal of plasmonic RPA increased as the target gene concentration increased. The electrophoresis results of liquid phase PCR and liquid phase RPA are shown in FIGS. 13 and 14 . The LOD for each target bacterial DNA according to each amplification method is summarized in Table 3.

DNA detection method	reaction Time	LOD (copies/reactions)
		S.pneumoniae	C. pneumoniae	H.influenzae	M.pneumoniae
Liquid phase multiplex PCR	1h 20 min	10 3	10 2	10 4	10 3
Liquid phasemultiplex RPA	30 min	10 2	10 1	10 4	10 4
Plasmonic RPAarray chip	30 min	10 1	10 1	10 4	10 3

In the case of the plasmonic RPA chip, a value obtained by adding 3 times the standard deviation to the mean of the negative control analysis results was calculated according to the IUPAC limit of detection (LOD) definition, and the minimum target gene concentration greater than this value was determined as the LOD.

Since the electrophoresis results are difficult to quantify, the minimum target gene concentration visually distinct from the negative control was determined as the LOD. In the case of S. pneumoniae , the LOD of the plasmonic RPA chip was 10 ¹ copies/rxn, which was 10 times lower than liquid phase RPA (10 ² copies/rxn) and 100 times lower than liquid phase PCR (10 ³ copies/rxn). In the case of C. pneumoniae , the LOD of the plasmonic RPA chip was 10 ¹ copies/rxn, similar to liquid phase RPA (10 ¹ copies/rxn) and 10 times lower than liquid phase PCR (10 ² copies/rxn). In the case of H. influenzae , the LOD of the plasmonic RPA chip was 10 ⁴ copies/rxn, which was similar to the LOD of liquid phase RPA (10 ⁴ copies/rxn) and liquid phase PCR (10 ⁴ copies/rxn). In the case of M. pneumoniae , the LOD of the plasmonic RPA chip was 10 ³ copies/rxn, which was 10 times lower than the liquid phase RPA (10 ⁴ copies/rxn) and similar to the liquid phase PCR (10 ³ copies/rxn). Based on these results, it was confirmed that the sensitivity of the plasmonic RPA array chip to 4 types of bacteria was more than 10 times higher than those of liquid phase multiplex RPA and PCR.

Example 8: Plasmonic RT-RPA on 3D Au NPOP Chip for Viral RNA Detection

A 4-plex RPA array chip was fabricated using a 3D Au NPOP substrate to detect CoV 229E, CoV OC43, CoV NL63 and hMPV viruses. The target genes were the S gene of CoV 229E , the M gene of CoV OC43 , the S gene of NL63, and the F gene of hMPV . To evaluate the specificity of the plasmonic RT-RPA array chip for virus detection, viral RNA extracted from CoV 229E, CoV OC43, CoV NL63 or hMPV was used as a target gene at a concentration of 10 ⁷ copies/rxn.

According to FIG. 15 , a specific fluorescence signal for the target virus was detected at the position where the forward primer for the target virus was fixed, and no fluorescence signal was detected at the position where the forward primer for the non-target virus was fixed. Therefore, it was confirmed that the plasmonic RT-RPA array chip is very specific for virus detection.

The sensitivity of the plasmonic RT-RPA array chip for virus detection was evaluated and compared with the sensitivity of conventional liquid phase multiplex RT-PCR and liquid phase multiplex RT-RPA. Target genes extracted from CoV 229E, CoV OC43, CoV NL63 and hMPV were serially diluted from 10 ⁷ copies/rxn to 10 ¹ copies/rxn. The amplification reaction time was 40 minutes for plasmonic RT-RPA and liquid phase RT-RPA, and 140 minutes for liquid phase RT-PCR. According to FIG. 16, the plasmonic RT-RPA array chip showed a distinguishable fluorescence signal in the presence of a target gene. CoV OC43, CoV NL63 and hMPV tended to increase the fluorescence signal with increasing target gene concentration, whereas CoV 229E showed saturation at concentrations above 10 ³ copies/rxn. The electrophoresis results of liquid phase RT-PCR and RT-RPA are shown in Figures 17 and 18, and the limit of detection (LOD) for each target viral gene according to the method used is summarized in Table 4.

RNA detection method	reaction Time	LOD (copies/reactions)
		CoV 229E	CoV OC43	CoV NL63	hMPV
Liquid phase multiplex RT-PCR	2h 20 min	10 4	10 4	10 5	10 4
Liquid phasemultiplex RT-RPA	40 min	10 4	10 4	10 5	10 4
Plasmonic RT-RPAarray chip	40 min	10 3	10 4	10 5	10 2

For CoV 229E and hMPV , the LOD of the plasmonic RT-RPA chip was 10 ³ copies/rxn, which was 10 times lower than that of liquid RT-RPA and liquid RT-PCR (10 ⁴ copies/rxn). For CoV OC43 and CoV NL63 , the plasmonic RT-RPA chip showed LODs of 10 ⁴ copies/rxn and 10 ⁵ copies/rxn, respectively, which were similar to those of liquid-phase RT-RPA and liquid-phase RT-PCR. Based on these results, it was confirmed that the sensitivity of the plasmonic RT-RPA array chip to four viruses was similar to or 10 times higher than that of multiple liquid phase RT-RPA and liquid phase RT-PCR. Compared to the LOD of bacterial DNA, the LOD of viral RNA was somewhat higher for all detection methods. This may be due to the difference in amplification efficiency depending on the primer and reverse transcriptase efficiency. However, the plasmonic RT-RPA chip was judged to show sensitivity corresponding to detection of liquid phase RT-RPA, confirming the feasibility of this platform. A more accurate primer design or enzyme optimization could further increase the speed and sensitivity of RT-RPA.

Plasmonic RPA and RT-RPA array chips showed high PEF for long DNA amplicons and showed sensitivity comparable to conventional liquid-phase-based amplification and detection, thereby overcoming the low amplification efficiency and sensitivity of solid-phase amplification. These results are very encouraging for solid-phase amplification and suggest the possibility of practical application of solid-phase amplification-based multiplex technology in clinical diagnosis.

Example 9: Preliminary clinical evaluation using clinical samples

Plasmon RT-RPA ArrayChip was used in clinical nasopharynx of patients tested positive or negative for CoV 229E (n = 3), CoV OC43 (n = 3), CoV NL63 (n = 3) and hMPV (n = 3). It was evaluated using a clinical nasopharyngeal swab sample.

After RNA extraction, plasmonic RT-RPA was performed according to the established procedure above. According to FIG. 19, the plasmonic RT-RPA array chip showed strong fluorescence signals for all positive samples at specific sites where primers for each virus were fixed. There was no cross-reactivity between the four viruses. There was no detectable fluorescence signal for the negative samples (n = 3). According to FIG. 20, it was confirmed that the virus positive sample was amplified and the negative sample was not amplified in gel electrophoresis of liquid phase multiple RPA. Therefore, it was confirmed that the use of the plasmonic RT-RPA array chip can successfully detect the virus from a patient's nasopharyngeal swab sample. In order to optimize the plasmonic RPA array chip for clinical applications, additional tests on many clinical samples are required, but the possibility of using the plasmonic RPA array chip for clinical multi-molecular diagnosis was confirmed. Since the plasmonic RPA array chip enables rapid, high-sensitivity, and high-level multi-diagnosis, it is expected to contribute to the development of on-site multi-molecular diagnostic systems in connection with automated pre-processing and analysis equipment.

Claims (12)

1. a substrate including nanopillars;

   a noble metal layer laminated on the surface of the substrate or nanopillars;

   an insulating layer laminated on the surface of the substrate, nano-pillars, or noble metal layer;

   noble metal nanoparticles bonded to the surface of the noble metal layer or the insulating layer; and

   Comprising a forward primer bonded to the surface of the substrate, nano-pillars, noble metal layer, insulating layer, or noble metal nanoparticles,

   Recombinase-polymerase isothermal amplification device.
2. According to claim 1,

   The forward primer comprises 10 thymine linkers,

   Recombinase-polymerase isothermal amplification device.
3. According to claim 1,

   The insulating layer is a hydrocarbon thiol (alkanethiol), a hydrocarbon disulfide (alkyldisulfide), a fluorocarbon thiol (fluorocarbon thiol), a fluorocarbon silane (fluorocarbon silane), a chlorocarbon silane (chlorocarbon silane), fluorocarbon carboxylic acid (fluorocarbon carboxylic acid) acid), at least one selected from fluorocarbon amines, fluorocarbon polymers, and derivatives thereof,

   Recombinase-polymerase isothermal amplification device.
4. According to claim 1,

   The noble metal is at least one selected from Au, Ag, Cu, Al, Pt, Pd, Ti, Rd, Ru, and alloys thereof,

   Recombinase-polymerase isothermal amplification device.
5. According to claim 1,

   The recombinant-enzyme polymerase isothermal amplification device performs plasmonic fluorescence amplification when an amplification product is generated by performing recombinant-enzyme polymerase isothermal amplification with a reverse primer labeled with a target gene and a fluorophore,

   Recombinase-polymerase isothermal amplification device.
6. A Recombinase-Polymerase Amplification (RPA) kit comprising the following devices and materials:

   a) a substrate comprising nanopillars; a noble metal layer laminated on the surface of the substrate or nanopillars; an insulating layer laminated on the surface of the substrate, nano-pillars, or noble metal layer; noble metal nanoparticles bonded to the surface of the noble metal layer or the insulating layer; And Recombinant enzyme-polymerase isothermal amplification device comprising a forward primer bound to the substrate, nano-pillars, noble metal layer, insulating layer, or the surface of the noble metal nanoparticles

   b) fluorophore-labeled reverse primer

   c) strand displacement polymerases, recombinases, and single-stranded DNA binding proteins.
7. 7. The method of claim 6,

   The forward and reverse primers consist of a sequence in which the size of the amplification product by these primer pairs is 162 to 231 bp,

   Recombinase-Polymerase Amplification Kit.
8. 7. The method of claim 6,

   The plasmon coupling effect of the plasmonic nucleic acid amplification device is maximized at the emission wavelength of the fluorophore,

   Recombinase-Polymerase Amplification Kit.
9. 7. The method of claim 6,

   The forward primer and the reverse primer,

   A pair of primers for detecting the Ply gene of Streptococcus pneumoniae consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2;

   A pair of primers for detecting the OmpA gene of Chlamydophila pneumoniae consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4;

   A pair of primers for detecting the P6 gene of Haemophilus influenzae consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6; and

   At least one selected from the group consisting of a primer pair for detecting the ITS1 gene of Mycoplasma pneumoniae consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8,

   Recombinase-Polymerase Amplification Kit.
10. 7. The method of claim 6,

    The forward primer and the reverse primer,

    A pair of primers for detecting the S gene of human coronavirus 229E (Human Coronavirus 229E) consisting of a forward primer of SEQ ID NO: 9 and a reverse primer of SEQ ID NO: 10;

    A pair of primers for detecting the M gene of human coronavirus OC43 (Human Coronavirus OC43) consisting of a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12;

    A pair of primers for detecting the S gene of human coronavirus NL63 (Human Coronavirus NL63) consisting of a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 14; and

    At least one selected from the group consisting of a primer pair for detecting the F gene of human metapneumovirus consisting of a forward primer of SEQ ID NO: 15 and a reverse primer of SEQ ID NO: 16,

    Recombinase-Polymerase Amplification Kit.
11. Regarding paragraph 6,

    The RPA reaction temperature of the kit is 37 ℃ to 42 ℃,

    Recombinase-Polymerase Amplification Kit.
12. Regarding paragraph 6,

    The RPA reaction time of the kit is 20 to 40 minutes,

    Recombinase-Polymerase Amplification Kit.

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