KR102659913B1 - Device for recombinase-polymerase amplification that generates plasmon-enhanced fluorescence signal at the same time as isothermal amplification - Google Patents

Abstract

The recombinant enzyme-polymerase isothermal amplification device of the present invention performs surface fluorescence amplification while performing isothermal amplification, so it has excellent sensitivity and specificity and can be miniaturized, so it can be used for pathogen diagnosis in clinical settings.

Description

{Device for recombinase-polymerase amplification that generates plasmon-enhanced fluorescence signal at the same time as 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, is the fourth leading cause of death across all age groups and was the leading cause of death among children under 5 worldwide in 2017. Lower respiratory tract infections are caused by various bacteria and viruses, but it is difficult to distinguish them based on symptoms. Therefore, in order to avoid unnecessary use of antibiotics and provide early treatment, technology is needed to quickly and accurately identify pathogens in the field.

PCR has excellent sensitivity and specificity, so it is mainly used to identify pathogens. However, PCR requires complex processes such as sophisticated temperature change devices, long amplification times, and confirmation by electrophoresis, so it is difficult to apply to on-site diagnosis.

Recently, various isothermal amplification technologies are being studied for on-site diagnosis. Isothermal amplification technology is attracting attention for use in point-of-care diagnostics because it enables miniaturization of temperature devices, but its amplification efficiency, detection sensitivity, and specificity do not reach those of PCR, which limits its clinical application.

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

Republic of Korea Public Gazette No. 10-2013-0011338 (2013.01.30)

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

One aspect includes a substrate containing nanopillars; A noble metal layer laminated on the surface of the substrate or nanopillar; an insulating layer laminated on the surface of the substrate, nanopillar, or noble metal layer; Precious metal nanoparticles bound to the surface of the noble metal layer or insulating layer; and a forward primer bound to the surface of the substrate, nanopillar, noble metal layer, insulating layer, or noble metal nanoparticle.

The solid-phase Recombinase Polymerase Amplification (RPA) device performs RPA in a solid-phase by immobilizing a forward primer on the surface of the substrate, noble metal layer, insulating layer, or noble metal nanoparticle. can do.

The present invention has the following advantages. First, since the forward primer is fixed to a substrate 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 on the reverse primer, a strong fluorescence signal can be obtained simultaneously with isothermal amplification. Although plasmon resonance is a previously known technology, as in the present invention, by performing recombinant enzyme-polymerase isothermal amplification (RPA) consisting of an immobilized forward primer and a fluorophore-labeled reverse primer, plasmonic fluorescence amplification is achieved simultaneously with isothermal amplification. There is no known solid-state isothermal amplification device that can generate a strong signal and detect the signal quickly.

The nanopillar is a pillar-shaped nanostructure with a diameter of about 10 nanometers. The nanopillars may be referred to as nanopillars.

The substrate including the nanopillars may have a plurality of nanopillars spaced apart from each other on the surface. The substrate may be 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 can be formed by depositing a Raman active material, for example, by thermal evaporation, vapor deposition, or solution process.

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

The insulating layer can be formed by depositing a precious metal layer, for example, by thermal evaporation, vapor deposition, or solution process.

According to one embodiment, the insulating layer is alkane thiol, alkyldisulfide, fluorocarbon thiol, fluorocarbon silane, chlorocarbon silane, fluorocarbon It may be one or more selected from fluorocarbon carboxylic acid, fluorocarbon amine, and fluorocarbon polymer, and according to an embodiment, it may be PFDT.

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

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

The recombinant enzyme-polymerase isothermal amplification device can be manufactured in the form of a microarray, microchip, etc., and can also be manufactured in the form of a multiple microarray that can detect multiple target genes simultaneously.

Another aspect is a Recombinase-Polymerase Amplification (RPA) kit comprising the following devices and materials, wherein the devices and materials include: a) a substrate containing nanopillars; A noble metal layer laminated on the surface of the substrate or nanopillar; an insulating layer laminated on the surface of the substrate, nanopillar, or noble metal layer; Precious metal nanoparticles bound to the surface of the noble metal layer or insulating layer; and a recombinant enzyme-polymerase isothermal amplification device comprising a forward primer bound to the noble metal layer, the insulating layer, or the surface of the noble metal nanoparticle, b) a fluorophore-labeled reverse primer, c) a strand displacement polymerase, a recombinase, and single-stranded DNA binding proteins.

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

According to one embodiment, the forward primer and reverse primer may be composed of sequences such that the size of the amplification product by these primer pairs is 162 to 231 bp.

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 one embodiment, the fluorophore is Cy5, and the recombinant enzyme-polymerase isothermal amplification device may have a maximum plasmon coupling effect at 650 nm. In particular, the present inventor confirmed that the plasmon coupling effect was maximized at 650 nm when the average thickness of the gold layer deposited on the nanofiller was 100 nm, the average thickness of the gold layer deposited after PFDT coating was 80 nm, and the nanoparticle diameter was 70 nm on average. did.

According to one embodiment, the forward primer and reverse primer are a primer pair 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 primer pair 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 primer pair 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 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 reverse primer are a primer pair for detecting the S gene of 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, consisting of a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12; A primer pair for detecting the S gene of 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 one or more selected from the group consisting of a primer pair for detecting the F gene of human metapneumovirus consisting of the forward primer of SEQ ID NO: 15 and the reverse primer of SEQ ID NO: 16.

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

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

Another aspect includes preparing an isothermal amplification device including a plasmonic substrate with a forward primer fixed to the surface of the nanopillar; Preparing a sample expected to contain a target gene; Performing a recombinase-polymerase amplification reaction by contacting the sample and the reverse primer labeled with a fluorophore with the forward primer; and detecting a plasmonic fluorescence amplified signal generated from the isothermal amplification device.

The device according to one embodiment has excellent sensitivity and specificity because surface fluorescence amplification occurs simultaneously with isothermal amplification.

The device according to one embodiment can be miniaturized and has better sensitivity and specificity than existing isothermal amplification technology, so it can quickly and accurately identify pathogens in the field.

Figures 1A and B show SEM images of the 3D NPOP substrate surface. Figure 1C and D show TEM images of nanopillars of a 3D NPOP substrate.
Figure 2A shows that the 3D NPOP substrate has a very strong intensity of Rayleigh scattering within the long red range wavelength (650-750 nm) of visible light, and the excitation spectrum (red dotted line) and emission spectrum (red dotted line) of the Cy5 fluorophore This indicates that it matches the red shaded area). B in Figure 2 is a far-field optical microscope (OM) image showing strong Rayleigh scattering in the 3D NPOP structure.
Figure 3 shows the results of binding short-length (25bp) double-stranded (ds) DNA to a 3D gold NPOP substrate and confirming PEF.
Figure 4 shows a plot of fluorescence intensity enhancer versus Cy5 labeled dsDNA concentration. The enhancement factor was determined by dividing the average spot intensity of the 3D gold NPOP substrate by the intensity of the corresponding normal Au substrate.
Figure 5 shows the results of confirming PEF after binding a short-length (25bp) single-stranded (ss) DNA to a 3D gold NPOP substrate and hybridizing it with the complementary strand.
Figure 6 shows the results of gel electrophoresis of the RPA product for the P6 gene of H.influenzae and a schematic diagram of the process of binding the RPA product to a 3D gold NPOP substrate.
Figure 7 shows the PEF results for the RPA product bound to a 3D gold NPOP substrate.
Figure 8 is a schematic diagram showing the process of solid-state RPA execution in a plasmonic chip combining a forward primer with a 3D gold NPOP substrate.
Figure 9 shows the results of optimization experiments on temperature and reaction time when running plasmonic RPA on a 3D gold NPOP substrate. A and C are agarose gel electrophoresis results of liquid RPA products, and B and D are fluorescence microscopy images and fluorescence intensity of solid-phase plasmonic RPA performed on a 3D gold NPOP substrate.
Figure 10 shows the results of an optimization experiment for reaction time when executing plasmonic RT-RPA on a 3D gold NPOP substrate. A is the agarose gel electrophoresis result of the liquid RT-RPA product, and B is the fluorescence image and fluorescence intensity of the solid-phase plasmonic RT-RPA performed on a 3D gold NPOP substrate.
Figure 11 shows the results and specificity of four types of bacterial DNA detection using a plasmonic RPA array chip made with a 3D gold NPOP substrate. The abbreviations for bacteria used in the drawing are S: Streptococcus pneumoniae, H: Haemophilus influenzae, C: Chlamydia pneumoniae, and M: Mycoplasma pneumoniae.
Figure 12 shows the results and detection sensitivity of four types of bacterial DNA detection using a plasmonic RPA array chip made with a 3D gold NPOP substrate.
Figure 13 shows the results of detection of four types of bacterial DNA by liquid phase multiplex PCR.
Figure 14 shows the results of detection of four types of bacterial DNA using liquid phase multiplex RPA.
Figure 15 shows the results and specificity of four types of viral RNA detection using a plasmonic RT-RPA array chip made with a 3D gold NPOP substrate. The abbreviations of the viruses used in the drawing are E: Corona virus 229E, O: Corona virus OC43, N: Corona virus NL63, h: human metapneumovirus.
Figure 16 shows the results and sensitivity of four types of viral RNA detection using a plasmonic RT-RPA array chip made with a 3D gold NPOP substrate.
Figure 17 shows the results of detection of four types of viral RNA by liquid phase multiplex RT-PCR.
Figure 18 shows the results of detection of four types of viral RNA using liquid phase multiplex RT-RPA.
Figure 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 viruses.
Figure 20 shows the results of liquid phase multiplex RT-RPA using nasopharyngeal swab samples from patients infected with four types of viruses.

Hereinafter, one or more embodiments will be described in more detail through examples. However, these examples are intended to illustrate 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 was fabricated, which had a protruding surface with nanopillars formed, a noble metal layer and an insulating layer were stacked, and noble metal nanoparticles were combined. (See Figure 1) PET polymer substrates (Toray industries Inc.) with a thickness of 188 μm were 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. The plasma power was maintained at 100 W for 2 minutes of etching time.

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

Then, 10 μl of 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 the 3D curved surface by depositing an 80 nm thick Au film on the PFDT-coated Au/PET nanopillar substrate at a deposition rate of 0.3 Å/s using a thermal evaporation system. . The AuNPs, PFDT coating, 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. Washed three times with nuclease-free water and once with PBS-Tween 1X buffer (Thermofisher). The cleaned substrate was immersed in 1X Elis Blocker (T & I, Gangwon-do, Korea) at room temperature for 2 hours. It was washed three times with nuclease-free water and fixed by spotting biotin-modified forward primer (1 μl, 1 μM) on the substrate. After incubation at room temperature for 1 hour, the substrate was washed three times with nuclease-free water and dried. The 3D Au NPOP chip with immobilized primers was sealed and refrigerated at 4°C until used in the RPA reaction.

Example 3: How to perform plasmonic RPA

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

Example 4: Characterization of 3D Au NPOP substrate

A 3D Au NPOP array formed with high-density spherical AuNPs was fabricated by coating Au nanopillars with slippery perfluorodecanethiol (PFDT) to improve the surface movement of Au adatoms deposited on them. Surface movement of gold adatoms 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 the gold atoms (γ _Au = 1.53 J/m ² ). This was improved, and spherical gold nanoparticles (AuNPs) were formed. Referring to the scanning electron microscopy (SEM) images of Figures 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 ㎛ ^-2 . Referring to the transmission electron microscope (TEM) photo in Figure 1C, a clear cross-section view of the 3D Au NPOP substrate was confirmed, and the entire curved surface of the Au nanopillars with AuNPs attached was confirmed. Depending on the distance between nanopillars, AuNPs formed junctions between AuNPs or bridges between nanopillars. Large AuNPs with a diameter of approximately 70 nm were formed on the top surface of the Au nanopillars by depositing an 80 nm thick Au film.

Multiscale structures in three-dimensional space (i.e., nanoscale junctions and particles with diameters of tens of nanometers on ~100 nm Au pillars) can be studied using surface-enhanced Raman spectroscopy (SERS) and plasmon-enhanced spectroscopy. , PEF) has proven to be very advantageous. Figure 2 shows the 3D NPOP structure and optical properties of Cy5 fluorophore. Enhanced light-matter interaction between 3D plasmonic nanostructures causes high Rayleigh scattering values in the long wavelength range of 650-750 nm in the visible light wavelength range (solid black line). According to the dark-field optical microscopy image in Figure 2, the 3D NPOP structure displayed 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 the plasmonic nanostructure, 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 the Cy5 fluorophore. .

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

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

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

To evaluate plasmon-enhanced fluorescence (PEF) for short lengths of DNA in the previously fabricated 3D Au NPOP nanostructure, Cy5-labeled and biotinylated 25 bp (approximately 8.5 nm) double-stranded DNA was incubated with streptavidin (approx. 5 nm) was bound to the coated 3D Au NPOP substrate at concentrations of 1 μM, 500 nM, 250 nM, and 100 nM (see Figure 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. Labeled Cy5 was located approximately 13.5 nm away from the substrate surface.

Figure 3B shows fluorescence images after the washing process for the three substrates, and Figure 3C shows the loading concentration versus fluorescence intensity as a bar graph. Compared with plain Au-coated PET substrates and plastic substrates, the 3D Au NPOP substrates exhibited greatly enhanced fluorescence signals over 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, although the plain Au-coated PET substrate showed a detectable fluorescence signal at a low concentration of 500 nM dsDNA, 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 with a regular Au-coated PET substrate after background subtraction. According to Figure 4, the enhancer showed a 19-fold difference at 1 μM, 15-fold at 500 nM, 35-fold at 250 nM, and 97-fold at 100 nM depending on dsDNA concentration. On the other hand, under the same conditions, the plastic substrate showed no detectable fluorescence signal at any dsDNA concentration.

Next, we evaluated the effect of DNA hybridization on 3D Au NPOP substrates. Biotinylated single-stranded DNA (25 mer) was immobilized on a streptavidin-coated 3D Au plasmonic substrate, a regular Au substrate, and a plastic substrate. (See Figure 5A) Cy5-labeled complementary DNA was then 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, labeled Cy5 was located approximately 13.5 nm from the substrate surface.

According to Figures 5B and C, the hybridized ssDNA-bound 3D Au NPOP substrate showed a fluorescence pattern similar to that of the previously tested dsDNA-bound substrates. Although the fluorescence intensity was reduced compared to dsDNA (1.5 to 2 times), the 3D Au NPOP substrate showed greatly improved fluorescence signals compared to regular Au-coated PET substrates and plastic substrates, and fluorescence signals could be detected even at concentrations of ssDNA as low as 100 nM. did. The decrease in fluorescence of the hybridized ssDNA-bound 3D Au NPOP substrate is most likely due to the reduced number of fluorophores bound to the surface, 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, plain Au-coated substrates and plastic substrates did not show detectable fluorescence signals even at concentrations of up to 1 μM ssDNA, which was similar to the results found in NTC. According to the above results, it was confirmed that high-efficiency PEF is possible even with hybridized ssDNA-bound 3D Au NPOP substrate.

To evaluate the PEF of RPA products of long DNA amplicon on 3D Au NPOP substrate, liquid-phase RPA was performed using biotin-labeled forward primer and Cy5-labeled reverse primer. , the RPA product was attached to a 3D Au NPOP substrate coated with streptavidin (approximately 5 nm). For comparison, a plain 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 (approximately 34 to 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 on the substrate was modified with a biotin-10 thymine linker to improve substrate immobilization and hybridization. Therefore, Cy5 displayed in the RPA product was located approximately 76 nm away from the substrate surface. Liquid-phase RPA reaction (10 ¹ copies/reaction(rxn) - 10 ⁷ copies/rxn) for H.influenzae genes 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 Figure 6, a 201bp RPA amplicon was confirmed by agarose gel electrophoresis, which could be detected at a target gene concentration as low as 10 ² copies/rxn.

The liquid RPA product was then loaded onto the 3D Au NPOP substrate, plain Au coated substrate, and plastic substrate. All three substrates were pre-coated with streptavidin. After incubation at room temperature for 10 minutes and washing with PBS-Tween 20 buffer, the surfaces of the three substrates were imaged by fluorescence microscopy. Figures 7A and B 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 the short DNA tested previously. The 3D Au NPOP substrate showed greatly improved fluorescence signals on the entire surface compared to regular Au-coated substrates and plastic substrates, and fluorescence detection was possible at a low target gene concentration of 10 ¹ copies/rxn. The regular 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, the 3D Au NPOP substrate showed sensitivity ¹⁰ times higher than that of a regular Au coated substrate and 10 times higher than that of gel electrophoresis. On the other hand, the plastic substrate showed no detectable fluorescence signal at any concentration. There was also no detectable fluorescence for NTC. Therefore, it was confirmed that the 3D Au NPOP structure exhibits high PEF even for long DNA amplicons. Since the 3D Au NPOP substrate has greatly increased sensitivity, it is expected to be able to overcome the low efficiency of solid-phase nucleic acid amplification.

Primers used in bacterial DNA detection experiments are listed 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-TTTTTTTTTTTCTCATTTAGGAGAAATCTAATGAACR: Cy5-GTGATGTCGTATTTATCAAAACC 201bp Mycoplasma pneumoniae ITS1 F: Biotin-TTTTTTTTTTGGTAATGGCTAGAGTTTGACTGR: Cy5-TTCACCGCTCCACATGAA 231bp

Primers used in the viral RNA detection experiment are listed in Table 2 below.

Virus Target gene Primer sequence Amplicon length Human Coronavirus 229E S F: Biotin-TTTTTTTTTTAGAAAATGTATTTGCTGTTGAGAR: Cy5-AAACATCTGAGGAAAAACCTT 230bp HumanCoronavirusOC43 M F: Biotin-TTTTTTTTTTTACAATGTATGTTAGGCCGATR: Cy5-ACATAAACAGCAAAACCACTA 215bp HumanCoronavirus 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 Figure 8, solid-phase RPA reaction (hereinafter referred to as plasmonic RPA) on a 3D Au NPOP substrate can 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) in Figure 8, the target gene (dsDNA) binds to the reverse primer contained in the solution or the forward primer immobilized on the surface of the substrate by recombinase and SSB (single-stranded DNA binding protein), and strand-displacement polymerase. An elongated primer is created by strand-displacing polymerase. According to 3) of FIG. 8, the reverse primer contained in the solution may be elongated by binding to the elongated forward primer on the surface of the substrate. On the other hand, according to 4) of FIG. 8, the extended reverse primer in the solution can be released and bind to the forward primer on the surface of the substrate. According to 5) of FIG. 8, the reverse primer extended as a result of the reaction may exist on the surface of the substrate.

To optimize the RPA reaction conditions on the 3D Au NPOP substrate , the H. influenzae P6 gene was used as a target gene. The forward primer modified with biotin and 10 thymine linkers was immobilized on a 3D Au NPOP substrate coated with streptavidin. Then, the RPA reaction mixture containing Cy5-labeled reverse primer, RPA reagent, and 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 stopped at the beginning of the washing step.

To optimize the reaction temperature, the RPA reaction was performed at 25°C, 37°C, 39°C, 42°C, and 45°C for 30 min. For comparison, a liquid-phase RPA reaction was also performed. According to Figure 9A, as seen in gel electrophoresis, the reaction of liquid RPA proceeded at 37°C to 42°C, but did not proceed at 25°C or 45°C. According to Figure 9B, similarly, in the case of the plasmonic RPA reaction, the fluorescence signal was detected at 37°C to 42°C. This means that the Cy5-labeled reverse primer hybridized with 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. For optimization, the signal intensity, standard deviation and non-specific signal of negative control (blank) were considered. We then tested the RPA response time. According to Figure 9C, the liquid RPA amplicon obtained by reacting for 10 minutes was weakly detected in gel electrophoresis, and the signal continued to increase as the reaction time increased up to 40 minutes. According to Figure 9D, in the plasmonic RPA reaction, a weak fluorescence signal was detected from 10 minutes after the start of the reaction, and a distinguishable fluorescence signal was detected from 20 minutes. The signal increased with reaction time up to 40 min. Solid-phase amplification may be more delayed than liquid-phase amplification due to the inherent steric hindrance of the solid phase. However, due to the high PEF of the 3D Au NPOP substrate, a distinct fluorescence signal was detected after 20 minutes of amplification.

Additionally, the reaction time of reverse transcription RPA (RT-RPA) was optimized for viral RNA detection. For 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 Figure 10A, liquid-phase amplification was weakly detected in gel electrophoresis starting after 10 min, and the signal increased with reaction time for up to 40 min, similar to that observed for liquid-phase RPA. According to Figure 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 was analyzed for respiratory infectious bacteria ( S. pneumoniae, C. pneumoniae, M. pneumoniae ) and viruses ( CoV OC43, CoV NL63, hMPV ) after reaction at the above-mentioned temperature and time. Considering all reaction efficiencies, the reaction temperature was optimized at 39°C, and the reaction time was optimized at 30 minutes for plasmonic RPA and 40 minutes for plasmonic RT-RPA. Plasmonic RPA of 3D Au NPOP substrate has excellent sensitivity and does not differ significantly from existing liquid RPA in the time required for amplification, so it can be used as an array chip for high-level multiplex detection and can be used for on-site clinical diagnosis of various respiratory infections. It can be useful.

Example 7: Detection of bacterial DNA by plasmonic RPA in 3D Au NPOP array chip

A 4-plex RPA array chip for detecting four types of bacteria , S. pneumoniae, C. pneumoniae, H. influenzae , and M. pneumoniae, was fabricated using a 3D Au NPOP substrate (plasmonic RPA array chip) and optimized in the above example. Detection was performed under the following 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 on. The immobilization was performed by dot spotting each primer solution (1 μL, 1 mM) on the substrate.

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

According to Figure 11, a specific fluorescence signal for the target bacterial DNA was detected at the position where the forward primer for that gene was fixed, and no discernible fluorescence signal was detected at the position where the forward primer for another gene was fixed. Therefore, we confirmed that the 4-plex plasmonic RPA array chip is highly specific for the detection of the investigated 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 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 for liquid phase RPA and plasmonic RPA was 30 minutes, and the amplification reaction time for 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 consisted of 10 μl of TwistAmp rehydration buffer, 2 μl of E-mix buffer, 1.8 mM dNTP, and multiplex primers (250 nM for S. pneumoniae and C. pneumoniae, 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 added at a concentration of 480 nM, 1 μl of reverse transcriptase (200U/μl), and 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 amplification products were 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 concentrations of primers and target genes 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 a final extension reaction at 72°C for 5 minutes. Multiplex RT-PCR was performed using AccuPower® RT-PCR PreMix. After cDNA synthesis at 42°C for 60 minutes, all amplification conditions were the same as for PCR. Amplification products were confirmed by 2% agarose gel electrophoresis.

According to Figure 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 Figures 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/reaction) S.pneumoniae C.pneumoniae H. influenzae M.pneumoniae Liquid phase
Multiplex PCR 1h 20min 10 ³ 10 ² 10 ⁴ 10 ³ Liquid phasemultiplex RPA 30min 10 ² 10 ¹ 10 ⁴ 10 ⁴ Plasmonic RPAarray chip 30min 10 ¹ 10 ¹ 10 ⁴ 10 ³

For the plasmonic RPA chip, according to the IUPAC limit of detection (LOD) definition, the average of the negative control analysis results plus 3 times the standard deviation was calculated, and the minimum target gene concentration greater than this value was determined as the LOD.

Because electrophoresis results are difficult to quantify, the LOD was determined as the minimum target gene concentration that was visually distinct from the negative control. In the case of S. pneumoniae, the LOD of the plasmonic RPA chip was 10 ¹ copies/rxn, 10 times lower than liquid RPA (10 ² copies/rxn) and 100 times lower than liquid 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 found to be 10 ⁴ copies/rxn, which was similar to the LOD of liquid 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 liquid phase RPA (10 ⁴ copies/rxn) and similar to liquid phase PCR (10 ³ copies/rxn). Based on these results, it was confirmed that the sensitivity of the plasmonic RPA array chip to four types of bacteria was more than 10 times higher than that 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 the target gene at a concentration of 10 ⁷ copies/rxn.

According to Figure 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 that 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 Figure 16, the plasmonic RT-RPA array chip showed a distinguishable fluorescence signal in the presence of the target gene. CoV OC43, CoV NL63 and hMPV tended to increase their fluorescence signals 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/reaction) CoV 229E CoV OC43 CoV NL63 hMPV Liquid phase
Multiplex RT-PCR 2h 20min 10 ⁴ 10 ⁴ 10 ⁵ 10 ⁴ Liquid phasemultiplex RT-RPA 40min 10 ⁴ 10 ⁴ 10 ⁵ 10 ⁴ Plasmonic RT-RPAarray chip 40min 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 the LODs 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 differences in amplification efficiency depending on primer and reverse transcriptase efficiency. However, the feasibility of this platform was confirmed by determining that the plasmonic RT-RPA chip showed sensitivity equivalent to liquid-phase RT-RPA detection. The speed and sensitivity of RT-RPA could be further increased by more accurate primer design or enzyme optimization.

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

Example 9: Preliminary clinical evaluation using clinical samples

The plasmonic RT-RPA arraychip was used to detect clinical nasopharyngeal tissue from patients who tested positive or negative for CoV 229E (n = 3), CoV OC43 (n = 3), CoV NL63 (n = 3), and hMPV (n = 3). It was assessed using clinical nasopharyngeal swab samples.

After RNA extraction, plasmonic RT-RPA was performed according to the established procedure above. According to Figure 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. No detectable fluorescent signal was present for the negative samples (n = 3). According to Figure 20, it was confirmed that virus positive samples were amplified and negative samples were not amplified in gel electrophoresis of liquid phase multiple RPA. Therefore, it was confirmed that the virus could be successfully detected from a patient's nasopharyngeal swab sample using the plasmonic RT-RPA array chip. Although further testing on many clinical samples is required to optimize the plasmonic RPA array chip for clinical applications, we confirmed the feasibility of using the plasmonic RPA array chip for clinical multimolecular diagnostics. The plasmonic RPA array chip is capable of rapid, highly sensitive, and high-level multiple diagnosis, so it is expected to contribute to the development of an on-site multiple molecular diagnostic system in conjunction with automated preprocessing and analysis equipment.

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> Device for recombinase-polymerase amplification that generates plasmon-enhanced fluorescence signal at the same time as isothermal amplification <130> PN200086 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Ply gene of Streptococcus pneumoniae <400> 1 ggtcaagttt ggttctgact ttg 23 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Ply gene of Streptococcus pneumoniae <400> 2 tcttgaacac atctcctgga tt 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for OmpA gene of Chlamydophila pneumoniae <400> 3 aactgcatgg aacccttctt ta 22 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for OmpA gene of Chlamydophila pneumoniae <400> 4 gagcttctgc agtaagtgac cat 23 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for P6 gene of Haemophilus influenzae <400> 5 ctcatttagg agaaatctaa tgaac 25 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for P6 gene of Haemophilus influenzae <400> 6 gtgatgtcgt atttatcaaa acc 23 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for ITS1 gene of Mycoplasma pneumoniae <400> 7 ggtaatggct agagtttgac tg 22 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for ITS1 gene of Mycoplasma pneumoniae <400> 8 ttcaccgctc cacatgaa 18 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for S gene of Human Coronavirus 229E <400> 9 agaaaatgta tttgctgttg aga 23 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for S gene of Human Coronavirus 229E <400> 10 aaacatctga ggaaaaacct t 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for M gene of Human Coronavirus OC43 <400> 11 acaatgtatg ttaggccgat 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for M gene of Human Coronavirus OC43 <400> 12 acataaacag caaaaccact a 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for S gene of Human Coronavirus NL63 <400> 13 actgtgaatg tcaccacaca 20 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for S gene of Human Coronavirus NL63 <400> 14 acaggccata aacaagttaa acg 23 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for F gene of Human Metapneumovirus <400> 15 taagaatgcc ctcaaaacg 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for F gene of Human Metapneumovirus <400> 16 tctgttgaat tgactgaagc 20

Claims (12)

A substrate containing nanopillars;
A noble metal layer laminated on the surface of the substrate or nanopillar;
an insulating layer laminated on the surface of the substrate, nanopillar, or noble metal layer;
Precious metal nanoparticles bound to the surface of the noble metal layer or insulating layer; and
Comprising a forward primer bound to the surface of the substrate, nanopillar, noble metal layer, insulating layer, or noble metal nanoparticle,
In a recombinant enzyme-polymerase isothermal amplification device, the forward primer contains biotin and streptavidin at the end,
Recombinant enzyme-polymerase isothermal amplification device. According to paragraph 1,
The forward primer contains 10 thymine linkers,
Recombinant enzyme-polymerase isothermal amplification device. According to paragraph 1,
The insulating layer includes alkanethiol, alkyldisulfide, fluorocarbon thiol, fluorocarbon silane, chlorocarbon silane, and fluorocarbon carboxylic acid. carboxylic acid), fluorocarbon amine, and fluorocarbon polymer,
Recombinant enzyme-polymerase isothermal amplification device. According to paragraph 1,
The noble metal is one or more selected from Au, Ag, Cu, Al, Pt, Pd, Ti, Rd, Ru, and alloys thereof,
Recombinant enzyme-polymerase isothermal amplification device. According to paragraph 1,
The recombinant enzyme - polymerase isothermal amplification device performs recombinant enzyme - polymerase isothermal amplification with a reverse primer labeled with a target gene and a fluorophore, and when an amplification product is generated, plasmonic fluorescence amplification occurs.
Recombinant enzyme-polymerase isothermal amplification device. Recombinase-Polymerase Amplification (RPA) kit comprising the following a) to c):
a) Substrate containing nanopillars; A noble metal layer laminated on the surface of the substrate or nanopillar; an insulating layer laminated on the surface of the substrate, nanopillar, or noble metal layer; Precious metal nanoparticles bound to the surface of the noble metal layer or insulating layer; And the primer bound to the surface of the substrate, nanopillar, noble metal layer, insulating layer, or noble metal nanoparticle is a recombinant enzyme-polymerase isothermal amplification device including a forward primer containing biotin and streptavidin at the end.
b) Fluorophore-labeled reverse primer
c) Strand displacement polymerase, recombinase, and single-stranded DNA binding protein. According to clause 6,
The forward primer and reverse primer are composed of sequences such that the size of the amplification product by these primer pairs is 162 to 231 bp,
Recombinase-polymerase amplification kit. According to clause 6,
The plasmon coupling effect of the kit is maximized at the emission wavelength of the fluorophore,
Recombinase-polymerase amplification kit. According to clause 6,
The forward primer and reverse primer are,
A primer pair 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 primer pair 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 primer pair 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. According to clause 6,
The forward primer and reverse primer are,
A primer pair for detecting the S gene of 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, consisting of a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12;
A primer pair for detecting the S gene of 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 the forward primer of SEQ ID NO: 15 and the reverse primer of SEQ ID NO: 16,
Recombinase-polymerase amplification kit. According to clause 6,
The RPA reaction temperature of the kit is 37°C to 42°C,
Recombinase-polymerase amplification kit. According to clause 6,
The RPA reaction time of the kit is 20 to 40 minutes,
Recombinase-polymerase amplification kit.