Plasmon photo-thermal PCR (Polymerase Chain Reaction) detection chip as well as preparation method and application thereof

Abstract

The invention discloses a plasmon photo-thermal PCR detection chip and a preparation method and application thereof.The plasmon photo-thermal PCR detection chip comprises a shell, black gold glass fibers, a PCR solution and a nucleic acid detection test strip, a cavity is formed in the shell, and the top of the cavity is communicated with the outside through an opening; the surface of the black gold glass fiber is coated with a silicon dioxide shell; the black gold glass fiber and the PCR detection solution are hermetically arranged at one end, close to the opening, in the cavity; the nucleic acid detection test strip is arranged in the cavity along the length direction, the top end of the nucleic acid detection test strip is overlapped with the photo-thermal material by 1.5-2.5 mm, a 6-FAM monoclonal antibody and BSA-Biotin are respectively fixed on a T line and a C line of the nucleic acid detection test strip, and openings are respectively formed in positions, corresponding to the T line and the C line of the nucleic acid detection test strip, in the cavity. Rapid detection can be carried out through the detection chip, the problems that in the prior art, laser serves as a light source, cost is high, operation is tedious, and detection time is long are solved, and application scenes of the detection chip include the fields of virus detection, bacterium detection and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of detection chips, and in particular to a plasmon photothermal PCR detection chip, a preparation method and an application thereof. Background Art

[0002] Currently, nucleic acid amplification tests based on polymerase chain reaction (PCR) are considered the gold standard for virus diagnosis due to their high sensitivity and accuracy. However, most PCR tests require professional operators, long amplification times, and rely on expensive machines. Therefore, traditional PCR technology is difficult to use as a rapid detection method in resource-limited areas or large-scale screening sites.

[0003] With the development of materials science, nanomaterials with photothermal effect have brought new breakthroughs in the field of rapid PCR research. Directly contacting nanomaterials with photothermal effect with PCR solution greatly improves the heating rate under the control of modulated light source. However, many rapid photothermal PCR systems rely on expensive lasers as excitation light sources, which increases the cost of the device. On the other hand, the detection of photothermal amplification products still relies on gel electrophoresis and fluorescence. These detection methods require additional post-processing steps and detection equipment, which are cumbersome to operate and prolong the detection time, obscuring the advantages of rapid thermal cycling of photothermal PCR. Summary of the invention

[0004] The present invention provides a plasmon photothermal PCR detection chip and a preparation method thereof. The plasmon photothermal PCR detection chip can use a low-cost white light LED as a plasmon photothermal excitation light source, which reduces the detection cost, is simple to operate, and has high detection efficiency. Rapid detection is performed using the plasmon photothermal PCR detection chip, which solves the problems of high cost, cumbersome operation, and long detection time in the prior art using laser as a light source. At the same time, the plasmon photothermal PCR detection chip has the characteristics of small size, fast detection rate, and simple operation. It is an ideal POCT device, which is conducive to expanding the scope of use of molecular diagnosis, enhancing the timeliness of PCR detection, and has application prospects for large-scale screening.

[0005] At the same time, the present invention also provides an application of the above-mentioned plasmon photothermal PCR detection chip in influenza A (H1N1) detection or in photothermal amplification-electrophoresis detection of Escherichia coli DNA. The above-mentioned plasmon photothermal PCR detection chip can achieve photothermal amplification-electrophoresis detection of influenza A (H1N1) virus RNA as low as 1 copy / μL, which is equivalent to the level of desktop equipment.

[0006] The technical solution provided by the present invention is as follows:

[0007] The first aspect of the present invention provides a plasmon photothermal PCR detection chip, comprising a housing, black gold glass fiber, a PCR solution, and a nucleic acid detection test strip;

[0008] A chamber is provided inside the shell, and the top of the chamber is connected to the outside through an opening;

[0009] The surface of the black gold glass fiber is wrapped with a silicon dioxide shell, and the black gold glass fiber and the PCR detection solution are sealed and arranged in the chamber near the opening end;

[0010] The nucleic acid detection test strip is arranged in the chamber along the length direction, the top of the nucleic acid detection test strip overlaps with the photothermal material by 1.5-2.5 mm, 6-FAM monoclonal antibody and BSA-Biotin are respectively fixed on the T line and C line of the nucleic acid detection test strip, and openings are respectively arranged at the positions corresponding to the T line and C line of the nucleic acid detection test strip in the chamber.

[0011] Furthermore, the nucleic acid detection test strip includes a sample pad, a conjugation pad, an NC membrane, an absorption pad and a back plate. The sample pad, conjugation pad, NC membrane and absorption pad are fixed on the back plate in sequence. During the fixing process, the two ends of all components are ensured to overlap by 1.5-2.5 mm. A nano-label is dripped on the conjugation pad and dried.

[0012] Furthermore, the nano-labels dripped onto the conjugate pad include gold nanoparticles, colored microspheres, quantum dots, magnetic nanoparticles or up-conversion nanoparticles.

[0013] Further, the PCR solution includes 5 μL PrimeScript 1Step Enzyme Mix, 1 μL influenza A H1N1 RNA, 0.4 μL forward primer, 0.4 μL reverse primer and 3.2 μL enzyme-free water;

[0014] The primer sequence of the forward primer is: Biotin-TAAAGACAAGACCAATCCTGTCACC; the primer sequence of the reverse primer is: 6-FAM-TCCCATTTAGGGCATTTTGGACAAAGC; or

[0015] The PCR solution includes 0.5 μL of E. coli RNA, 0.3 μM of forward primer, 0.3 μM of reverse primer, 0.75 U TaKaRa Z-Taq, 1 μL of 10× Z-Taq Buffer and 7.5 μL of enzyme-free water;

[0016] The primer sequence of the forward primer is: Biotin-GCTACAATGGCGCATACAAA;

[0017] The primer sequence of the reverse primer is: 6-FAM-TTCATGGAGTCGAGTTGCAG.

[0018] Furthermore, the black gold glass fiber is prepared from a mixture of glass fiber, chloroauric acid and trisodium citrate;

[0019] The black gold glass fiber is compounded with at least one of graphene, carbon black or titanium nitride.

[0020] A second aspect of the present invention provides a method for preparing a plasmon photothermal PCR detection chip, which is used to prepare the plasmon photothermal PCR detection chip as described above, comprising the following steps:

[0021] S1. Prepare black gold glass fiber with a silica shell on the surface;

[0022] S2, fix the sample pad, conjugate pad, NC membrane and absorption pad on the back plate in sequence, and ensure that the two ends of each component overlap by 1.5-2.5mm during the assembly process, and then fix the 6-FAM monoclonal antibody and BSA-Biotin on the T line and C line respectively for drying, and after drying, drop the nano-label on the test strip conjugate pad and dry it again to obtain a nucleic acid detection test strip;

[0023] S3, preparing PCR solution;

[0024] S4. Prepare a plastic shell with a chamber, with different openings at the top and bottom of the chamber respectively, seal and fix the PCR solution and the black gold glass fiber at the front of the plastic shell, fix the nucleic acid detection test strip at the middle and rear part of the chip and ensure that it overlaps with the BGGF by 1.5-2.5mm, and place the T line and C line of the nucleic acid detection test strip directly opposite the lower opening of the chamber respectively.

[0025] Furthermore, in step S1, the preparation of the black gold glass fiber with a surface coated with a silica shell is specifically as follows:

[0026] S101, cutting the glass fiber into a size of 25 mm*25 mm, and then cleaning it with deionized water, and soaking the dried glass fiber in a mixture of 1 ml 25 mM chloroauric acid and 1 ml 100 mg / ml trisodium citrate for 1 hour, until the glass fiber changes from white to black, thereby obtaining black gold glass fiber;

[0027] S102, soaking the black gold glass fiber in a 2% by volume APTMS ethanol solution overnight, washing away excess APTMS, and then immersing the black gold glass fiber in a 0.054wt% sodium silicate solution with a pH of 10.5 and heating at 95°C for 1h to obtain a black gold glass fiber with a silica shell on the surface.

[0028] Further, in step S2, the 6-FAM monoclonal antibody and the BSA-Biotin are both 1 mg / ml;

[0029] The nanotag is gold nanoparticles, and the gold nanoparticles are prepared specifically as follows: 1 ml of gold nanoparticles is taken, centrifuged at 7000 rpm for 10 min, the supernatant is removed and the solution is resuspended in 0.5 ml of deionized water, 8 μL of 1 mg / ml streptavidin is added thereto and incubated for 30 min, 50 μL of 10% BSA is added to block the residual active sites for 2 h, the solution is centrifuged at 7000 rpm for 10 min, the supernatant is removed and the solution is resuspended in 50 μL of 20 mM Tris-HCl to obtain the gold nanoparticles.

[0030] Further, in step S3, the total volume of the PCR solution is 10 μL, and the PCR solution includes 5 μL PrimeScript 1 Step Enzyme Mix, 1 μL influenza A H1N1 RNA, 0.4 μL forward primer, 0.4 μL reverse primer and 3.2 μL enzyme-free water;

[0031] The primer sequence of the forward primer is: Biotin-TAAAGACAAGACCAATCCTGTCACC; the primer sequence of the reverse primer is: 6-FAM-TCCCATTTAGGGCATTTTGGACAAAGC; or

[0032] In step S3, the PCR solution includes 0.5 μL of E. coli RNA, 0.3 μM of forward primer, 0.3 μM of reverse primer, 0.75 U of TaKaRa Z-Taq, 1 μL of 10×Z-Taq Buffer and 7.5 μL of enzyme-free water;

[0033] The primer sequence of the forward primer is: Biotin-GCTACAATGGCGCATACAAA;

[0034] The primer sequence of the reverse primer is: 6-FAM-TTCATGGAGTCGAGTTGCAG.

[0035] A third aspect of the present invention provides a use of the above-mentioned plasmon photothermal PCR detection chip in photothermal amplification-electrophoresis detection of influenza A (H1N1) virus RNA or in photothermal amplification-electrophoresis detection of Escherichia coli DNA.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. Compared with the prior art, the present invention has developed a plasmonic photothermal material BGGF with a wide spectrum and strong absorption, which can achieve plasmonic photothermal excitation using white light LEDs. Using low-cost white light LEDs to replace expensive, high-maintenance lasers as excitation light sources significantly reduces the cost of photothermal PCR equipment, which is conducive to the promotion of various detection scenarios.

[0038] 2. Compared with the prior art, after the completion of the plasmon photothermal reverse transcription-PCR, the amplified product is released onto the nucleic acid test strip, and the target nucleic acid H1N1 RNA can be efficiently detected by visual inspection. No additional equipment such as gel electrophoresis instrument, fluorescent excitation light source, and other additional operation steps such as electrophoresis sample addition are required. The invention has the characteristics of fast detection rate and simple operation. It is an ideal POCT device, which is conducive to expanding the scope of use of molecular diagnosis, enhancing the timeliness of PCR detection, and has the application prospect of large-scale screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 (a) is a physical picture of the plasmon photothermal PCR detection chip, and (b) is a schematic diagram of the photothermal reverse transcription-PCR principle;

[0040] Figure 2 Figure 1 is a characterization diagram of the BGGF photothermal material in the embodiment of the present invention, wherein (a) is a SEM image of BGGF, (b) is a UV-visible absorption spectrum of BGGF, (c) is a temperature rise effect of GF and BGGF under white light LED excitation, and (d) is a temperature rise effect of BGGF under PWM wave control of LED with different duty cycles;

[0041] Figure 3 For example, SiO 2 The inhibitory effect of coated BGGF on PCR reaction, Lane 1: DNA marker; Lane 2: PCR product; Lane 3: PCR product on GF; Lane 4: PCR product on BGGF; Lane 5: PCR product on BGGF wrapped with SiO2 coating;

[0042] Figure 4 The plasmon photothermal Comsol simulation of BGGF in an embodiment of the present invention (a) and the temperature distribution in BGGF (b) are vertical temperature gradient diagrams in BGGF;

[0043] Figure 5 This is a diagram showing the temperature variation process of reverse transcription-PCR in an embodiment of the present invention;

[0044] Figure 6Figure 1 is a graph showing the results of photothermal amplification-electrophoresis detection of influenza A (H1N1) RNA in an embodiment of the present invention, wherein (a) shows the electrophoresis results of different concentrations of influenza A (H1N1) RNA photothermal reverse transcription-PCR, (b) shows the test strip detection results of different concentrations of influenza A (H1N1) RNA photothermal reverse transcription-PCR, and (c) shows the results of 10 5 copy / ml influenza A H1N1 RNA six times photothermal reverse transcription-PCR test strip test results;

[0045] Figure 7 This is a diagram showing the results of photothermal amplification-electrophoresis detection of Escherichia coli DNA in an embodiment of the present invention. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the embodiments described below are part of the embodiments of the present application, rather than all of the embodiments. The components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.

[0047] The invention provides a plasmon photothermal PCR detection chip, comprising a shell, black gold glass fiber, a PCR solution and a nucleic acid detection test strip.

[0048] A chamber is arranged inside the shell, and the top of the chamber is communicated with the outside through an opening.

[0049] The surface of the black gold glass fiber is wrapped with a silica shell, and the black gold glass fiber and the PCR detection solution are sealed and arranged in the chamber near the opening end.

[0050] The nucleic acid test strip is arranged in the chamber along the length direction, and the top of the nucleic acid test strip overlaps with the photothermal material by 1.5-2.5 mm. 6-FAM monoclonal antibody and BSA-Biotin are respectively fixed on the T line and C line of the nucleic acid test strip, and openings are respectively arranged at the positions corresponding to the T line and C line of the nucleic acid test strip in the chamber.

[0051] Figure 1 (a) is a physical picture of the plasmon photothermal PCR detection chip, and (b) is a schematic diagram of the photothermal reverse transcription-PCR principle.

[0052] Figure 2The characterization of black gold glass fiber (BGGF) photothermal material is shown; wherein (a) is the SEM image of BGGF, (b) is the UV-visible absorption spectrum of black gold glass fiber (BGGF), (c) is the heating effect of glass fiber (GF) and black gold glass fiber (BGGF) under white light LED excitation, and (d) is the heating effect of black gold glass fiber (BGGF) under PWM wave control of LED with different duty cycles.

[0053] Black gold glass fiber is a photothermal material with a wide spectrum and strong absorption. Figure 4 The plasmon photothermal Comsol simulation of black gold glass fiber (a) and the temperature distribution in the black gold glass fiber (b) are shown. The vertical temperature gradient in BGGF.

[0054] Optionally, the nucleic acid test strip includes a sample pad, a conjugation pad, an NC membrane, an absorption pad and a back plate. The sample pad, the conjugation pad, the NC membrane and the absorption pad are fixed on the back plate in sequence. During the fixing process, ensure that both ends of all components overlap by 1.5-2.5 mm. Add a nano-label on the conjugation pad and dry it.

[0055] Optionally, the nano-labels dropped onto the conjugate pad include gold nanoparticles, colored microspheres, quantum dots, magnetic nanoparticles or up-conversion nanoparticles.

[0056] Optionally, the PCR solution includes 5 μL PrimeScript 1Step Enzyme Mix, 1 μL influenza A (H1N1) RNA, 0.4 μL forward primer, 0.4 μL reverse primer and 3.2 μL enzyme-free water.

[0057] The primer sequence of the forward primer is: Biotin-TAAAGACAAGACCAATCCTGTCACC; the primer sequence of the reverse primer is: 6-FAM-TCCCATTTAGGGCATTTTGGACAAAGC.

[0058] In other embodiments, the PCR solution includes 0.5 μL E. coli RNA, 0.3 μM forward primer, 0.3 μM reverse primer, 0.75 U TaKaRa Z-Taq, 1 μL 10×Z-Taq Buffer and 7.5 μL enzyme-free water. The primer sequence of the forward primer is: Biotin-GCTACAATGGCGCATACAAA, and the primer sequence of the reverse primer is: 6-FAM-TTCATGGAGTCGAGTTGCAG. Optionally, the black gold glass fiber is prepared from a mixture of glass fiber and trisodium citrate.

[0059] Black gold glass fiber can be compounded with graphene, carbon black or titanium nitride.

[0060] The present invention also provides a method for preparing a plasmon photothermal PCR detection chip, which is used to prepare the above-mentioned plasmon photothermal PCR detection chip, comprising the following steps:

[0061] S1. Prepare black gold glass fiber with a silica shell on the surface.

[0062] Black gold glass fiber (BGGF) was prepared by in situ reduction of gold nanoparticles on the surface of glass fiber (GF), and then a silicon dioxide (SiO2) coating was coated on the surface of the gold nanoparticles to reduce the inhibitory effect of BGGF on the PCR reaction. Figure 3 shows that the SiO 2 Investigation of the inhibitory effect of coated BGGF on PCR reaction, Lane 1: DNA marker; Lane 2: PCR product; Lane 3: PCR product on GF; Lane 4: PCR product on BGGF; Lane 5: PCR product on BGGF coated with SiO2 coating.

[0063] S2. Fix the sample pad, conjugate pad, NC membrane and absorption pad on the back plate in sequence. During the assembly process, ensure that the two ends of each component overlap by 1.5-2.5 mm. Then fix the 6-FAM monoclonal antibody and BSA-Biotin on the T line and C line respectively for drying. After drying, drop the nano-label onto the test strip conjugate pad and dry it again to obtain a nucleic acid detection test strip.

[0064] S3. Prepare PCR solution.

[0065] S4. Prepare a plastic shell with a chamber, with different openings at the top and bottom of the chamber respectively. Seal the PCR solution and black gold glass fiber at the front of the plastic shell, fix the nucleic acid test strip at the middle and rear of the chip and ensure that it overlaps with BGGF by 1.5-2.5mm, and place the T line and C line of the nucleic acid test strip directly opposite the lower opening of the chamber.

[0066] Specifically, black gold glass fiber (BGGF) is glued to a PET high-temperature tape, and then the PCR solution is dripped onto the BGGF, which is then sealed with another piece of PET tape. In order to achieve the integration of photothermal amplification and electrophoresis detection of target nucleic acids, the sealed BGGF and nucleic acid detection test strips are assembled into a 3D printed plastic shell in sequence to form a plasmon photothermal PCR detection chip.

[0067] The chamber of the plasmon photothermal PCR detection chip prepared by the present invention is a three-dimensional (3D) printed plastic shell. The BGGF containing 10 μL of PCR solution is sealed with two pieces of PET high-temperature tape and assembled on the front of the chip, and the nucleic acid detection test strip is fixed to the middle and rear part of the chip and ensures that it overlaps with the BGGF by 2 mm. Among them, the front opening of the chip exposes the BGGF so that the excitation light source directly irradiates the BGGF and then heats the PCR solution through the plasmon photothermal effect. At the same time, the chip also has openings at the positions of the T line and the C line of the nucleic acid detection test strip so that the results can be visually read after the detection is completed.

[0068] Optionally, in step S1, the preparation of black gold glass fiber with a surface coated with a silica shell is specifically as follows:

[0069] S101. Cut the glass fiber into 25 mm*25 mm size, clean it with deionized water, and soak the dried glass fiber in 1 ml 25 mM HAuCl 4 (chloroauric acid) and 1 ml of 100 mg / ml trisodium citrate mixture were reacted for 1 h until the glass fiber changed from white to black to obtain black gold glass fiber.

[0070] S102, soaking the black gold glass fiber in a 2% by volume APTMS ethanol solution overnight, washing away excess APTMS, and then immersing the black gold glass fiber in a 0.054wt% sodium silicate solution with a pH of 10.5 and heating at 95°C for 1h to obtain a black gold glass fiber with a silica shell on the surface.

[0071] Optionally, in step S2, the concentrations of 6-FAM monoclonal antibody and BSA-Biotin are both 1 mg / ml.

[0072] The nanotag is gold nanoparticles. The preparation of gold nanoparticles is specifically as follows: 1 ml of gold nanoparticles is taken, centrifuged at 7000 rpm for 10 min, the supernatant is removed and resuspended in 0.5 ml of deionized water, 8 μL of 1 mg / ml streptavidin is added thereto and incubated for 30 min, 50 μL of 10% BSA is added to block the residual active sites for 2 h, centrifuged at 7000 rpm for 10 min, the supernatant is removed and resuspended in 50 μL of 20 mM Tris-HCl to obtain gold nanoparticles.

[0073] The specific operations of step S2 are:

[0074] S201. Fix the sample pad, conjugate pad, NC membrane and absorption pad on the back plate in sequence. During the assembly process, ensure that the two ends of each component overlap by about 2 mm to ensure that the liquid can flow continuously under the action of capillary force. 6-FAM monoclonal antibody (1 mg / ml) and BSA-Biotin (1 mg / ml) are fixed on the T line and C line respectively. Then, dry the test strip at 37°C for 1 hour, cut into single test strips of 3 mm width, and store them in a desiccator for subsequent use.

[0075] S202, 15nm gold nanoparticles were prepared by reducing chloroauric acid with trisodium citrate. Take 1ml of gold nanoparticles, centrifuge at 7000rpm for 10min, remove the supernatant and resuspend in 0.5ml deionized water. Then, add 8μL 1mg / ml streptavidin and incubate for 30min. Then add 50μL 10% BSA to block the residual active sites for 2h. Centrifuge the coupled gold nanoparticles at 7000rpm for 10min. Remove the supernatant and resuspend in 50μL 20mM Tris-HCl (containing 1% BSA, 15% sucrose and 0.2% Triton X-100) to obtain a nanotag. Finally, the gold nanotag coupled with streptavidin is added dropwise to the test strip conjugate pad and dried.

[0076] Optionally, in step S3, the total volume of the PCR solution is 10 μL, and the PCR solution includes 5 μL Prime Script 1 Step Enzyme Mix, 1 μL influenza A (H1N1) RNA, 0.4 μL forward primer, 0.4 μL reverse primer and 3.2 μL enzyme-free water.

[0077] The primer sequence of the forward primer is: Biotin-TAAAGACAAGACCAATCCTGTCACC; the primer sequence of the reverse primer is: 6-FAM-TCCCATTTAGGGCATTTTGGACAAAGC.

[0078] The benchtop reverse transcription-PCR was performed on an Applied Biosystems SimpliAmp PCR instrument (Thermo Fisher Scientific). The reverse transcription-PCR program was: reverse transcription at 52°C for 5 min, 95°C for 10 s, and 60°C for 30 s for a total of 40 cycles.

[0079] The present invention has developed a BGGF with a wide spectrum and strong absorption, and at the same time, SiO is wrapped on the outer surface. 2The coating gives the material good biocompatibility. Since BGGF has the characteristics of wide spectrum and strong absorption, low-cost white light LED can be used to replace expensive and high-maintenance laser as the excitation light source. Using white light LED as the plasmon photothermal excitation light source can significantly reduce the equipment cost and facilitate its promotion to various detection scenarios.

[0080] In this embodiment, the chip prepared above is used for photothermal amplification-electrophoresis detection of influenza A (H1N1) virus RNA, specifically as follows: white light LED is used to irradiate BGGF to excite plasmon photothermal effect, k-type thermocouple monitors the temperature of PCR reagent in BGGF, and feeds back to the controller to adjust the output light intensity of LED to realize the preset reverse transcription-PCR program. After the reverse transcription-PCR is completed, the amplification product sealed in the PET tape is released onto the nucleic acid detection test strip, and the T line color development result is observed.

[0081] The hardware components required in this embodiment include: white light LED, LED constant current drive board, STM32F407 development board, ultra-fine k-type thermocouple temperature measuring wire, MAX31855 thermocouple module and fan; compile the software and burn it into the development board to realize the controllable change output of the white light LED and realize the photothermal reverse transcription-PCR process. The k-type thermocouple is fixed to the surface of the BGGF with Kapton tape to feedback the temperature of the PCR solution in real time. A white light LED is fixed on a copper heat sink with a cooling fan as a plasmon photothermal light source. The front opening of the photothermal PCR detection chip is directly facing the LED, and the white light LED directly irradiates the BGGF and then heats the PCR solution through the plasmon photothermal effect. A 1kHz pulse width modulation (PWM) signal is used to control the intensity of the LED. The fan is fixed obliquely above the BGGF to accelerate the cooling rate in the temperature cycle. The microcontroller (STM32F407) accurately controls all functions such as plasmon photothermal cycle, temperature monitoring and fan cooling. The embedded software automatically executes the reverse transcription-PCR program and displays the real-time temperature. The temperature change process of reverse transcription-PCR is as follows: Figure 5 shown.

[0082] After the reverse transcription-PCR is completed, a sharp needle is used to pierce the PET tape wrapped around BGGF, and the amplified product is released from BGGF and flows to the nucleic acid detection test strip. The amplified product flows through the sample pad, the binding pad, the T line, and the C line in sequence. When the amplified product is present, the product DNA binds to the gold nanoparticles to form a streptavidin-gold nanoparticle-product DNA complex; the reaction solution continues to flow forward, and at the T line, the anti-F-FAM antibody captures the 6-FAM labeled with the reverse primer to form a streptavidin-gold nanoparticle-product DNA-anti-6-FAM complex; when the amplified product is not present, the complex cannot be captured on the C line, and the gold nanoparticles are captured at the T line. Therefore, it is easy to determine whether H1N1 nucleic acid exists in the sample by observing whether the T line is colored.

[0083] Figure 6 The results of photothermal amplification-electrophoresis of influenza A (H1N1) RNA; (a) shows the electrophoresis results of different concentrations of influenza A (H1N1) RNA photothermal reverse transcription-PCR, (b) shows the test strip detection results of different concentrations of influenza A (H1N1) RNA photothermal reverse transcription-PCR, (c) shows 10 5 The test strip test results after six times of photothermal reverse transcription-PCR of influenza A (H1N1) RNA copy / ml.

[0084] By using the chip prepared as described above for photothermal amplification-electrophoresis detection of influenza A (H1N1) virus RNA, photothermal amplification-electrophoresis detection of influenza A (H1N1) virus RNA as low as 1 copy / μL can be achieved, which is comparable to the level of desktop equipment.

[0085] Through reasonable primer design, the plasmon photothermal PCR chip can also be applied to the field of bacterial detection.

[0086] Optionally, the PCR solution includes 0.5 μL E. coli RNA, 0.3 μM forward primer, 0.3 μM reverse primer, 0.75 U TaKaRa Z-Taq, 1 μL 10× Z-Taq Buffer and 7.5 μL enzyme-free water.

[0087] The primer sequence of the forward primer is: Biotin-GCTACAATGGCGCATACAAA and the primer sequence of the reverse primer is: 6-FAM-TTCATGGAGTCGAGTTGCAG.

[0088] The results of Escherichia coli DNA photothermal amplification-electrophoresis detection are as follows Figure 7 shown.

[0089] The above is only the best specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions within the technical scope disclosed in the present application shall be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A plasmon photothermal PCR detection chip, characterized in that: Including shell, black gold glass fiber, PCR solution and nucleic acid test strips; A chamber is provided inside the shell, and the top of the chamber is connected to the outside through an opening; The surface of the black gold glass fiber is wrapped with a silicon dioxide shell, and the black gold glass fiber and the PCR detection solution are sealed and arranged in the chamber near one end of the opening; The nucleic acid detection test strip is arranged in the chamber along the length direction, the top of the nucleic acid detection test strip overlaps with the photothermal material by 1.5-2.5 mm, 6-FAM monoclonal antibody and BSA-Biotin are respectively fixed on the T line and C line of the nucleic acid detection test strip, and openings are respectively arranged at the positions corresponding to the T line and C line of the nucleic acid detection test strip in the chamber.

2. The plasmon photothermal PCR detection chip according to claim 1, characterized in that: The nucleic acid detection test strip comprises a sample pad, a conjugation pad, an NC membrane, an absorption pad and a back plate. The sample pad, the conjugation pad, the NC membrane and the absorption pad are fixed on the back plate in sequence. During the fixing process, the two ends of all components are ensured to overlap by 1.5-2.5 mm. A nano-label is dripped on the conjugation pad and dried.

3. The plasmon photothermal PCR detection chip according to claim 2, characterized in that: The nano-labels dripped onto the conjugate pad include gold nano-particles, colored micro-spheres, quantum dots, magnetic nano-particles or up-conversion nano-particles.

4. The plasmon photothermal PCR detection chip according to any one of claims 1 to 3, characterized in that: The PCR solution includes 5 μL PrimeScript 1Step Enzyme Mix, 1 μL influenza A H1N1 RNA, 0.4 μL forward primer, 0.4 μL reverse primer and 3.2 μL enzyme-free water; The primer sequence of the forward primer is: Biotin-TAAAGACAAGACCAATCCTGTCACC; the primer sequence of the reverse primer is: 6-FAM-TCCCATTTAGGGCATTTTGGACAAAGC; or The PCR solution includes 0.5 μL of E. coli RNA, 0.3 μM of forward primer, 0.3 μM of reverse primer, 0.75 U of TaKaRaZ-Taq, 1 μL of 10×Z-Taq Buffer and 7.5 μL of enzyme-free water; The primer sequence of the forward primer is: Biotin-GCTACAATGGCGCATACAAA; The primer sequence of the reverse primer is: 6-FAM-TTCATGGAGTCGAGTTGCAG.

5. The plasmon photothermal PCR detection chip according to claim 4, characterized in that: The black gold glass fiber is prepared from a mixture of glass fiber, chloroauric acid and trisodium citrate; The black gold glass fiber is compounded with at least one of graphene, carbon black or titanium nitride.

6. A method for preparing a plasmon photothermal PCR detection chip, for preparing the plasmon photothermal PCR detection chip according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Prepare black gold glass fiber with a silica shell on the surface; S2, fix the sample pad, conjugate pad, NC membrane and absorption pad on the back plate in sequence, and ensure that the two ends of each component overlap by 1.5-2.5mm during the assembly process, and then fix the 6-FAM monoclonal antibody and BSA-Biotin on the T line and C line respectively for drying, and after drying, drop the nano-label on the test strip conjugate pad and dry it again to obtain a nucleic acid detection test strip; S3, preparing PCR solution; S4. Prepare a plastic shell with a chamber, with different openings at the top and bottom of the chamber respectively, seal and fix the PCR solution and the black gold glass fiber at the front of the plastic shell, fix the nucleic acid detection test strip at the middle and rear part of the chip and ensure that it overlaps with the BGGF by 1.5-2.5mm, and place the T line and C line of the nucleic acid detection test strip directly opposite the lower opening of the chamber respectively.

7. The preparation method according to claim 6, characterized in that: In step S1, the preparation of the black gold glass fiber with a silicon dioxide shell on the surface is specifically as follows: S101, cutting the glass fiber into a size of 25 mm*25 mm, and then cleaning it with deionized water, and soaking the dried glass fiber in a mixture of 1 ml 25 mM chloroauric acid and 1 ml 100 mg / ml trisodium citrate for 1 hour, until the glass fiber changes from white to black, thereby obtaining black gold glass fiber; S102, soaking the black gold glass fiber in a 2% by volume APTMS ethanol solution overnight, washing away excess APTMS, and then immersing the black gold glass fiber in a 0.054wt% sodium silicate solution with a pH of 10.5 and heating at 95°C for 1h to obtain a black gold glass fiber with a silica shell on the surface.

8. The preparation method according to claim 6, characterized in that: In step S2, the 6-FAM monoclonal antibody and the BSA-Biotin are both 1 mg / ml; The nanotag is gold nanoparticles, and the gold nanoparticles are prepared specifically as follows: 1 ml of gold nanoparticles is taken, centrifuged at 7000 rpm for 10 min, the supernatant is removed and the solution is resuspended in 0.5 ml of deionized water, 8 μL of 1 mg / ml streptavidin is added thereto and incubated for 30 min, 50 μL of 10% BSA is added to block the residual active sites for 2 h, the solution is centrifuged at 7000 rpm for 10 min, the supernatant is removed and the solution is resuspended in 50 μL of 20 mM Tris-HCl to obtain the gold nanoparticles.

9. The preparation method according to claim 6, characterized in that: In step S3, the total volume of the PCR solution is 10 μL, and the PCR solution includes 5 μL PrimeScript 1 Step Enzyme Mix, 1 μL influenza A H1N1 RNA, 0.4 μL forward primer, 0.4 μL reverse primer and 3.2 μL enzyme-free water; The primer sequence of the forward primer is: Biotin-TAAAGACAAGACCAATCCTGTCACC; the primer sequence of the reverse primer is: 6-FAM-TCCCATTTAGGGCATTTTGGACAAAGC; or In step S3, the PCR solution includes 0.5 μL of E. coli RNA, 0.3 μM of forward primer, 0.3 μM of reverse primer, 0.75 U of TaKaRa Z-Taq, 1 μL of 10×Z-Taq Buffer and 7.5 μL of enzyme-free water; The primer sequence of the forward primer is: Biotin-GCTACAATGGCGCATACAAA; The primer sequence of the reverse primer is: 6-FAM-TTCATGGAGTCGAGTTGCAG.

10. Use of the plasmon photothermal PCR detection chip as claimed in any one of claims 1 to 5 in photothermal amplification-electrophoresis detection of influenza A (H1N1) virus RNA or photothermal amplification-electrophoresis detection of Escherichia coli DNA.

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