Material of photosensitive amplification-photo-thermal detection test paper for tacA, tacB and tacC and application

Abstract

The invention discloses a material of light-triggered amplification-photo-thermal detection test paper for tacA, tacB and tacC. A sample is irradiated by a coaxial reflection LED light source outside 504nm, a photosensitizer CsYF4 (at) Yb2O3 provides photoelectrons and heat for aRPA through light triggering, a target gene in a positive sample is used as a template to initiate an amplification reaction, three single-stranded DNA amplification products are obtained, and the three single-stranded DNA amplification products are used for detecting the tacA, the tacB and the tacC; a photosensitizer CsYF4 (at) Yb2O3 is modified by three nucleic acid chains to obtain three signal probes, a detection region captures an amplification product and the signal probes in a sandwich form, a photo-thermal temperature change signal positively related to the content of the amplification product is given, and tacA, tacB and tacC in a sample are quantified according to the temperature change signal. Photoelectron and photothermal effects are generated by using the photoactivity of the photosensitizer CsYF4 (at) Yb2O3, the temperature is rapidly controlled, the photosensitizer is in direct contact with an amplification system on a molecular level, photothermal detection test paper detection is carried out, and the photothermal efficiency is remarkably improved.

Description

Technical Field

[0001] The invention relates to a material for a photosensitive amplification-photothermal detection test paper for tacA, tacB and tacC and application thereof, belonging to the technical field of inspection and detection. Background Art

[0002] Lodging resistance genes are genes that enable plants to survive the threat of drastic climate change. Rapid identification of lodging resistance genes through molecular biological methods can help simplify field management and reduce labor costs. In addition, genetic testing can also help breeders optimize breeding strategies and provide regulatory agencies with a scientific basis to evaluate the safety and compliance of genetically modified crops. Currently, the commonly used methods for detecting genetically modified crops have problems such as high cost, cumbersome operation, and difficulty in on-site detection. Therefore, there is an urgent need to develop on-site detection technology for the simultaneous detection of lodging resistance genes.

[0003] The detection methods of lodging resistance genes are mainly based on nucleic acid amplification, including polymerase chain reaction (PCR) and isothermal amplification technology. Because PCR amplification takes a long time and relies on professionals and equipment, it is not suitable for rapid on-site detection.

[0004] Recombinase Polymerase Amplification (RPA) is a new type of nucleic acid amplification method. Its amplification principle mainly relies on the synergistic effect of multiple enzymes to specifically amplify the target DNA or RNA sequence under isothermal conditions. The main enzymes and proteins involved in the RPA amplification process include:

[0005] (1) Recombinase - responsible for recognizing and binding to specific DNA sequences. In the RPA reaction, the recombinase binds to the primer to form nucleoprotein filaments, which can recognize and bind to the target region of double-stranded DNA;

[0006] (2) Single-stranded DNA binding protein (SSB) - stabilizes the opened DNA double helix and prevents it from reforming into a double-stranded structure. In the RPA reaction, SSB binds to the displaced DNA chain and stabilizes the resulting D-loop structure.

[0007] (3) Strand displacement DNA polymerase - Using the primer as the starting point, it synthesizes a new DNA chain along the template. In the RPA reaction, the strand displacement DNA polymerase extends the primer on the single-stranded template, thereby achieving DNA amplification.

[0008] The RPA amplification process can be summarized into the following steps: recombinase and primer binding, strand displacement and primer insertion, polymerase-mediated DNA synthesis, and cyclic amplification.

[0009] The target gene is amplified by RPA reaction combined with asymmetric amplification at a constant temperature of 37-42°C. No rapid temperature change instrument such as PCR instrument is required. It is suitable for portable rapid nucleic acid detection and can amplify the target DNA by 10 within 10-20 minutes. 9 The reaction time is shorter, and different concentrations of primers are used for reaction. High-concentration primers are used as non-restrictive primers. When low-concentration primers are exhausted, single-stranded DNA is generated to meet downstream operation or detection requirements. It is expected to be widely used in disease diagnosis, pathogen detection, genetically modified crop detection and food safety detection.

[0010] Light-triggered amplification is achieved by adding photosensitizers to the amplification system, using light-emitting diodes (LEDs) or lasers as light sources, and evenly dispersing photothermal materials in the amplification system. After LED excitation, it provides energy supply for nucleic acid amplification.

[0011] There are significant differences in the reaction conditions between RPA and LAMP, especially in terms of temperature. On the one hand, RPA is usually carried out under lower constant temperature conditions, while LAMP can operate over a wider temperature range. This temperature difference may affect the performance and stability of the photosensitizer, thereby affecting its light-controlled effect in RPA.

[0012] On the other hand, there are also differences in the enzymes used by RPA and LAMP. RPA relies on the action of recombinase and polymerase, while LAMP mainly uses DNA polymerase. Different enzymes may have different sensitivities to photosensitizers and their products, thus affecting the effect of light-controlled amplification.

[0013] On the other hand, the reaction mechanisms of RPA and LAMP are also different. RPA achieves nucleic acid amplification through recombinase-mediated primer exchange and polymerase-mediated chain extension, while LAMP initiates and maintains the amplification reaction by forming a specific stem-loop structure. This mechanistic difference may lead to significant differences in the light-triggered amplification mode of photosensitizers in RPA and LAMP.

[0014] In addition, the challenges of using photosensitizer-mediated, light-triggered RPA amplification include compatibility issues (the need to ensure that the nanomaterials are compatible with the RPA components), photoexcitation accuracy issues (the need to precisely control photoexcitation conditions to avoid interfering with the reaction), stability maintenance (the need to optimize reaction conditions to maintain amplification stability), and detection method innovation (new detection methods need to be developed or optimized to adapt to photoexcitation conditions).

[0015] There are significant differences between the external coaxial reflective LED light source and other LED light sources in many aspects, mainly reflected in the structural design, light characteristics, application scenarios and optical efficiency. The external coaxial reflective LED light source is mainly composed of a lateral light source and a beam splitter. The light emitted by the light source first passes through the beam splitter and then is reflected parallel to the object to be measured. Other LED light sources such as ring light sources, strip light sources, and linear light sources have different structural designs, but usually do not include a beam splitter component. For example, the ring light source directly illuminates the top of the object to be measured, while the strip light source provides a variety of illumination effects such as direct, oblique and photometric. Due to the use of a beam splitter design, the light of the external coaxial reflective LED light source can be evenly illuminated on the surface of the object after passing through the beam splitter, reducing the interference caused by shadows and reflections. At the same time, this light source usually has high brightness and uniformity, which can highlight the contour features of the object. Although other LED light sources also have their own light characteristics, such as the ring light source can solve the problem of diagonal illumination shadows, and the strip light source can adjust the illumination angle and installation at will, but in comparison, the external coaxial reflective LED light source has more advantages in light uniformity and reducing shadows. The external coaxial reflection LED light source beam splitter design reduces light loss and thus improves optical efficiency.

[0016] At present, there is no report on the simultaneous detection of multiple lodging resistance genes using external coaxial reflection LED light source excitation-light-triggered asymmetric recombinase polymerase isothermal amplification combined with photothermal detection test paper technology. Summary of the invention

[0017] Technical issues: The existing isothermal amplification technology of recombinase polymerase for lodging resistance genes has the following limitations: the efficiency of existing light-triggered amplification photosensitizers is not ideal; light-triggered amplification technology has not yet been applied to the simultaneous amplification of multiple lodging resistance genes; although the external coaxial reflective LED light source has the advantages of light uniformity and reduced shadows, it has not yet been combined with light-triggered amplification and photothermal detection test paper technology for the detection of lodging resistance genes. The challenges of using photosensitizer-mediated, light-triggered RPA amplification include compatibility issues (need to ensure that the nanomaterials are compatible with the RPA components), light excitation accuracy issues (need to precisely control light excitation conditions to avoid interfering with the reaction), stability maintenance (need to optimize reaction conditions to maintain amplification stability), and detection method innovation (need to develop or optimize new detection methods to adapt to light excitation conditions).

[0018] Technical solution: The present invention adopts the following technical solution.

[0019] The first object of the present invention is to provide a material for light-triggered amplification-photothermal detection test paper for tacA, tacB, and tacC, wherein the material for light-triggered amplification-photothermal detection test paper for tacA, tacB, and tacC is a photosensitizer CsYF4@Yb2O3, and the preparation method of the photosensitizer CsYF4@Yb2O3 comprises the following steps:

[0020] (1) Add 140-160 mg of EDTA to 20-30 mL of ultrapure water and stir for 5-10 min to obtain a mixed solution;

[0021] (2) Add Y(NO3)3 and Yb2O3 with a concentration of 0.6-0.8M to the mixed solution and stir rapidly for 30-40 minutes;

[0022] (3) Add 0.8-1.2M NH4F and CsF to the mixed solution, stir for 30-40 minutes, and heat in a reactor at 180-220°C for 23-25 ​​hours. After the reaction is completed, the initial product is obtained;

[0023] (4) After cooling, washing and drying, the initial product is obtained as photosensitizer CsYF4@Yb2O3.

[0024] As an optional embodiment, the volume of Y(NO3)3 used in step (2) is 0.8-1.0 mL, and the volume of Yb2O3 used is 0.1-0.3 mL, preferably 1 mL Y(NO3)3 and 0.2 mL Yb2O3.

[0025] As an optional embodiment, the volume of NH4F and CsF used in step (3) is 2-4 mL, preferably 3 mL.

[0026] As an optional embodiment, the drying comprises drying in an oven at 65-75° C. for 2-3 h, preferably drying at 70° C. for 2 h.

[0027] The second purpose of the present invention is to provide a method for light-triggered aRPA and detection of lodging resistance genes tacA, namely Ta-CCR, tacB, namely TaCM, and tacC, namely TaCAD, using the photothermal effect of photosensitizer CsYF4@Yb2O3. The method includes two links: light-triggered amplification and photothermal detection test paper detection. First, a 504nm external coaxial reflection LED light source is used to irradiate the amplification system to excite the photosensitizer CsYF4@Yb2O3 in the amplification system to produce photoelectrons and heat, provide energy for aRPA, and Ta-CCR, TaCM, and TaCAD are used as templates to trigger the amplification reaction to obtain a single-stranded amplification product of the DNA template; then, the amplification product and the signal probe are captured in a sandwich form by photothermal detection test paper, and a photothermal temperature change signal positively correlated with the content of the amplification product is given to achieve quantitative detection of the target gene. The isothermal amplification system includes the following components: a primer set, a photothermal material CsYF4@Yb2O3 and a DNA template, upstream primers and downstream primers of varying concentrations, and other necessary components.

[0028] Specifically, the present invention provides a method for simultaneously detecting Ta-CCR, TaCM, and TaCAD, comprising the following steps:

[0029] S1. Light-triggered amplification

[0030] Under the irradiation of 504nm external coaxial reflection LED light source, the sample to be tested is amplified by aRPA isothermal amplification system to obtain an amplification product, wherein:

[0031] The aRPA isothermal amplification system includes the following components: a primer set, a photosensitizer CsYF4@Yb2O3, a DNA template, a lyophilized powder, and a buffer solution, wherein the DNA template is the lodging resistance gene tacA, namely Ta-CCR, tacB, namely TaCM, and tacC, namely TaCAD;

[0032] S2. Photothermal test paper test

[0033] Capture probes were fixed on the T and C regions of the test strip to obtain a multi-target photothermal detection test strip for the target genes Ta-CCR, TaCM, and TaCAD light-triggered aRPA products;

[0034] The photosensitizer CsYF4@Yb2O3 was modified with nucleic acid chains of Ta-CCR, TaCM, and TaCAD to obtain photothermal signal probes;

[0035] The amplification product is detected using the photothermal signal probe and the photothermal detection test paper. The detection area of ​​the photothermal detection test paper captures the amplification product and the photothermal signal probe in a sandwich form, and gives a photothermal temperature change signal positively correlated with the content of the amplification product under the irradiation of a 504nm external coaxial reflection LED light source. The Ta-CCR, TaCM, and TaCAD in the sample to be tested are qualitatively and / or quantitatively determined based on the temperature change signal.

[0036] As an optional embodiment, in the aRPA isothermal amplification system, the concentration of each group of DNA templates is 4%-6% v / v.

[0037] As an optional embodiment, the primer set includes three sets of primers, each set of primers includes a front primer (F) and a rear primer (R).

[0038] As an optional embodiment, in the light-triggered aRPA system, the ratio of upstream and downstream primers in each set of primers is 1:3-1:9 or 3:1-9:1, and when the amplification system is 50 μL, the total amount of upstream and downstream primers is 40 nmol-60 nmol, and the concentration of each primer in each set of primers is adjustable within a certain range.

[0039] As an optional embodiment, the aRPA isothermal amplification system also includes the following components: lyophilized powder, amplification buffer solution, ddH2O and other components.

[0040] As an optional embodiment, the photothermal signal probe is used independently of the test paper body, or is dropped and dried on the conjugate pad and the conjugate pad is integrated on the test paper.

[0041] As an optional implementation, the irradiation parameters of the 504nm external coaxial reflection LED light source are power 30-60W and irradiation time 10-60min.

[0042] Beneficial Effects

[0043] The photosensitive material for anti-lodging genes provided by the present invention utilizes the photoactivity of the photosensitizer CsYF4@Yb2O3 to generate photoelectrons and photothermal effects, can quickly control temperature and directly contact the amplification system at the molecular level, and is significantly more efficient than the traditional water bath. aRPA is used to amplify the target gene, and the amplified product is a single-stranded DNA, which does not require additional modification or enzyme cleavage, and is directly combined with the signal probe and capture probe according to the principle of base complementary pairing, thereby reducing the detection cost. The 504nm external coaxial reflection LED light source has more advantages in light uniformity and shadow reduction, and has greater advantages for light-controlled reactions and photothermal detection test strips. In addition, the photosensitizer is used as a signal probe for the photothermal detection test strip, providing a sensitive and quantifiable signal. The whole set of methods can quickly achieve efficient aRPA amplification and detection of multiple target DNAs. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a schematic diagram of the working process of the present invention;

[0045] Figure 2 The photothermal temperature rise of the photothermal material of the present invention;

[0046] Figure 3 The electrophoresis diagram of the products amplified by the light-triggered aRPA and conventional RPA of the present invention on negative and positive samples;

[0047] Figure 4 This is a photo of the colorimetric and photothermal detection test strips of the light-triggered aRPA of the present invention;

[0048] Figure 5 The light-triggered aRPA of the present invention is combined with a lateral flow chromatography test paper for photothermal quantification;

[0049] Figure 6 The electrophoresis diagrams of the aRPA of the present invention in a water bath for 10 minutes and light-triggered amplification at different times;

[0050] Figure 7 The present invention uses a scattered light LED and a 504nm external coaxial reflection LED light source to respectively perform electrophoresis of the products of light-triggered aRPA. DETAILED DESCRIPTION

[0051] The present invention can be better understood according to the following examples. However, the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0052] The challenges of using photosensitizer-mediated, light-triggered RPA amplification include compatibility issues (need to ensure that the nanomaterials are compatible with the RPA components), light excitation accuracy issues (need to accurately control light excitation conditions to avoid interfering with the reaction), stability maintenance (need to optimize reaction conditions to maintain amplification stability), and detection method innovation (need to develop or optimize new detection methods to adapt to light excitation conditions). Based on this, the present invention discloses a photothermal material for anti-lodging genes Ta-CCR, TaCM, and TaCAD, and a method for using it in light-triggered asymmetric recombinase polymerase isothermal amplification, i.e., aRPA combined with photothermal detection paper for detecting anti-lodging genes. The method includes two steps: light-triggered aRPA amplification and photothermal detection paper detection:

[0053] First, light-triggered aRPA amplification was performed, specifically including: simultaneously irradiating the sample with a 504nm external coaxial reflection LED light source, wherein the photosensitizer CsYF4@Yb2O3 provided photoelectrons and heat for aRPA after light triggering, and the target gene in the positive sample served as a template to trigger the amplification reaction, thereby obtaining a large amount of single-stranded DNA amplification products.

[0054] Secondly, the photothermal detection test paper is used for detection, which specifically includes: modifying CsYF4@Yb2O3 with three nucleic acid chains, Ta-CCR, TaCM, and TaCAD, to obtain three signal probes. The detection area of ​​the photothermal detection test paper captures the amplification product and the signal probe in a sandwich form, and gives a photothermal temperature change signal that is positively correlated with the content of the amplification product. According to the temperature change signal, Ta-CCR, TaCM, and TaCAD in the original sample are further quantified.

[0055] The present invention uses the photoactivity of the photosensitizer CsYF4@Yb2O3 to generate photoelectrons and photothermal effects, quickly controls the temperature and directly contacts the amplification system at the molecular level, and then performs color development detection with a photothermal detection paper, thereby significantly improving the photothermal efficiency.

[0056] Example

[0057] The following embodiment of the present application discloses a method for realizing a light-triggered aRPA system and detection of resistance genes Ta-CCR, TaCM and TaCAD by using the photothermal effect of photosensitizer CsYF4@Yb2O3, including the following steps:

[0058] (1) Preparation of photosensitizer CsYF4@Yb2O3:

[0059] Add 140-160mg of EDTA to a 50mL beaker, add 25mL of ultrapure water and stir rapidly for 5min. Then add 0.8-1.0mL of Y(NO3)3 with a molar concentration of 0.6-0.8mol / L, 0.1-0.3mL of Yb2O3 with a molar concentration of 0.6-0.8mol / L and stir rapidly for 30min. Subsequently, add 2-4mL of NH4F with a molar concentration of 0.8-1.2mol / L and 2-4mL of CsF with a molar concentration of 0.8-1.2mol / L, and stir continuously for 30min until a milky white solution is produced. After transferring the solution to a 200℃ autoclave, react for 24h, cool to room temperature, wash alternately with ultrapure water and alcohol 2-3 times, and dry at 70℃ for 2h to obtain CsYF4@Yb2O3 nanocomposite material.

[0060] (2) Primer sequence design:

[0061] According to the aRPA principle, taking a target gene sequence as an example, an amplification primer set is designed for the target gene sequence, and the primer set includes a front primer (F) and a rear primer (R).

[0062] (3) Plasmid DNA extraction:

[0063] The microorganisms collected from the sample or the preserved strains containing the target genes (Ta-CCR, TaCM and TaCAD) are cultured in LB medium for 24 hours or not, and plasmid DNA is extracted. The specific method is: plasmid DNA is extracted using a DNA extraction kit, and the kit includes RNase A, Buffer S, Solution I, Solution II, Solution III, Wash Solution, Elution Buffer, EZ-10 Column, and Collection Tube.

[0064] As an optional embodiment, the plasmid DNA extraction method can also be any one of the adsorption column method, magnetic bead method, CTAB method, etc.

[0065] (4) Construction of optically triggered aRPA system:

[0066] A light-triggered aRPA system was constructed using the extracted plasmid DNA as a template. The amplification system included the following components: a primer set, a photosensitizer CsYF4@Yb2O3 (referring to the photosensitizer CsYF4@Yb2O3 solution prepared in step (1)), a lyophilized powder, an amplification buffer solution, ddH2O, and the DNA template extracted in step (3).

[0067] (5) External coaxial reflection LED light source excitation:

[0068] The amplification system is irradiated with a 504nm external coaxial reflection LED to excite the photosensitizer in the amplification system and provide the energy required for aRPA.

[0069] In one example, the wavelength band of the 504nm external coaxial reflection LED light source is 500-510nm.

[0070] In one example, the irradiation parameters of the 504nm external coaxial reflection LED light source are power 30-60W and irradiation time 10-30min.

[0071] (6) Construction and application of photothermal detection test strips:

[0072] Three types of photothermal signal probes were obtained by modifying the photosensitizer CsYF4@Yb2O3 with three nucleic acid chains, and T probe and C probe were fixed in the detection area and quality control area of ​​the test paper respectively to obtain multi-target photothermal detection test paper for aRPA products of target genes Ta-CCR, TaCM, and TaCAD. The amplified products were detected using photothermal detection test paper, and the temperature signal was read by a smartphone for quantification.

[0073] The principle of the present invention is as follows Figure 1 As shown. The method is efficient, convenient and accurate, and is suitable for the detection of Ta-CCR, TaCM and TaCAD. The present invention can realize the efficient on-site amplification of lodging resistance genes, and the amplification is driven by the photothermal effect of the photosensitizer CsYF4@Yb2O3 excited by a 504nm external coaxial reflection LED.

[0074] The following examples use Ta-CCR, TaCM, and TaCAD as examples of resistance genes to fully and in detail illustrate the entire amplification method.

[0075] Embodiment 1:

[0076] A method for light-triggering aRPA using a photosensitizer CsYF4@Yb2O3, the method comprising the following steps:

[0077] (1) Preparation of photosensitizer CsYF4@Yb2O3:

[0078] Add 150mg of EDTA to a 50mL beaker, add 25mL of ultrapure water and stir rapidly for 5min. Then add 1.0mL of Y(NO3)3 with a molar concentration of 0.6mol / L and 0.1mL of Yb2O3 with a molar concentration of 0.6mol / L and stir rapidly for 30min. Subsequently, add 3mL of NH4F with a molar concentration of 1mol / L and 4mL of CsF with a molar concentration of 1mol / L, and stir continuously for 30min until a milky white solution is produced. After transferring the solution to a 200℃ autoclave, react for 24h, cool to room temperature, wash it alternately with ultrapure water and alcohol 3 times, and dry it at 70℃ for 2h to obtain CsYF4@Yb2O3 nanocomposite material.

[0079] The photosensitizer CsYF4@Yb2O3 solution was irradiated with a 504nm laser and compared with the solutions of CsYF4 and Yb2O3 with the same concentration. The photothermal heating curves of the three are as follows: Figure 2 As shown, it can be seen that the heating efficiency of the CsYF4@Yb2O3 complex is significantly improved compared with that of a single substance, which is beneficial to improving its efficiency as a photosensitizer.

[0080] (2) Primer sequence design:

[0081] According to the aRPA principle, an amplification primer set is designed for the target gene sequence. The primer set includes a front primer (F) and a rear primer (R). The primer sequences are shown in Table 1:

[0082] Table 1 Primer sequences of the light-triggered aRPA system of the present invention

[0083]

[0084] (3) Plasmid DNA extraction:

[0085] The preserved bacteria containing the target genes (Ta-CCR, TaCM, TaCAD) were cultured in LB medium for 24 hours, and plasmid DNA was extracted. The specific method was: plasmid DNA was extracted using a DNA extraction kit, and the kit included RNase A, Buffer S, Solution I, Solution II, Solution III, Wash Solution, Elution Buffer, EZ-10 Column, and Collection Tube.

[0086] (4) Construction of light-triggered aRPA system: The amplification system includes the following components: a primer set, a photosensitizer CsYF4@Yb2O3 (referring to the photosensitizer CsYF4@Yb2O3 solution prepared in step (1)), a lyophilized powder, an amplification buffer solution, ddH2O and a DNA template extracted in step (3).

[0087] Under the premise of maintaining the final volume at 25 μL, the concentrations of the components and the reaction volume of the amplification system are shown in Table 2 below.

[0088] Table 2 Concentrations of components and reaction volumes of the light-triggered aRPA system of the present invention

[0089]

[0090]

[0091] (5) External coaxial reflection LED light source excitation:

[0092] The aRPA amplification system was irradiated with a 504nm external coaxial reflection LED light source to excite the photothermal material in the aRPA amplification system to provide the energy required for aRPA. The excitation power was 30W and the excitation time was 10min.

[0093] (6) Construction and application of photothermal detection test strips:

[0094] Three photothermal signal probes were obtained by modifying the photosensitizer CsYF4@Yb2O3 with three nucleic acid chains. The specific method was as follows: 20 μL of three nucleic acid chains were added to 2 mL of photosensitizer CsYF4@Yb2O3 respectively, 50 μL of sodium citrate buffer was added after oscillation and mixing, and the mixture was incubated at 37°C for 30 min. Then, 100 μL of HEPES buffer was added and the mixture was incubated at 37°C for 30 min. The mixture was centrifuged and 150 μL of photothermal reconstitution solution was added to dissolve the mixture, thereby obtaining three photothermal signal probes of photosensitizer CsYF4@Yb2O3 modified with three nucleic acid chains.

[0095] T probe and C probe were fixed in the detection area and quality control area of ​​the test strip, respectively, to obtain a multi-target photothermal detection test strip for aRPA products of target genes Ta-CCR, TaCM, and TaCAD.

[0096] The probe sequences of the photothermal detection test strip for detecting aRPA amplification products are shown in Table 3 below.

[0097] Table 3 Probe sequences of amplified products detected by the photothermal test strip in this embodiment

[0098]

[0099] The constructed multi-target photothermal test strip is used to detect the amplified product and provide it with photoheat, thereby stimulating the photothermal effect of the photosensitizer CsYF4@Yb2O3 in the photothermal signal probe, and realizing the detection efficacy of the photothermal test strip; and the photothermal signal of the T zone on the photothermal test strip is read by a smartphone equipped with an infrared thermal imaging accessory, and the presence or absence of the target object is judged according to the change phenomenon of the photothermal signal, and a qualitative test is performed; and a quantitative test can be further performed according to the intensity of the temperature change signal, where the standard curve used in the quantitative test can be characterized and drawn according to the concentration gradient method.

[0100] (7) Specificity verification

[0101] After adding all reagents except primers and template to 6 light-triggered aRPA test tubes, add

[0102] ①tacA template, tacB primer, tacC primer;

[0103] ②tacA template, tacA primer, tacB primer, tacC primer;

[0104] ③tacB template, tacC primer;

[0105] ④tacB template, tacB primer, tacC primer;

[0106] ⑤tacC template, tacB primer;

[0107] ⑥tacC template, tacB primer, tacC primer;

[0108] Amplification and detection result analysis:

[0109] Gel electrophoresis images such as Figure 3 As shown, the electrophoresis of the positive sample showed the target gene band, and there were amplification products of the anti-lodging resistance gene; the electrophoresis of the negative sample did not show any amplification products; in addition, compared with the single amplification band of conventional RPA, the image of the aRPA positive sample will show two bands with different molecular weights in the same lane, the larger molecular weight is double-stranded DNA (dsDNA), and the smaller molecular weight band is single-stranded DNA (ssDNA). Once again, the method of using photosensitizer-mediated, light-triggered RPA amplification needs to solve the following problems: compatibility issues (need to ensure that the nanomaterials are compatible with the RPA components), light excitation accuracy issues (need to accurately control the light excitation conditions to avoid interfering with the reaction), and stability maintenance (need to optimize the reaction conditions to maintain amplification stability).

[0110] Figure 4 The photo of the test paper shows that the test paper can specifically detect the amplification products of specific target genes in the aRPA system in colorimetric and photothermal modes respectively.

[0111] Figure 5 This is a concentration gradient detection experiment for the three target genes Ta-CCR, TaCM and TaCAD, and a quantitative curve is drawn for the three target genes based on the photothermal signal of the test paper. The above results verify that the present invention can successfully aRPA amplify and quantitatively detect the three target genes Ta-CCR, TaCM, and TaCAD. After experimental verification, the detection limit of the three target genes Ta-CCR, TaCM and TaCAD all reached 10 copies / μL.

[0112] Comparative Example 1: Water Bath aRPA

[0113] This example compares the method of realizing light-triggered aRPA using the photothermal effect of the photosensitizer CsYF4@Yb2O3 with the effect of traditional water bath aRPA to verify the advancement of the method of the present invention in terms of amplification effect.

[0114] The water bath ring-mediated isothermal amplification method comprises the following steps:

[0115] (1) Primer sequence design:

[0116] Same as Example 1

[0117] (2) Plasmid DNA extraction:

[0118] Same as Example 1

[0119] (3) Construction of aRPA isothermal amplification system:

[0120] The water bath aRPA system was constructed. The water bath aRPA system included the following components: primer set, lyophilized powder, amplification buffer solution, ddH2O and DNA template.

[0121] Under the premise of maintaining the final volume at 50 μL, the concentrations of the components and the reaction volume of the water bath aRPA system are shown in Table 4 below:

[0122] Table 4 Concentrations of components and reaction volumes of water bath aRPA system

[0123]

[0124] (4) Amplification process:

[0125] Add the components of the water bath aRPA system into a 1.5 mL centrifuge tube and place it in a 40 °C water bath for 10 min.

[0126] (5) Amplification product analysis:

[0127] The amplified products of Example 1 and Comparative Example 1 were purified and the amplification results were characterized by gel electrophoresis.

[0128] like Figure 6 As shown, the water bath aRPA group of comparative example 1 showed a bright amplification band only after 10 minutes of amplification, while the light-triggered aRPA group showed a clear and bright amplification band at 5 minutes, showing a higher amplification efficiency. From the comparison results, the light-triggered aRPA amplification method provided in Example 1 of the present invention has amplification products of 0.6 times and 2.1 times that of water bath aRPA (10 minutes) at 5 minutes and 10 minutes, respectively. Therefore, the light-triggered aRPA amplification has a better amplification effect than the water bath aRPA at the same time.

[0129] In addition, the scattered light LED and the external coaxial reflection LED were used as the light source for light-triggered aRPA, and their amplification effects were compared. Figure 7 As shown in the figure, only the external coaxial reflection LED can give a bright amplification band, that is, a high-efficiency amplification, while the scattered light LED does not induce effective amplification. The reason may be that when the scattered light LED is working, it not only generates light energy but also heat energy. Part of the heat energy may be absorbed by the scattering material or structure and converted into other forms of energy (such as internal energy), thereby reducing the heat dissipation efficiency; while the external coaxial reflection LED mainly converts electrical energy into light energy, generates relatively less heat energy, and can be dissipated more efficiently.

[0130] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A material for light-triggered amplification-photothermal detection test strips for tacA, tacB, and tacC, characterized in that: The material of the light-triggered amplification-photothermal detection test paper for tacA, tacB, and tacC is the photosensitizer CsYF4@Yb2O3, and the preparation method of the photosensitizer CsYF4@Yb2O3 comprises the following steps: (1) Add 140-160 mg of EDTA to 20-30 mL of ultrapure water and stir for 5-10 min to obtain a mixed solution; (2) Add Y(NO3)3 and Yb2O3 with a concentration of 0.6-0.8 M to the mixed solution and stir rapidly for 30-40 min; (3) Add 0.8-1.2M NH4F and CsF to the mixed solution, stir for 30-40 minutes, and heat in a reactor at 180-220°C for 23-25 ​​hours. After the reaction is completed, the initial product is obtained; (4) After cooling, washing and drying, the initial product is obtained as photosensitizer CsYF4@Yb2O3.

2. The photosensitive material for tacA, tacB and tacC according to claim 1, characterized in that: The volume of Y(NO3)3 used in step (2) is 0.8-1.0 mL, and the volume of Yb2O3 used is 0.1-0.3 mL.

3. The photosensitive material for tacA, tacB and tacC according to claim 1, characterized in that: The volume of NH4F and CsF used in step (3) is 2-4 mL.

4. The photosensitive material for tacA, tacB and tacC according to claim 1, characterized in that: The drying in step (4) includes drying in an oven at 65-75° C. for 2-3 hours.

5. A method for simultaneously detecting tacA, tacB, and tacC, characterized in that: Using the photosensitive material CsYF4@Yb2O3 for tacA, tacB, and tacC as described in any one of claims 1 to 4, the method comprises the following steps: S1. Light-triggered multiplex aRPA amplification Under the irradiation of 504nm external coaxial reflection LED light source, the sample to be tested is amplified by aRPA isothermal amplification system to obtain an amplification product, wherein: The aRPA isothermal amplification system includes the following components: a primer set, a photosensitizer CsYF4@Yb2O3, a DNA template, and a buffer, wherein the DNA template is the lodging resistance gene tacA, namely Ta-CCR, tacB, namely TaCM, and tacC, namely TaCAD; S2. Photothermal test paper test Capture probes were fixed on the T and C regions of the test strip to obtain a multi-target photothermal detection test strip for the target genes Ta-CCR, TaCM, and TaCAD light-triggered aRPA products; The photosensitizer CsYF4@Yb2O3 was modified with nucleic acid chains of Ta-CCR, TaCM, and TaCAD to obtain photothermal signal probes; The amplification product is detected using the photothermal signal probe and the photothermal detection test paper. The detection area of ​​the photothermal detection test paper captures the amplification product and the photothermal signal probe in a sandwich form, and gives a photothermal temperature change signal positively correlated with the content of the amplification product under the irradiation of a 504nm external coaxial reflection LED light source. The Ta-CCR, TaCM, and TaCAD in the sample to be tested are qualitatively and / or quantitatively determined based on the temperature change signal.

6. The method according to claim 5, characterized in that In the light-triggered aRPA system, the concentration of each group of DNA templates is 4%-6% v / v.

7. The method according to claim 5, characterized in that The primer set includes three sets of primers, and each set of primers includes a front primer (F) and a rear primer (R).

8. The method according to claim 5, characterized in that In the aRPA isothermal amplification system, the ratio of upstream and downstream primers in each set of primers is 1:3-1:9 or 3:1-9:1, and when the amplification system is 50 μL, the total amount of upstream and downstream primers is 40 nmol-60 nmol; Preferably, the aRPA isothermal amplification system further comprises the following components: lyophilized powder and ddH2O.

9. The method according to claim 5, characterized in that The irradiation parameters of the 504nm external coaxial reflection LED light source are power 30-60W and irradiation time 10-60min.

10. The method according to claim 5, characterized in that The photothermal signal probe is used independently of the test paper body, or is dropped and dried on a conjugate pad and the conjugate pad is integrated on the test paper.

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