Material for photon amplification-photothermal analysis of cat1, floR and cmlA and application thereof

Abstract

The invention discloses a material for photon amplification-photothermal analysis of cat1, floR and cmlA and application of the material. A sample is irradiated by a coaxial reflection LED light source outside 808nm, a photoresponsive agent Bi2MoO6 (at) NiS is driven by photons to provide photoelectrons and heat for LAMP, a target gene is used as a template to initiate an amplification reaction, a large number of double-chain structure DNA photon amplification products are obtained, Bi2MoO6 (at) NiS is modified by a nucleic acid chain to obtain a signal probe, a detection area of photothermal analysis test paper captures the amplification products and the signal probe in a sandwich form, and the detection area of the photothermal analysis test paper captures the amplification products and the signal probe. A photo-thermal temperature change signal in positive correlation with the content of a photon amplification product is given, and cat1, floR and cmlA in the sample are detected according to the temperature change signal. Photoelectron and photothermal effects are generated by using the photoinduced activity of the photoresponse agent Bi2MoO6 at NiS, the temperature is rapidly controlled, the Bi2MoO6 at NiS is in direct contact with an amplification system on a molecular level, photothermal analysis test paper detection is carried out, and the photothermal rate is remarkably improved.

Description

Technical Field

[0001] The invention relates to a material for photon amplification-photothermal analysis of cat1, floR and cmlA and application thereof, belonging to the technical field of analysis and detection. Background Art

[0002] Antibiotic resistance genes refer to genes present in the genomes of bacteria or other microorganisms that make them resistant to antibiotics. These genes encode a series of proteins or other molecular mechanisms that enable bacteria to develop resistance to antibiotics. The existence and spread of antibiotic resistance genes pose certain hazards to public health, the environment, and food safety. Chloramphenicol resistance is mainly mediated by plasmids, which can carry genes encoding resistance and spread between bacteria through horizontal gene transfer. The presence of chloramphenicol resistance genes may be transmitted to humans through the food chain, thereby increasing resistance to chloramphenicol. Therefore, in order to meet the challenges posed by chloramphenicol resistance genes, it is urgent to develop on-site rapid detection methods for chloramphenicol antibiotic resistance genes.

[0003] The detection of antibiotic resistance genes mainly relies on nucleic acid amplification techniques, such as polymerase chain reaction (PCR) and isothermal amplification. However, the PCR process is time-consuming and highly dependent on professionals and equipment, which is not conducive to rapid on-site detection. Loop-mediated isothermal amplification stands out for its high efficiency, simplicity and high specificity under constant temperature conditions, without the need for expensive thermal cycling equipment. LAMP technology uses 4 primers (2 outer primers and 2 inner primers) that recognize 6 specific fragments of conserved sequence DNA, and a strand displacement DNA polymerase to perform amplification reactions under isothermal conditions.

[0004] Photon amplification is activated by photothermal nanomaterials or photoresponsive agents under LED or laser irradiation to provide energy for nucleic acid amplification, which can significantly improve the amplification efficiency. It has been successfully combined with single-plex PCR, LAMP, and RCA amplification technologies, showing good combined effects and becoming a promising on-site detection method. Currently, there are no reports showing that it can simultaneously amplify multiple antibiotic resistance genes.

[0005] 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, bar 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 bar light source provides a variety of illumination effects such as direct, oblique and photometric. The external coaxial reflective LED light source uses a specific optical design of a beam splitter to make the light shine vertically on the observed object, thereby reducing the impact of light scattering and reflection. And this light source usually has high brightness and high uniformity, which can reduce shadows and reflections. 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 bar 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.

[0006] Currently, there is no report on the simultaneous detection of chloramphenicol genes using external coaxial reflection LED light source excitation-photon amplification combined with photothermal analysis test paper technology. Summary of the invention

[0007] Technical issues: The efficiency of existing photon amplification photoresponses is not ideal; photon amplification technology has not yet been applied to the simultaneous amplification of multiple 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-driven LAMP amplification and photothermal analysis test paper technology for the detection of chloramphenicol resistance genes.

[0008] Technical solution: The present invention adopts the following technical solution.

[0009] The first object of the present invention is to provide a material for photon amplification-photothermal analysis of cat1, floR, and cmlA, wherein the material is Bi2MoO6@NiS, and the preparation method of the photoresponse agent Bi2MoO6@NiS comprises the following steps:

[0010] (1) dissolving nickel nitrate and CH4N2S in water at a molar ratio of 1:4-1:5, stirring for 1-2 hours to obtain a mixed solution;

[0011] (2) injecting the mixed solution into a Bi2MoO6 aqueous solution, wherein the molar ratio of Bi2MoO6 to nickel nitrate is 1:2-1:4, and heating in a reactor at 160-180° C. for 1-2 h. After the reaction is completed, a primary product is obtained;

[0012] (3) After centrifugation, washing and drying, the initial product is obtained to obtain the photoresponsive agent Bi2MoO6@NiS;

[0013] In the present invention, the photoresponse agent Bi2MoO6@NiS mediates photon amplification of cat1, floR, and cmlA and photothermal analysis of cat1, floR, and cmlA.

[0014] As an optional embodiment, in step (1), the concentration of nickel nitrate dissolved in water is 0.4-0.5 mM, preferably 0.4 mM.

[0015] As an optional embodiment, in step (2), the concentration of Bi2MoO6 dissolved in water is 0.1-0.25 mM, preferably 0.2 mM.

[0016] As an optional embodiment, in step (3), the centrifugation includes centrifugation at 10000-12000 rpm for 10-15 min, preferably centrifugation at 10000 rpm for 15 min.

[0017] As an optional embodiment, the drying includes drying in an oven at 50-55° C. for 20-24 hours, preferably drying in an oven at 50° C. for 24 hours.

[0018] The second object of the present invention is to provide a method for realizing a photon-driven multiple loop-mediated isothermal amplification system of cat1, floR, and cmlA using the photothermal effect of the photoresponsive agent Bi2MoO6@NiS. Specifically, the present invention provides a method for simultaneously detecting cat1, floR, and cmlA, comprising the following steps:

[0019] S1. Photon Amplification

[0020] Under the irradiation of 808nm external coaxial reflection LED light source, the sample to be tested undergoes amplification reaction through the LAMP photon amplification system to obtain LAMP photon amplification products, wherein:

[0021] The LAMP photon amplification system includes the following components: a primer set, a photoresponse agent Bi2MoO6@NiS, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer, wherein the DNA template is the resistance genes cat1, floR, and cmlA;

[0022] S2. Photothermal analysis test paper detection

[0023] Capture probes were fixed on the T and C regions of the test strip to obtain a multi-target photothermal analysis test strip for the target gene cat1, floR, cmlA LAMP photon amplification products;

[0024] The photothermal signal probe was obtained by modifying the photoresponse agent Bi2MoO6@NiS with nucleic acid chains of cat1, floR, and cmlA respectively;

[0025] The LAMP photon amplification product is detected using the photothermal signal probe and the photothermal analysis test paper. The detection area of ​​the photothermal analysis test paper captures the LAMP photon 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 LAMP photon amplification product under the irradiation of an 808nm external coaxial reflection LED light source. Cat1, floR, and cmlA in the sample to be tested are qualitatively and / or quantitatively determined based on the temperature change signal.

[0026] As an optional implementation, in the LAMP photon amplification system, the concentration of each group of DNA templates is 4%-8% v / v.

[0027] As an optional embodiment, the primer set includes three sets of primers, each set of primers includes two external primers F3 and B3, two internal primers FIP and BIP, and two loop primers loop F and loop B.

[0028] As an optional embodiment, in the LAMP photon amplification system, the concentration of the two external primers F3 and B3 in each set of primers is 2%-6% v / v, the concentration of the two internal primers FIP and BIP is 2%-6% v / v, and the concentration of the two loop primers loop F and loop B is 0%-4% v / v.

[0029] As an optional embodiment, the LAMP photon amplification system further comprises the following components: BSA, dNTPs, betaine and ddH2O.

[0030] As an optional embodiment, the LAMP isothermal amplification buffer includes Tris-HCl, (NH4)2SO4, KCl, MgSO4, and Tween-20.

[0031] 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.

[0032] As an optional implementation, the irradiation parameters of the 808nm external coaxial reflection LED light source are power 4.5-6W and irradiation time 10-30min.

[0033] Beneficial effects: The photon amplification-photothermal analysis material and its application provided by the present invention use the photoactive agent Bi2MoO6@NiS to generate photoelectrons and photothermal effects, which can quickly control the temperature and directly contact the LAMP photon amplification system at the molecular level, which is significantly more efficient than traditional water baths and metal baths. At the same time, the photoresponse agent is used as a signal probe for photothermal analysis test paper, providing a sensitive and quantifiable signal. The whole set of methods can quickly achieve efficient LAMP photon amplification and specific detection of cat1, floR, and cmlA target DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the working process of the present invention;

[0035] Figure 2 The scanning electron microscope and X-ray photoelectron spectrum of the photoresponse agent Bi2MoO6@NiS of the present invention;

[0036] Figure 3 The photothermal temperature rise of the photothermal material of the present invention;

[0037] Figure 4 It is the verification of the light-driven multiple LAMP photon amplification primers of the present invention;

[0038] Figure 5 The electrophoresis photos, color development and photothermal analysis test paper photos of the light-driven single-plex and multiple-plex LAMP photon amplification products of the present invention are shown;

[0039] Figure 6 The LAMP photon amplification of the present invention is combined with the photothermal analysis test paper for photothermal quantification;

[0040] Figure 7 The electrophoresis diagram of the product of LAMP photon amplification of cat1 using an 808nm external coaxial reflection LED light source and a scattered light LED respectively;

[0041] Figure 8 The present invention uses gel electrophoresis of light-controlled multiplex LAMP and water-bath LAMP amplification products. DETAILED DESCRIPTION

[0042] 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.

[0043] The present invention discloses a novel light-driven amplification-photothermal analysis material and its analytical application for simultaneously detecting chloramphenicol resistance genes cat1, floR, cmlA by light-driven loop-mediated isothermal amplification, i.e., LAMP combined with photothermal analysis test paper. The method includes two steps: LAMP photon amplification and photothermal analysis test paper detection:

[0044] (1) LAMP photon amplification

[0045] N sample tubes were irradiated simultaneously with an 808nm external coaxial reflection LED light source. The photoresponse agent Bi2MoO6@NiS was driven by light to provide photoelectrons and heat for LAMP. The target gene in the positive sample was used as a template to trigger the amplification reaction, and a large number of double-stranded DNA LAMP photon amplification products were obtained.

[0046] (2) Photothermal analysis test paper detection

[0047] Bi2MoO6@NiS was modified with three nucleic acid chains, cat1, floR, and cmlA, to obtain three signal probes. The detection area of ​​the photothermal analysis test paper captured the LAMP photon amplification products and signal probes in a sandwich form, and gave a photothermal temperature change signal that was positively correlated with the content of the LAMP photon amplification products. According to the temperature change signal, cat1, floR, and cmlA in the original sample were further quantified.

[0048] The present invention uses the photoactivity of the photoresponsive agent Bi2MoO6@NiS to generate photoelectrons and photothermal effects, which mediate the photon amplification of cat1, floR, and cmlA and the photothermal analysis of cat1, floR, and cmlA, realize the rapid temperature control of the present invention and directly contact with the LAMP photon amplification system at the molecular level, and then perform color development detection with a photothermal analysis test paper, thereby significantly improving the photothermal efficiency.

[0049] Embodiment 1:

[0050] The principle of the present invention is as follows Figure 1 As shown, a method for driving multiple LAMP photon amplification and detection using the photothermal effect of a photoresponsive agent Bi2MoO6@NiS, the method comprising the following steps:

[0051] (1) Preparation of photoresponsive agent Bi2MoO6@NiS:

[0052] Nickel nitrate and CH4N2S were dissolved in deionized water at a molar ratio of 1:5 and stirred for 1 hour. Then, they were injected into an aqueous solution of Bi2MoO6 with a molar concentration of 1 / 2 of nickel and heated at 165°C in a reactor for 1.5 hours. After the reaction, the product was centrifuged at 10,000 rpm for 15 minutes and washed with deionized water. After centrifugal washing three times, it was dried in an oven at 50°C for 24 hours.

[0053] Scanning electron microscopy and X-ray photoelectron spectroscopy were used to measure the photoresponse of Bi2MoO6@NiS. Figure 2 shown.

[0054] The photoresponse agent Bi2MoO6@NiS solution was irradiated with an 808nm external coaxial reflection LED light source in the integrated device and compared with the solutions of Bi2MoO6 and NiS with the same concentration. The photothermal heating curves of the three are shown in Figure 3 As shown, it can be seen that the heating efficiency of the Bi2MoO6@NiS composite is significantly improved compared with that of a single substance, which is beneficial to improving its efficiency as a photoresponse agent.

[0055] (2) Primer sequence design:

[0056] According to the LAMP principle, amplification primer sets were designed for six different regions of the target gene cat1, floR and cmlA sequences. The primer sets included two external primers (F3 and B3), two internal primers (FIP and BIP) and two loop primers (loop F and loop B). Some primer sequences are shown in Table 1:

[0057] Table 1 Primer sequences of the light-driven multiple LAMP system of the present invention

[0058]

[0059] (3) Plasmid DNA extraction:

[0060] The preserved bacteria containing the target genes (cat1, floR, cmlA) 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, which included RNaseA, BufferS, SolutionⅠ, SolutionⅡ, SolutionⅢ, Wash Solution, Elution Buffer, EZ-10 Column, and Collection Tube.

[0061] In other embodiments of the present invention, the plasmid DNA extraction method may also be any one of an adsorption column method, a magnetic bead method, a CTAB method, and the like.

[0062] (4) Construction of light-driven multiple LAMP system:

[0063] A multiple LAMP photon amplification system was constructed using cat1, floR, and cmlA in the extracted plasmid DNA as templates, respectively. The multiple LAMP photon amplification system included the following components: a primer set, a photoresponse agent Bi2MoO6@NiS (referring to the photoresponse agent Bi2MoO6@NiS solution prepared in step (1)), BSA, MgSO4, an isothermal amplification buffer (Tris-HCl, (NH4)2SO4, KCl, MgSO4, Tween-20), dNTPs, betaine, Bst 3.0 DNA polymerase, ddH2O, and a DNA template.

[0064] Under the premise of maintaining the final volume at 25 μL, the concentrations of the components and the reaction volumes of the multiple LAMP photon amplification system are shown in Table 2 below.

[0065] Table 2 Concentrations and reaction volumes of components in the multiple LAMP photon amplification system of this embodiment

[0066]

[0067] (5)LED light source excitation:

[0068] The LAMP photon amplification system was irradiated with an 808nm external coaxial reflection LED light source to excite the photoresponse agent in the LAMP photon amplification system to provide the energy required for multiple LAMPs. The excitation power was 4.5W and the excitation time was 20min.

[0069] In the embodiment of the present invention, the 808nm external coaxial reflection LED light source is provided by the existing 808nm external coaxial reflection LED light source device. There are no excessive restrictions in the present invention, so the technical solution of providing an 808nm external coaxial reflection LED light source can be realized, which all belongs to the protection scope of the present invention.

[0070] (6) LAMP photon amplification and detection result analysis:

[0071] Three photothermal signal probes were obtained by modifying the photoresponsive agent Bi2MoO6@NiS with three nucleic acid chains. The specific method was as follows: 20 μL of three nucleic acid chains were added to 2 mL of photoresponsive agent Bi2MoO6@NiS 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 the photoresponsive agent Bi2MoO6@NiS modified with three nucleic acid chains.

[0072] Capture probes were fixed on the T and C regions of the test strip to obtain a multi-target photothermal analysis test strip targeting the target gene cat1, floR, and cmlA LAMPLAMP photon amplification products.

[0073] The probe sequences of the photothermal analysis test paper for detecting LAMP photon amplification products are shown in Table 3 below.

[0074] Table 3 Probe sequences of LAMP photon amplification products detected by photothermal analysis test paper in this embodiment

[0075]

[0076] The constructed photothermal analysis test paper is used to detect the amplified product and provide it with photoheat, thereby stimulating the photothermal effect of the photoresponsive agent Bi2MoO6@NiS in the photothermal signal probe, and realizing the detection efficacy of the photothermal analysis test paper; and the photothermal signal of the T zone on the photothermal analysis test paper 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.

[0077] (7) Specificity verification

[0078] After adding all reagents except primers and templates to 6 LAMP photon amplification tubes, add

[0079] ①Cat1 template, floR primer, cmlA primer;

[0080] ②Cat1 template, Cat1 primer, floR primer, cmlA primer;

[0081] ③FloR template, cmlA primer;

[0082] ④FloR template, FloR primer, cmlA primer;

[0083] ⑤CmlA template, floR primer;

[0084] ⑥CmlA template, floR primer, cmlA primer;

[0085] Figure 4 The electrophoresis photograph shows that only when the primers and templates of the same gene coexist, the gel electrophoresis image shows diffuse bands, indicating that the method has good selectivity for Cat1, FloR, and CmlA.

[0086] Figure 5 The electrophoresis photograph shows that the coexisting primers and templates do not interfere with the amplification of other target genes, and the test paper photograph shows that the test paper can specifically detect the multiple LAMP photon amplification products of specific target genes in the multiple LAMP photon amplification system in colorimetric and photothermal modes, respectively.

[0087] Figure 6 This is a concentration gradient detection experiment for the three target genes cat1, floR and cmlA, 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 amplify and quantitatively detect the three target genes cat1, floR and cmlA. After experimental verification, the detection limit of the three target genes cat1, floR and cmlA reached 10 copies / μL.

[0088] Comparative Example 1: Effectiveness Verification of Scattered Light LED Light Source

[0089] In this comparative example, an 808nm scattered light LED light source and an 808nm external coaxial reflection LED light source are used as the light sources for LAMP photon amplification, and their amplification effects are compared, in order to explore the effectiveness of different light sources for the photothermal effect of the photoresponsive agent Bi2MoO6@NiS.

[0090] The results are as follows Figure 7 As shown in the figure, only the 808nm external coaxial reflection LED light source can give a bright diffuse band, that is, a high-efficiency amplification, while the 808nm scattered light LED light source does not induce effective amplification. The reason may be that when the scattered light LED light source 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 light source mainly converts electrical energy into light energy, generates relatively less heat energy, and can be dissipated more efficiently.

[0091] Comparative Example 2: Water Bath LAMP

[0092] This comparative example compares the method of realizing multiple LAMP photon amplification by the photothermal effect of the photoresponsive agent Bi2MoO6@NiS with the traditional water bath LAMP effect to verify the advancement of the method of the present invention in terms of amplification effect.

[0093] The water bath LAMP method includes the following steps:

[0094] (1) Primer sequence design:

[0095] Same as Example 1

[0096] (2) Plasmid DNA extraction:

[0097] Same as Example 1

[0098] (3) Construction of isothermal amplification system:

[0099] The water bath LAMP system was constructed. The water bath loop-mediated amplification system included the following components: primer set, MgSO4, isothermal amplification buffer (Tris-HCl, (NH4)2SO4, KCl, MgSO4, Tween-20), dNTPs, betaine, Bst 3.0 DNA polymerase, ddH2O and DNA template.

[0100] Under the premise of maintaining the final volume at 25 μL, the concentrations of the components and the reaction volume of the water bath LAMP system are shown in Table 4 below:

[0101] Table 4 Concentrations of components and reaction volumes of water bath LAMP system

[0102]

[0103] (4) Amplification process:

[0104] Add the components of the water bath LAMP system into a 1.5 mL centrifuge tube and place it in a 65 °C water bath for 60 min.

[0105] (5) Amplification product analysis:

[0106] The amplified products were analyzed by agarose gel electrophoresis.

[0107] Gel electrophoresis images such as Figure 8 As shown, the negative group image without template DNA only shows primers and no diffuse bands, while the image of the positive group with template DNA successfully amplified shows diffuse bands. At the same time, the water bath LAMP group of comparative example 1 only shows bright diffuse bands after 60 minutes of amplification, while the LAMP photon amplification group shows clear and bright diffuse bands at 20 minutes, indicating that the amplification efficiency of the LAMP photon amplification group is better than that of the water bath LAMP group.

[0108] 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 photon amplification-photothermal analysis of cat1, floR, cmlA, characterized in that: The material is a photoresponse agent Bi2MoO6@NiS, and the preparation method of the photoresponse agent Bi2MoO6@NiS comprises the following steps: (1) dissolving nickel nitrate and CH4N2S in deionized water at a molar ratio of 1:4-1:5, and stirring for 1-2 hours to obtain a mixed solution; (2) injecting the above mixed solution into a Bi2MoO6 aqueous solution, wherein the molar ratio of Bi2MoO6 to nickel nitrate is 1.2-1.4, and heating in a reactor at 160-180° C. for 1-2 h. After the reaction is completed, a primary product is obtained; (3) After centrifugation, washing and drying, the initial product is obtained to obtain the photoresponsive agent Bi2MoO6@NiS; The photoresponse agent Bi2MoO6@NiS mediates photon amplification of cat1, floR, and cmlA and photothermal analysis of cat1, floR, and cmlA.

2. The photosensitive material for cat1, floR, cmlA according to claim 1, characterized in that: In step (1), the concentration of nickel nitrate dissolved in water is 0.4-0.5 mM.

3. The photosensitive material for cat1, floR, cmlA according to claim 1, characterized in that: In step (2), the concentration of Bi2MoO6@NiS dissolved in water is 0.1-0.25 mM.

4. The photosensitive material for cat1, floR, cmlA according to claim 1, characterized in that: In step 3, the centrifugation includes centrifugation at 10000-12000 rpm for 10-15 min; Preferably, the drying comprises drying in an oven at 50-55° C. for 20-24 hours.

5. A method for simultaneously detecting cat1, floR, and cmlA, characterized in that: Using the material for photon amplification-photothermal analysis of cat1, floR, cmlA as described in any one of claims 1 to 4, the method comprises the following steps: S1. Photon Amplification Under the irradiation of 808nm external coaxial reflection LED light source, the sample to be tested undergoes an amplification reaction through the LAMP photon amplification system to obtain a LAMP photon amplification product, wherein: The LAMP photon amplification system includes the following components: a primer set, a light-responsive agent Bi2MoO6@NiS, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer, wherein the DNA template is the resistance genes light cat1, floR, and cmlA; S2. Photothermal analysis test paper detection Capture probes were fixed on the T and C regions of the test strip to obtain a multi-target photothermal analysis test strip for the target genes cat1, floR, and cmlA light-driven LAMP products; The photothermal signal probe was obtained by modifying the photoresponse agent Bi2MoO6@NiS with nucleic acid chains of cat1, floR, and cmlA respectively; The LAMP photon amplification product is detected using the photothermal signal probe and the test paper. The detection area of ​​the photothermal analysis test paper captures the LAMP photon 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 LAMP photon amplification product under the irradiation of an 808nm external coaxial reflection LED light source. The cat1, floR, and cmlA 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 LAMP photon amplification system, the concentration of each group of DNA templates is 4%-8% v / v.

7. The method according to claim 5, characterized in that The primer set includes three sets of primers, each set of primers includes two external primers F3 and B3, two internal primers FIP and BIP, and two loop primers loop F and loop B; Preferably, in the LAMP photon amplification system, the concentration of the two external primers F3 and B3 in each set of primers is 2-6% v / v, the concentration of the two internal primers FIP and BIP is 2-6% v / v, and the concentration of the two loop primers loop F and loop B is 0-4% v / v.

8. The method according to claim 5, characterized in that The LAMP photon amplification system also includes the following components: BSA, dNTPs, betaine ddH2O; Preferably, the LAMP photon amplification buffer comprises Tris-HCl, (NH4)2SO 4， KCl, MgSO4, Tween-20.

9. The method according to claim 5, characterized in that The irradiation parameters of the 808nm external coaxial reflection LED light source are power 4.5-6W and irradiation time 10-30min.

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

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