Method and device for light-driven amplification-photothermal analysis of sul1, sul2 and sul3

Abstract

The invention discloses a method and a device for light-driven amplification-photothermal analysis of sul1, sul2 and sul3, a sample is irradiated by a coaxial reflection LED light source outside 808nm, a light activator Sb2MoO6 at-NiFeS2 provides photoelectrons and heat for LAMP through light driving, a target gene is used as a template to initiate an amplification reaction, and a large number of double-stranded DNA amplification products are obtained. Sb2MoO6-coated NiFeS2 is modified by a nucleic acid chain to obtain a signal probe, an amplification product and the signal probe are captured by a photo-thermal lateral flow chromatography test paper detection area in a sandwich form, a photo-thermal temperature change signal positively correlated with the content of the amplification product is given, and sul1, sul2 and sul3 in a sample are quantified according to the photo-thermal temperature change signal. Photoelectron and photothermal effects are generated by using the photoactivity of the photoactivator Sb2MoO6-coated NiFeS2, the temperature is quickly controlled, the photoactivator is in direct contact with an LAMP system on a molecular level, and then photothermal lateral flow chromatography test paper color development detection is performed, so that the photothermal efficiency is remarkably improved.

Description

Technical Field

[0001] The invention relates to a method and a device for light-driven amplification-photothermal analysis of sul1, sul2 and sul3, belonging to the technical field of analysis and detection. Background Art

[0002] Antibiotic resistance genes are genes that encode a series of proteins or other molecular mechanisms that enable bacteria to develop resistance to antibiotics. Their existence and spread pose a serious threat to public health, food safety and environmental safety. Among them, sulfonamide antibiotic resistance genes are genes that bacteria and other microorganisms acquire through gene mutation or horizontal gene transfer when facing sulfonamide antibiotic pressure, which can give bacteria resistance to sulfonamide antibiotics. With the widespread use of sulfonamide antibiotics, their residues are ubiquitous in the environment and are transmitted through the food chain, causing bacteria and pathogens in the human body to develop resistance to sulfonamide antibiotics, which seriously threatens human health. Therefore, in order to cope with the challenges brought by sulfonamide resistance genes, it is urgent to develop on-site rapid detection methods for sulfonamide resistance genes.

[0003] The detection methods of antibiotic 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 depends on professionals and equipment, it is not suitable for rapid on-site detection. Loop-mediated isothermal amplification, or LAMP technology, can use chain displacement DNA polymerase (such as Bst DNA polymerase) and a set of specific primers under isothermal conditions (usually 60°C to 65°C) to complete the rapid amplification of the target nucleic acid through self-circulating chain displacement DNA synthesis. LAMP technology designs 4 specific primers for 6 specific regions of the target gene. These 4 primers are FIP (forward inner primer), BIP (reverse inner primer), F3 (forward outer primer) and B3 (reverse outer primer). These primers play a key role in the amplification reaction and can ensure the specificity and efficiency of amplification.

[0004] The LAMP amplification process is mainly divided into two stages:

[0005] (1) Initialization stage: In this stage, both inner and outer primers participate in the reaction. They bind to specific regions of the target DNA and are extended under the action of DNA polymerase to form a double dumbbell structure DNA. This structure provides a template for subsequent cyclic amplification.

[0006] (2) Cyclic amplification stage: After entering this stage, only the inner primer participates in the reaction. The inner primer continuously produces new stem-loop structure DNA products through chain displacement DNA synthesis reaction. These products can continue to participate in the reaction as templates, thereby achieving exponential amplification of the target sequence.

[0007] Light-driven amplification, by adding various photoactivators to the amplification system, using light-emitting diodes (LEDs) or lasers as light sources, the photoactivators are evenly dispersed in the amplification system, and are excited by LEDs to provide energy supply for nucleic acid amplification. Chen et al. established a light-driven LAMP mediated by polypyrrole-coated iron oxide particles, which can detect as low as 8 CFU of malB gene in Escherichia coli in about 1 hour (Biosensors and Bioelectronics 2022, 215, 114574).

[0008] At present, there are no reports on the use of light-driven amplification technology to achieve simultaneous LAMP amplification of multiple genes. The challenge of using photoactivator-mediated, light-driven LAMP is that it requires photoactivators with higher activity, and the operational difficulty is that the photoactivator needs to be compatible with the LAMP system and not interfere with the reaction; there needs to be a precisely controllable light source and light-driven conditions to avoid affecting the stability of amplification; and a highly sensitive and specific detection method needs to be developed.

[0009] 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.

[0010] At present, there is no report on the simultaneous detection of sulfonamide genes using external coaxial reflection LED light source excitation-light-driven multiple LAMP amplification combined with photothermal test paper technology. Summary of the invention

[0011] Technical issues: The efficiency of existing light-driven amplification photoactivators is not ideal; light-driven 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 amplification and photothermal test paper technology to detect sulfonamide resistance genes. The challenges and operational difficulties of using photoactivator-mediated, light-driven LAMP are that the photoactivator must be compatible with the LAMP system and not interfere with the reaction; there must be a precisely controllable light source and light-driven amplification conditions to avoid affecting the stability of amplification; and a highly sensitive and specific detection method must be developed.

[0012] Purpose of the invention: In order to overcome the above-mentioned deficiencies and defects of the prior art, the present invention provides a method and device for light-driven amplification-photothermal analysis of sul1, sul2, and sul3, using a photoactivator Sb2MoO6@NiFeS2 as a photothermal material, and utilizing its photoactivity to generate photoelectrons and photothermal effects. Under the irradiation of an 808nm external coaxial reflective LED light source, rapid temperature control is achieved and direct contact with the LAMP amplification system at the molecular level is achieved, and the photothermal efficiency is significantly improved. Then, photothermal test paper colorimetric detection is performed, thereby realizing intensive and efficient amplification and detection of the two links of light-driven amplification and photothermal lateral flow chromatography test paper detection.

[0013] Technical solution: The present invention adopts the following technical solution.

[0014] The first purpose of the present invention is to provide a method for realizing light-driven LAMP and detection of resistance genes sul1, sul2 and sul3 using a light activator Sb2MoO6@NiFeS2 and a photothermal lateral flow chromatography test paper. The method includes two steps: light-driven amplification and photothermal lateral flow chromatography test paper detection. Firstly, an 808nm external coaxial reflection LED light source is used to irradiate the light-driven amplification system to excite the photoactivator Sb2MoO6@NiFeS2 in the light-driven amplification system to produce photoelectrons and heat, providing energy for LAMP, so that sul1, sul2 and sul3 are used as templates to initiate the amplification reaction and obtain a large number of double-stranded DNA amplification products; the photoactivator Sb2MoO6@NiFeS2 is modified with three nucleic acid chains to obtain three photothermal signal probes, and the T probe and C probe are fixed in the detection area and quality control area of ​​the photothermal lateral flow chromatography test paper respectively to obtain a multi-target photothermal lateral flow chromatography test paper for the light-driven LAMP products of the target genes sul1, sul2, and sul3; then, the photothermal lateral flow chromatography test paper is used to capture the amplification products and signal probes in a sandwich form, giving a photothermal temperature change signal positively correlated with the content of the amplification products, thereby realizing the quantitative detection of the target genes. The LAMP isothermal amplification system includes the following components: a primer set, a photoactivator Sb2MoO6@NiFeS2, a DNA template, Bst3.0 DNA polymerase and an isothermal amplification buffer.

[0015] The challenge of using photoactivator-mediated, light-driven LAMP is that photoactivators with higher activity are required. The operational difficulty is that the photoactivator needs to be compatible with the LAMP system and not interfere with the reaction. Precisely controllable light sources and light-driven conditions are required to avoid affecting the stability of amplification. Highly sensitive and specific detection methods need to be developed.

[0016] Specifically, the present invention provides a method for light-driven amplification-photothermal analysis of sul1, sul2, and sul3, comprising the following steps:

[0017] S1. Preparation of photoactivator Sb2MoO6@NiFeS2

[0018] S2, light-driven amplification

[0019] Under the mediation of the photoactivator Sb2MoO6@NiFeS2, the sample to be tested is amplified by the light-driven ring-mediated isothermal amplification, i.e., the light-driven LAMP isothermal amplification system, using 808nm external coaxial reflection LED light source to obtain the amplified product.

[0020] The LAMP isothermal amplification system includes the following components: a primer set, a photoactivator Sb2MoO6@NiFeS2, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer, wherein the DNA template is the resistance genes sul1, sul2, and sul3;

[0021] S3, Photothermal test paper detection

[0022] Capture probes were fixed on the T and C regions of the test strip to obtain a multi-target photothermal lateral flow chromatography test strip targeting the light-driven LAMP products of the target genes sul1, sul2, and sul3;

[0023] The photothermal signal probe was obtained by modifying the photoactivator Sb2MoO6@NiFeS2 with the nucleic acid chains of sul1, sul2, and sul3 respectively;

[0024] The amplification product is detected using the photothermal signal probe and the photothermal lateral flow chromatography test paper. The detection area of ​​the photothermal lateral flow chromatography 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 an 808nm external coaxial reflection LED light source. The sul1, sul2, and sul3 in the sample to be tested are qualitatively and / or quantitatively determined based on the temperature change signal.

[0025] 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.

[0026] As an optional embodiment, the preparation method of the photoactivator Sb2MoO6@NiFeS2 comprises the following steps:

[0027] (1) Dissolve Ni(NO3)2·6H2O, FeCl2 and CH4N2S in deionized water at a molar ratio of 1:1:8-1:1:10 and stir for 1-2 hours to obtain a mixed solution.

[0028] (2) injecting the mixed solution into an aqueous solution of Sb2MoO6, wherein the molar ratio of Sb2MoO6 to nickel nitrate is 1:2-1:4, and heating in a reactor at 160-180°C for 1-2h. After the reaction is completed, a primary product is obtained;

[0029] (3) The product is centrifuged, washed and dried to obtain Sb2MoO6@NiFeS2.

[0030] 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.

[0031] As an optional embodiment, in step (2), the concentration of Sb2MoO6 dissolved in water is 0.1-0.25 mM, preferably 0.2 mM.

[0032] 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.

[0033] 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.

[0034] As an optional embodiment, in the LAMP isothermal amplification system, the concentration of each group of DNA templates is 4%-8% v / v.

[0035] 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.

[0036] As an optional embodiment, in the LAMP isothermal 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.

[0037] As an optional embodiment, the LAMP isothermal amplification system further includes the following components: BSA, dNTPs, betaine and ddH2O.

[0038] As an optional embodiment, the LAMP isothermal amplification buffer includes Tris-HCl, (NH4)2SO4, KCl, MgSO4, and Tween-20.

[0039] As an optional embodiment, the photothermal signal probe is used independently of the photothermal lateral flow chromatography test paper body, or is dropped and dried on the conjugate pad and the conjugate pad is integrated on the test paper.

[0040] The second object of the present invention is to provide a device for light-driven amplification-photothermal analysis of sul1, sul2, and sul3, which is used to realize the integrated and convenient operation of the above method. Specifically, the device includes an 808nm external coaxial reflection LED light source, a light-driven amplification device, and a photothermal lateral flow chromatography test paper assembly, wherein:

[0041] The light-driven amplification device comprises: a light-proof and heat-insulating shell, and a light-driven amplification component arranged inside the light-proof and heat-insulating shell, wherein:

[0042] The light-driven amplification component includes a sample holder, which is provided with N PCR tube channels for placing PCR tubes, wherein N≥1; the PCR tubes are used to hold a light-driven amplification system, the light-driven amplification system contains a primer set, a light activator Sb2MoO6@NiFeS2, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer, wherein the DNA template is resistance genes sul1, sul2, and sul3;

[0043] The front part of the light-proof and heat-insulating shell has a transversely arranged sample channel, and the light-driven amplification component is inserted and fixed in the sample channel; the 808nm external coaxial reflection LED light source is located at the rear part of the light-proof and heat-insulating shell, and is arranged toward the light-driven amplification component and the photothermal lateral flow chromatography test paper component;

[0044] The photothermal lateral flow chromatography test strip assembly comprises a photothermal lateral flow analysis test strip and a photothermal signal probe arranged vertically or horizontally, wherein the photothermal signal probe is obtained by modifying the photoactivator Sb2MoO6@NiFeS2 with the nucleic acid chains of sul1, sul2 and sul3, respectively.

[0045] When the photothermal lateral flow analysis test paper is arranged vertically, specifically, a vertical test paper groove is arranged at the inner bottom of the front side of the light-proof and heat-insulating shell, and the photothermal lateral flow analysis test paper is vertically inserted and fixed in the test paper groove;

[0046] When the photothermal lateral flow analysis test paper is arranged horizontally, specifically, the photothermal test paper assembly includes a test paper holder and a photothermal lateral flow analysis test paper, the test paper holder is inserted into and fixed in the sample channel, the test paper holder contains a rectangular groove, and the photothermal lateral flow analysis test paper is fixed in the rectangular groove.

[0047] As an optional embodiment, the 808nm external coaxial reflection LED light source includes an LED light source, a beam splitter and a plano-convex cylindrical lens. The plano-convex cylindrical lens is vertically arranged inside the light-proof and heat-insulating outer shell with its flat side facing forward. The LED light source is horizontally placed at the bottom position of the rear side of the plano-convex cylindrical lens. The beam splitter is arranged at a 45° angle between the plano-convex cylindrical lens and the LED light source. After passing through the beam splitter and the plano-convex cylindrical lens, the LED light source is gathered into a linear light spot to constitute an external coaxial reflection LED light source.

[0048] As an optional implementation, the wavelength band of the LED light source is 800-810nm.

[0049] As an optional implementation, the irradiation parameters of the LED light source are power 4.5-6W and irradiation time 10-30min.

[0050] As an optional implementation, two sides of the light-proof and heat-insulating housing are provided with beam splitter grooves and plano-convex cylindrical lens grooves for inserting beam splitters and plano-convex cylindrical lenses, respectively.

[0051] As an optional embodiment, an observation channel is provided on the top of the light-proof and heat-insulating shell, which is located on the front side of the plane of the plano-convex cylindrical lens and is used to observe the light-driven amplification component and / or the photothermal lateral flow analysis test paper.

[0052] Beneficial effects: The light-driven amplification-photothermal analysis method and device provided by the present invention uses the photoactivation of the photoactivator Sb2MoO6@NiFeS2 to generate photoelectrons and photothermal effects, which can quickly control the temperature and directly contact the amplification system at the molecular level, significantly improving the efficiency compared with traditional water baths and metal baths. At the same time, the photoactivator is used as a signal probe for the photothermal test paper, providing a sensitive and quantifiable signal. The whole set of methods can quickly achieve efficient amplification and detection of multiple target DNAs. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a schematic diagram of the working process of the present invention;

[0054] Figure 2 It is a schematic diagram of the device of the present invention;

[0055] Figure 3 A schematic diagram of a self-assembly device of the present invention;

[0056] Figure 4The scanning electron microscope and X-ray photoelectron spectrum of the photoactivator Sb2MoO6@NiFeS2 of the present invention;

[0057] Figure 5 The photothermal temperature rise of the photothermal material of the present invention;

[0058] Figure 6 It is the light-driven multiplex LAMP primer verification of the present invention;

[0059] Figure 7 The electrophoresis photos, color development and photothermal test paper photos of the light-driven single and multiple LAMP products of the present invention are shown;

[0060] Figure 8 The light-driven LAMP of the present invention is combined with lateral flow chromatography test paper for photothermal quantification;

[0061] Fig. 9 The electrophoresis diagram of the products of light-driven LAMP on sul1 using the device of the present invention and using a scattered light LED respectively;

[0062] Fig.10 The present invention uses gel electrophoresis of light-driven multiplex LAMP and water-bath LAMP amplification products.

[0063] Figure 2 and Figure 3 In the figure, 1-sample channel, 2-observation channel, 3-light-proof and heat-insulating shell, 4-plano-convex cylindrical lens channel, 5-test paper channel, 6-spectrometer channel, 7-LED light source, 8-light-driven amplification component, 9-photothermal lateral flow chromatography test paper component, 10-plano-convex cylindrical lens, 11-spectrometer, 12-photothermal lateral flow analysis test paper, 13-self-assembled 808nm external coaxial reflection LED light source, 14-external coaxial reflection LED light source. DETAILED DESCRIPTION

[0064] 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.

[0065] The present invention discloses a method and device for light-driven loop-mediated isothermal amplification of sulfonamide resistance genes sul1, sul2, and sul3, namely LAMP combined with photothermal test paper analysis. The method includes two links: light-driven amplification and photothermal lateral flow chromatography test paper detection. N sample tubes are irradiated simultaneously with an 808nm external coaxial reflection LED light source, wherein the photoactivator Sb2MoO6@NiFeS2 is driven by light to provide photoelectrons and heat for LAMP, and the target gene in the positive sample is used as a template to trigger an amplification reaction, thereby obtaining a large amount of double-stranded DNA amplification products. Three nucleic acid chains, sul1, sul2, and sul3, are used to modify Sb2MoO6@NiFeS2 respectively to obtain three signal probes, and the detection area of ​​the photothermal lateral flow chromatography 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, and further quantifies sul1, sul2, and sul3 in the original sample according to the temperature change signal. The present invention uses the photoactivator Sb2MoO6@NiFeS2 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 test paper, thereby significantly improving the photothermal efficiency.

[0066] The following embodiment of the present application discloses a method for realizing a light-driven multiple LAMP system and detection of resistance genes sul1, sul2 and sul3 using the photothermal effect of the photoactivator Sb2MoO6@NiFeS2, including the following steps:

[0067] (1) Primer sequence design:

[0068] According to the LAMP principle, taking a target gene sequence as an example, an amplification primer set was designed for six different regions of the target gene sequence, and the primer set included two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (loop F and loop B).

[0069] (2) Plasmid DNA extraction:

[0070] The microorganisms collected from the sample or the preserved bacteria containing the target gene (sul1, sul2, sul3) are cultured in LB medium for 24 hours to extract plasmid DNA. 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.

[0071] As an optional embodiment, the plasmid DNA extraction method in step (2) can also be any one of the adsorption column method, magnetic bead method, CTAB method, etc.

[0072] (3) Preparation of photoactivator Sb2MoO6@NiFeS2:

[0073] Dissolve Ni(NO3)2·6H2O, FeCl2 and CH4N2S in deionized water at a molar ratio of 1:1:8-1:1:10, and stir for 1-2 hours to obtain a mixed solution. Inject the mixed solution into an aqueous solution of Sb2MoO6, with a molar ratio of Sb2MoO6 to nickel nitrate of 1:2-1:4, and heat at 160-180°C in a reactor for 1-2 hours. After the reaction is completed, the initial product is obtained. The product is centrifuged, washed and dried to obtain Sb2MoO6@NiFeS2.

[0074] (4) Construction of light-driven multiple LAMP system:

[0075] Using sul1, sul2, and sul3 in the extracted plasmid DNA as templates, a light-driven multiple LAMP system was constructed. The amplification system included the following components: a primer set, a photoactivator Sb2MoO6@NiFeS2 (i.e., the photoactivator Sb2MoO6@NiFeS2 solution prepared in step (3)), 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.

[0076] (5)LED light source excitation:

[0077] The amplification system is illuminated by an 808nm external coaxial reflection LED to excite the photoactivator in the amplification system to provide the energy required for multiple LAMPs.

[0078] In an embodiment of the present invention, a device for simultaneously detecting sul1, sul2, and sul3 is also provided, which integrates the two links of light-driven amplification and photothermal test paper detection together with the light source into a collective device, further enhancing the portability of the detection system of the present invention. Figure 2 As shown, the device includes an 808nm external coaxial reflection LED light source, a light-driven amplification device and a photothermal test paper assembly 9, wherein:

[0079] The optically driven amplification device comprises: a light-proof and heat-insulating housing 3, and an optically driven amplification component 8 disposed inside the light-proof and heat-insulating housing 3, wherein:

[0080] The light-driven amplification component 8 includes a sample holder, which is provided with N PCR tube channels for placing PCR tubes, wherein N≥1; the PCR tubes are used to hold a light-driven amplification system, and the light-driven amplification system contains a primer set, a light activator Sb2MoO6@NiFeS2, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer;

[0081] The front part of the light-proof and heat-insulating housing 3 has a transversely arranged sample channel 1, and the light-driven amplification component 8 is inserted and fixed in the sample channel 1; the 808nm external coaxial reflection LED light source is located at the rear part of the light-proof and heat-insulating housing 3, and is arranged toward the light-driven amplification component 8 and the photothermal test paper component 9 to provide a light source for them;

[0082] The photothermal test strip assembly 9 includes a photothermal lateral flow analysis test strip 12 arranged vertically or horizontally, wherein:

[0083] When the photothermal lateral flow analysis test paper 12 is arranged vertically, specifically, a vertical test paper groove 5 is provided at the inner bottom of the front side of the light-proof and heat-insulating shell 3, and the photothermal lateral flow analysis test paper 12 is vertically inserted and fixed in the test paper groove 5;

[0084] When the photothermal lateral flow analysis test paper 12 is arranged horizontally, specifically, the photothermal test paper assembly 9 includes a test paper holder and a photothermal lateral flow analysis test paper 12, the test paper holder is inserted into and fixed in the sample channel 1, the test paper holder contains a rectangular groove, and the photothermal lateral flow analysis test paper 12 is fixed in the rectangular groove.

[0085] In one example, if Figure 2 As shown in d, the 808nm external coaxial reflection LED light source is provided by the existing external coaxial reflection LED light source 14 with a wavelength of 808nm, which is not specifically limited in the present invention and all belong to the protection scope of the present invention.

[0086] In one example, the present invention also provides a self-assembled 808nm external coaxial reflection LED light source, such as Figure 3 As shown, the self-assembled 808nm external coaxial reflection LED light source includes an LED light source 7, a beam splitter 11 and a plano-convex cylindrical lens 10, the plano-convex cylindrical lens 10 is vertically arranged inside the light-proof and heat-insulating shell 3, with its flat side facing forward, the LED light source 7 is horizontally placed at the bottom position of the rear side of the plano-convex cylindrical lens 10, the beam splitter 11 is arranged at a 45° angle between the plano-convex cylindrical lens 10 and the LED light source 7, and the LED light source 7 is gathered into a linear light spot after passing through the beam splitter 11 and the plano-convex cylindrical lens 10, forming a self-assembled external coaxial reflection LED light source 13.

[0087] The diffuse light emitted by the LED light source is filtered by a beam splitter to obtain uniform parallel light. The parallel light is gathered into a horizontal linear light spot after passing through a plano-convex cylindrical mirror. The power of the LED light source can be adjusted to meet the energy required for light-driven amplification and photothermal test paper detection under different room temperature conditions. The size of the light spot in the amplification container (0.2mL PCR tube) and on the test paper can be adjusted by adjusting the distance between the light-driven amplification component, the photothermal test paper component and the plano-convex cylindrical mirror to cope with amplification systems of different volumes and different T zones on the test paper.

[0088] In one example, the wavelength band of the LED light source 7 is 800-810 nm.

[0089] In one example, the illumination parameters of the LED light source 7 are power 4.5-6W and illumination time 10-30min.

[0090] In one example, two sides of the light-shielding and heat-insulating housing 3 are oppositely provided with a beam splitter groove 6 and a plano-convex cylindrical lens groove 4 for inserting a beam splitter 11 and a plano-convex cylindrical lens 10, respectively.

[0091] In one example, an observation channel 2 is provided on the top of the light-proof and heat-insulating shell 3 , which is located on the front side of the plane of the plano-convex cylindrical lens 10 and is used to observe the light-driven amplification component 8 and / or the photothermal lateral flow analysis test paper 12 .

[0092] In one example, the diameter of the PCR tube channel is 5.38 mm, and the center distance between adjacent PCR tube channels is 9 mm.

[0093] In one example, among the N penetrating PCR tube channels, N≥2.

[0094] In one example, the PCR tube is a 200 μL PCR single tube and / or a 200 μL eight-tube strip.

[0095] In one example, the distances between the bottom end of the beam splitter and the bottom ends of the LED light source and the plano-convex cylindrical lens are 1 cm and 15 cm respectively.

[0096] In one example, the beam splitter is made of AR glass, and has a size of length×width×thickness=141 mm×104 mm×2 mm.

[0097] In one example, the material of the plano-convex cylindrical lens is N-SF11, the dimensions are length×width×center thickness=100mm×104mm×50mm, the focal length is 60mm, and the edge thickness is 0mm.

[0098] In one example, the sample channel is 45 mm away from the plane of the plano-convex cylindrical lens and 60 mm away from the inner bottom of the light-proof and heat-insulating shell.

[0099] In one example, the depth of the rectangular groove on the test paper holder is 3 mm.

[0100] (6) Photothermal test paper detection:

[0101] The photoactivator Sb2MoO6@NiFeS2 was modified with three nucleic acid chains to obtain three photothermal signal probes. The capture probes were fixed in the T and C regions of the test paper to obtain multi-target photothermal test paper for the light-driven LAMP products of the target genes sul1, sul2, and sul3. The amplified products were detected using the photothermal test paper, and the temperature signal was read by a smartphone for quantification.

[0102] The principle of the present invention is as follows Figure 1 As shown. This method is efficient, convenient and accurate, and is suitable for the detection of sul1, sul2 and sul3. The present invention can achieve efficient on-site amplification of sul1, sul2 and sul3, and drive amplification by the photothermal effect of the 808nm external coaxial reflection LED excitation light activator Sb2MoO6@NiFeS2. The challenges and operational difficulties of using photoactivator-mediated and light-driven multiple LAMP are that the photoactivator must be compatible with the LAMP system and not interfere with the reaction; there must be a precisely controllable light source and light-driven conditions to avoid affecting the amplification stability; and a high-sensitivity and high-specificity detection method matching light-driven multiple LAMP needs to be developed.

[0103] The following examples use sul1, sul2 and sul3 as examples of resistance genes to fully illustrate the entire amplification method.

[0104] Embodiment 1:

[0105] A method for driving multiple LAMPs and performing photothermal paper detection and analysis using the photothermal effect of a photoactivator Sb2MoO6@NiFeS2, the method comprising the following steps:

[0106] (1) Primer sequence design:

[0107] According to the LAMP principle, amplification primer sets were designed for six different regions of the target gene sul1, sul2 and sul3 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:

[0108] Table 1 Primer sequences of the light-driven multiple LAMP system of the present invention

[0109]

[0110] (2) Plasmid DNA extraction:

[0111] The preserved bacteria containing the target genes (sul1, sul2, sul3) 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 RNase A, Buffer S, Solution I, Solution II, Solution III, Wash Solution, Elution Buffer, EZ-10 Column, and Collection Tube.

[0112] (3) Preparation of photoactivator Sb2MoO6@NiFeS2:

[0113] Ni(NO3)2·6H2O, FeCl2 and CH4N2S were dissolved in deionized water at a molar ratio of 1:1:8 and stirred for 1 hour to obtain a mixed solution. The mixed solution was injected into an aqueous solution of Sb2MoO6, with a molar ratio of Sb2MoO6 to nickel nitrate of 1:2, and heated in a reactor at 160°C for 1.5 hours. After the reaction was completed, the product was centrifuged at 10000 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 to obtain Sb2MoO6@NiFeS2.

[0114] The scanning electron microscopy and X-ray photoelectron spectroscopy of the photoactivator Sb2MoO6@NiFeS2 were measured. Figure 4 shown.

[0115] The photoactivator Sb2MoO6@NiFeS2 solution was irradiated with an 808nm external coaxial reflection LED light source in an integrated device and compared with the solutions of Sb2MoO6 and NiFeS2 with the same concentration. The photothermal heating curves of the three are shown in Figure 2. Figure 5 As shown, it can be seen that the heating efficiency of the Sb2MoO6@NiFeS2 composite is significantly improved compared with that of a single substance, which is beneficial to improving its efficiency as a photoactivator.

[0116] (4) Construction of light-driven multiple LAMP system:

[0117] The amplification system includes the following components: a primer set, a photoactivator Sb2MoO6@NiFeS2 (referring to the photoactivator Sb2MoO6@NiFeS2 solution prepared in step (3)), 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.

[0118] 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.

[0119] Table 2 Concentrations of components and reaction volumes of the light-driven multiple LAMP system in this example

[0120]

[0121]

[0122] (5)LED light source excitation:

[0123] In this example, the two links of light-driven amplification and photothermal test strip detection, together with the light source, are integrated into a collective device to study the detection effect of the integrated device on the three target genes sul1, sul2 and sul3. Figure 2 , 3 As shown, the device includes an 808nm external coaxial reflection LED light source, a light-driven amplification device and a photothermal test paper assembly 9, wherein:

[0124] The optically driven amplification device comprises: a light-proof and heat-insulating housing 3, and an optically driven amplification component 8 disposed inside the light-proof and heat-insulating housing 3, wherein:

[0125] The light-driven amplification component 8 includes a sample holder, which is provided with 10 through-holes for PCR tubes for placing PCR tubes, the PCR is a 200 μL PCR single tube, the diameter of the tube hole is 5.38 mm, and the center distance between adjacent PCR tube holes is 9 mm;

[0126] The PCR tube is used to hold the light-driven amplification system. As described in the above steps, the light-driven amplification system contains a primer set, a photoactivator Sb2MoO6@NiFeS2, a DNA template, Bst3.0 DNA polymerase, and an isothermal amplification buffer;

[0127] The front part of the light-proof and heat-insulating housing 3 has a transversely arranged sample channel 1, and the light-driven amplification component 8 is inserted and fixed in the sample channel 1; the 808nm external coaxial reflection LED light source is located at the rear part of the light-proof and heat-insulating housing 3, and is arranged toward the light-driven amplification component 8 and the photothermal lateral flow chromatography test paper component 9 to provide a light source for them;

[0128] The photothermal lateral flow chromatography test strip assembly 9 includes a photothermal lateral flow analysis test strip 12 arranged vertically or horizontally. An observation channel 2 is provided on the top of the light-proof and heat-insulating outer shell 3, which is located on the front side of the plane of the plano-convex cylindrical lens 10 and is used to observe the light-driven amplification assembly 8 and / or the photothermal lateral flow analysis test strip 12.

[0129] In one example, when the photothermal lateral flow analysis test paper 12 is arranged vertically, specifically, a vertical test paper groove 5 is provided at the inner bottom of the front side of the light-shielding and heat-insulating shell 3, and the photothermal lateral flow analysis test paper 12 is vertically inserted and fixed in the test paper groove 5;

[0130] In another example, when the photothermal lateral flow analysis test paper 12 is arranged horizontally, specifically, the photothermal lateral flow chromatography test paper assembly 9 includes a test paper holder and a photothermal lateral flow analysis test paper 12, the test paper holder is inserted into and fixed in the sample channel 1, and the test paper holder contains a rectangular groove with a depth of 3 mm, and the photothermal lateral flow analysis test paper 12 is fixed in the rectangular groove.

[0131] like Figure 3 As shown, the self-assembled 808nm external coaxial reflection LED light source of this embodiment includes an LED light source 7, a beam splitter 11 and a plano-convex cylindrical lens 10. The plano-convex cylindrical lens 10 is vertically arranged inside the light-proof heat-insulating shell 3, with its flat side facing forward. The LED light source 7 is horizontally arranged at the bottom position of the rear side of the plano-convex cylindrical lens 10. The beam splitter 11 is arranged between the plano-convex cylindrical lens 10 and the LED light source 7 at a 45° inclination. After passing through the beam splitter 11 and the plano-convex cylindrical lens 10, the LED light source 7 is gathered into a linear light spot to form an external coaxial reflection LED light source 13. The two sides of the light-proof heat-insulating shell 3 are relatively provided with a beam splitter groove 6 and a plano-convex cylindrical lens groove 4, which are used to insert the beam splitter 11 and the plano-convex cylindrical lens 10 respectively. The material of the beam splitter is AR glass, and the size is length×width×thickness=141mm×104mm×2mm. The material of the plano-convex cylindrical lens is N-SF11, the size is length×width×center thickness=100mm×104mm×50mm, the focal length is 60mm, and the edge thickness is 0mm. The distances between the bottom end of the beam splitter and the bottom ends of the LED light source and the plano-convex cylindrical lens are 1cm and 15cm respectively.

[0132] One of the challenges and operational difficulties of using photoactivator-mediated, light-driven multiple LAMP is that a precisely controllable light source and light-driven conditions are required to avoid affecting the stability of amplification. The diffuse light emitted by the LED light source is filtered by a beam splitter to obtain uniform parallel light, which is then focused into a horizontal linear light spot after passing through a plano-convex cylindrical mirror. By adjusting the power of the LED light source, the energy required for light-driven amplification and photothermal test strip detection under different room temperature conditions can be met. By adjusting the distance between the light-driven amplification component, the photothermal test strip component and the plano-convex cylindrical mirror, the size of the light spot in the amplification container (0.2mL PCR tube) and the test strip can be adjusted to cope with amplification systems of different volumes and different T zones on the test strip.

[0133] The amplification system is irradiated by an 808nm external coaxial reflection LED in the integrated device to excite the light activator in the amplification system to provide the energy required for multiple LAMPs, achieve amplification, and obtain amplification products. In this embodiment, the excitation power of the 808nm external coaxial reflection LED is 4.5W, and the excitation time is 20min.

[0134] (6) Analysis of amplification results:

[0135] Three 20 μL nucleic acid chains were added to 2 mL photoactivator Sb2MoO6@NiFeS2 respectively, and 50 μL sodium citrate buffer was added after oscillation and mixing, and the mixture was incubated at 37°C for 30 min. Then, 100 μL HEPES buffer was added, and the mixture was incubated at 37°C for 30 min. After centrifugation, 150 μL photothermal reconstitution solution was added to dissolve the mixture, and three photothermal signal probes modified with three nucleic acid chains on photoactivator Sb2MoO6@NiFeS2 were obtained.

[0136] Capture probes were fixed in the T and C regions of the test paper to obtain a multi-target photothermal test paper targeting the light-driven LAMP products of the target genes sul1, sul2, and sul3.

[0137] The probe sequences of the photothermal test paper for detecting amplification products are shown in Table 3 below.

[0138] Table 3 Probe sequences of amplification products detected by photothermal test paper in this embodiment

[0139]

[0140] The photothermal lateral flow chromatography test paper assembly 9 of the integrated device is used to detect the amplification product obtained in the above steps. The specific method is as follows: In this embodiment, a vertical test paper groove 5 is provided on the inner bottom of the front side of the light-proof heat-insulating shell 3, and the photothermal lateral flow analysis test paper 12 is vertically inserted and fixed in the test paper groove 5, such as Figure 3 As shown in g; turn on the LED light source 7, the LED light source is focused into a linear light spot after passing through the beam splitter 11 and the plano-convex cylindrical lens 10, forming an external coaxial reflective LED light source 13, which is irradiated onto the photothermal lateral flow analysis test paper 12 in the front to provide it with photothermal heat, thereby stimulating the photothermal effect of the photoactivator Sb2MoO6@NiFeS2 in the photothermal signal probe, and realizing the detection efficacy of the photothermal test paper; and read the photothermal signal of the T zone on the photothermal lateral flow analysis test paper 12 through a smart phone equipped with an infrared thermal imaging accessory, and judge the presence or absence of the target object according to the change phenomenon of the photothermal signal, and perform a qualitative test; and can further perform a quantitative test according to the intensity of the temperature change signal, wherein the standard curve used in the quantitative test can be characterized and drawn according to the concentration gradient method.

[0141] (7) Specificity verification

[0142] After adding all reagents except primers and templates to 6 light-driven LAMP test tubes, add

[0143] ①Sul1 template, Sul2 primer, Sul3 primer;

[0144] ②Sul1 template, Sul1 primer, Sul2 primer, Sul3 primer;

[0145] ③Sul2 template, Sul3 primer;

[0146] ④Sul2 template, Sul2 primer, Sul3 primer;

[0147] ⑤Sul3 template, Sul2 primer;

[0148] ⑥Sul3 template, Sul2 primer, Sul3 primer;

[0149] Figure 6 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 Sul1, Sul2, and Sul3.

[0150] Figure 7 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 amplification products of specific target genes in the multiple LAMP system in colorimetric and photothermal modes, respectively.

[0151] Figure 8 This is a concentration gradient detection experiment for the three target genes sul1, sul2 and sul3, 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 sul1, sul2 and sul3. After experimental verification, the detection limit of the three target genes sul1, sul2 and sul3 reached 10 copies / μL.

[0152] In addition, the scattered light LED and the external coaxial reflection LED were used as the light-driven LAMP light source to compare their amplification effects. Fig. 9 As shown in the figure, only the external coaxial reflection LED can give a bright diffuse strip, 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.

[0153] Comparative Example 1: Water Bath LAMP

[0154] This example compares the method of realizing light-driven multiple LAMP using the photothermal effect of the photoactivator Sb2MoO6@NiFeS2 with the effect of the traditional water bath LAMP to verify the advancement of the method of the present invention in terms of amplification effect.

[0155] The water bath LAMP method includes the following steps:

[0156] (1) Primer sequence design:

[0157] Same as Example 1

[0158] (2) Plasmid DNA extraction:

[0159] Same as Example 1

[0160] (3) Construction of isothermal amplification system:

[0161] 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.

[0162] 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:

[0163] Table 4 Concentrations of components and reaction volumes of water bath LAMP system

[0164]

[0165] (4) Amplification process:

[0166] 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.

[0167] (5) Amplification product analysis:

[0168] The amplified products were analyzed by agarose gel electrophoresis.

[0169] Gel electrophoresis images such as Fig.10 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 showed bright diffuse bands after 60 minutes of amplification, while the light-driven LAMP group showed clear and bright diffuse bands at 20 minutes, indicating that the amplification efficiency of the light-driven LAMP group is better than that of the water bath LAMP group.

[0170] 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 method for light-driven amplification-photothermal analysis of sul1, sul2, and sul3, characterized in that: The following steps are involved: S1. Preparation of photoactivator Sb2MoO6@NiFeS2; S2, light-driven LAMP amplification Under the mediation of the photoactivator Sb2MoO6@NiFeS2, the sample to be tested is amplified by the light-driven ring-mediated isothermal amplification, i.e., the light-driven LAMP isothermal amplification system, using 808nm external coaxial reflection LED light source to obtain the amplified product. The LAMP isothermal amplification system includes the following components: a primer set, a photoactivator Sb2MoO6@NiFeS2, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer, wherein the DNA template is the resistance genes sul1, sul2, and sul3; S3, Photothermal test paper detection Capture probes were fixed on the T and C regions of the test strip to obtain a multi-target photothermal lateral flow chromatography test strip targeting the light-driven LAMP products of the target genes sul1, sul2, and sul3; The photothermal signal probe was obtained by modifying the photoactivator Sb2MoO6@NiFeS2 with the nucleic acid chains of sul1, sul2, and sul3 respectively; The amplification product is detected using the photothermal signal probe and the photothermal lateral flow chromatography test paper. The detection area of ​​the photothermal lateral flow chromatography 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 an 808nm external coaxial reflection LED light source. The sul1, sul2, and sul3 in the sample to be tested are qualitatively and / or quantitatively determined based on the temperature change signal.

2. The method according to claim 1, 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.

3. The method according to claim 1, characterized in that The preparation method of the photoactivator Sb2MoO6@NiFeS2 comprises the following steps: (1) Dissolve Ni(NO3)2·6H2O, FeCl2 and CH4N2S in deionized water at a molar ratio of 1:1:8-1:1:10, and stir for 1-2 hours to obtain a mixed solution; (2) injecting the mixed solution into an aqueous solution of Sb2MoO6, wherein the molar ratio of Sb2MoO6 to nickel nitrate is 1:2-1:4, and heating in a reactor at 160-180°C for 1-2h. After the reaction is completed, a primary product is obtained; (3) The product is centrifuged, washed and dried to obtain Sb2MoO6@NiFeS2.

4. The method according to claim 3, characterized in that In step (1), the concentration of nickel nitrate dissolved in water is 0.4-0.5 mM.

5. The method according to claim 3, characterized in that: In step (2), the concentration of Sb2MoO6 dissolved in water is 0.1-0.25mM.

6. The method according to claim 3, 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.

7. The method according to claim 1, characterized in that In the LAMP isothermal amplification system, the concentration of each group of DNA templates is 4%-8% v / v; Preferably, in the LAMP isothermal 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; Preferably, the LAMP isothermal amplification system further comprises the following components: BSA, dNTPs, betaine and ddH2O; Preferably, the LAMP isothermal amplification buffer comprises Tris-HCl, (NH4)2SO4, KCl, MgSO4, and Tween-20.

8. The method according to claim 1, 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.

9. A device for light-driven amplification-photothermal analysis of sul1, sul2, and sul3, characterized in that: It comprises an 808nm external coaxial reflection LED light source, a light-driven amplification device and a photothermal lateral flow chromatography test paper assembly (9), wherein: The light-driven amplification device comprises: a light-proof and heat-insulating shell (3), and a light-driven amplification component (8) arranged inside the light-proof and heat-insulating shell (3), wherein: The light-driven amplification component (8) comprises a sample holder, on which N PCR tube channels are provided for placing PCR tubes, wherein N≥1; the PCR tubes are used to hold a LAMP light-driven amplification system, wherein the LAMP light-driven amplification system contains a primer set, a light activator Sb2MoO6@NiFeS2, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer, wherein the DNA template is resistance genes sul1, sul2, and sul3; The front part of the light-proof heat-insulating housing (3) has a transversely arranged sample channel (1), and the light-driven amplification component (8) is inserted into and fixed in the sample channel (1); the 808nm external coaxial reflection LED light source is located at the rear part of the light-proof heat-insulating housing (3), and is arranged toward the light-driven amplification component (8) and the photothermal lateral flow chromatography test paper component (9); The photothermal lateral flow chromatography test strip assembly (9) comprises a photothermal lateral flow analysis test strip (12) arranged vertically or horizontally and a photothermal signal probe, wherein the photothermal signal probe is obtained by modifying the photoactivator Sb2MoO6@NiFeS2 with the nucleic acid chains of sul1, sul2 and sul3, respectively, wherein: When the photothermal lateral flow analysis test paper (12) is arranged vertically, it specifically comprises that a vertical test paper groove (5) is arranged on the inner bottom of the front side of the light-proof and heat-insulating shell (3), and the photothermal lateral flow analysis test paper (12) is vertically inserted and fixed in the test paper groove (5); When the photothermal lateral flow analysis test paper (12) is arranged horizontally, the photothermal lateral flow chromatography test paper assembly (9) comprises a test paper holder and a photothermal lateral flow analysis test paper (12), the test paper holder is inserted into and fixed in the sample channel (1), the test paper holder comprises a rectangular groove, and the photothermal lateral flow analysis test paper (12) is fixed in the rectangular groove.

10. The device according to claim 9, characterized in that The 808nm external coaxial reflection LED light source comprises an LED light source (7), a beam splitter (11) and a plano-convex cylindrical lens (10); the plano-convex cylindrical lens (10) is vertically arranged inside the light-shielding and heat-insulating shell (3) with its flat side facing forward; the LED light source (7) is horizontally arranged at the bottom position of the rear side of the plano-convex cylindrical lens (10); the beam splitter (11) is arranged between the plano-convex cylindrical lens (10) and the LED light source (7) at an angle of 45°; the LED light source (7) is gathered into a linear light spot after passing through the beam splitter (11) and the plano-convex cylindrical lens (10), thereby forming an external coaxial reflection LED light source (13); Preferably, the wavelength of the LED light source (7) is between 800-810 nm; and / or Preferably, the irradiation parameters of the LED light source (7) are power 4.5-6W, irradiation time 10-30min; and / or Preferably, the light-shielding and heat-insulating housing (3) has a beam splitter groove (6) and a plano-convex cylindrical lens groove (4) on two sides thereof, for inserting a beam splitter (11) and a plano-convex cylindrical lens (10) respectively; and / or Preferably, an observation channel (2) is provided on the top of the light-proof and heat-insulating housing (3), located on the front side of the plane of the plano-convex cylindrical lens (10), for observing the light-driven amplification component (8) and / or the photothermal lateral flow analysis test paper (12).

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