Photosensitive material for lin B, erm B and ee D and detection method thereof

Abstract

The invention discloses a photosensitive material for simultaneously detecting lin B, erm B and ee D and a detection method of the photosensitive material. A sample is simultaneously irradiated by a coaxial reflection LED light source outside 532nm, a sensitizer Ag-PDA / Cu3P2O8 mediated light regulates multiple LAMP reactions, and an amplification reaction of a target gene in a positive sample is initiated, so that a large number of double-stranded DNA amplification products are obtained. A photothermal signal probe is obtained by modifying Ag-PDA / Cu3P2O8 with a nucleic acid chain, a detection region of photothermal test paper captures an amplification product and the signal probe in a sandwich form, a photothermal temperature change signal positively related to the content of the amplification product is given, and the genes lin B, erm B and ee D in a sample are quantified according to the temperature change signal. Photoelectron and photothermal effects are generated by using the photoinduced activity of the sensitizer Ag-PDA / Cu3P2O8, the temperature is quickly controlled, the sensitizer is in direct contact with an amplification system on a molecular level, and then photothermal test paper color development detection is performed, so that the photothermal efficiency is remarkably improved.

Description

Technical Field

[0001] The invention relates to a photosensitive material for macrolide resistance genes lin B, erm B and ere D and a detection method thereof, belonging to the technical field of analysis and detection. Background Art

[0002] The irrational use of antibiotics has led to a large amount of antibiotics flowing into the environment, which in turn has induced the generation and widespread development of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). ARGs can be spread through vertical gene transfer and horizontal gene transfer, posing a serious threat to the ecological environment and human health. The abuse of antibiotics in various industries in my country is very serious, including aquaculture, livestock and poultry farming, and fruit and vegetable farming. In the livestock and poultry farming industry, the use of macrolide antibiotics accounts for 26% of all antibiotics. Macrolide antibiotics have strong antibacterial effects, stable structures, are difficult to degrade, and are easy to accumulate. Macrolide resistance genes are acquired and developed by bacteria and other microorganisms under the pressure of macrolide antibiotics through gene mutation or horizontal gene transfer, which can make microorganisms have corresponding macrolide resistance. With the widespread use of macrolide antibiotics, the discharge of bacterial residue waste and sewage has made them ubiquitous in the environment. Macrolides and macrolide resistance genes may enter animals and humans through direct ingestion, food chains, etc. If the resistance genes are integrated into pathogenic bacteria and successfully express antibiotic resistance, causing antibiotics to lose their therapeutic effect, they will become a major threat to human health. Therefore, in order to effectively prevent and control the formation and spread of macrolide ARGs, it is crucial to develop methods to simultaneously and rapidly detect multiple macrolide resistance genes.

[0003] The detection methods of antibiotic resistance genes are mainly based on nucleic acid amplification, including polymerase chain reaction (PCR), real-time fluorescence quantitative PCR technology (qPCR), metagenomics, and isothermal amplification technology. Because PCR amplification takes a long time, relies on professionals and equipment, and is cumbersome to operate, it is not suitable for rapid on-site detection. Among them, loop-mediated isothermal amplification technology LAMP uses a DNA polymerase with chain displacement activity and a set of specially designed primers to amplify the target gene at a constant temperature of 60-65°C. It is efficient, sensitive, and easy to operate. It is widely used in many fields such as food safety and pathogenic microorganism detection.

[0004] Photo-regulated amplification is to add various photoactive nanomaterials or sensitizers to the amplification system, use light-emitting diodes (LEDs) or lasers as light sources, and evenly disperse the sensitizers in the amplification system. After being excited by the light source, photoelectric energy and heat are supplied for nucleic acid amplification. Jin et al. developed a photothermal LAMP driven by a paper-based device made of a carbon black-poly(dimethylsiloxane) mixture (PDMS), which can rapidly amplify the rfbE gene of the foodborne pathogen Escherichia coli O157:H7 within 15 minutes and visualize the results, with a detection limit of 10 2 CFU·mL -1 (Analytical Methods 2022, 14, 2569). Light-regulated amplification improves amplification efficiency and has been combined with amplification technologies such as single-plex PCR, LAMP, and RCA. It has good amplification specificity and sensitivity and is a promising on-site detection technology.

[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, 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.

[0006] At present, there is no report on the use of external coaxial reflection LED light source excitation-light regulation of multiple LAMP reactions combined with photothermal test paper technology to simultaneously detect multiple antibiotic resistance genes. Summary of the invention

[0007] Technical issues: The efficiency of existing photo-regulated amplification sensitizers is not ideal; photo-regulated 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 photo-regulated amplification and photothermal test paper technology for the detection of macrolide resistance genes.

[0008] Purpose of the invention: In order to overcome the shortcomings and defects of the above-mentioned prior art, the present invention provides a photosensitive material for lin B, ermB, and ere D and a detection method thereof, adopting the sensitizer Ag-PDA / Cu3P2O8 as the photothermal material, utilizing its photoactivity to generate photoelectrons and photothermal effects, and directly contacting with the LAMP amplification system at the molecular level under the irradiation of a 532nm external coaxial reflection LED light source to achieve rapid temperature control and significantly improve the photothermal efficiency, and then performing photothermal test paper colorimetric detection, thereby realizing intensive and efficient amplification and detection of the two links of light-regulated amplification and photothermal test paper detection.

[0009] Technical solution: The present invention adopts the following technical solution.

[0010] The first object of the present invention is to provide a sensitizer for lin B, erm B, and ere D, wherein the sensitizer for lin B, erm B, and ere D is Ag-PDA / Cu3P2O8.

[0011] In the present invention, the preparation of Ag-PDA / Cu3P2O8 nanomaterial is prepared by co-modifying Cu3P2O8 powder with Ag / PDA nanoparticles. Specifically, the preparation method of Ag-PDA / Cu3P2O8 nanomaterial comprises the following steps:

[0012] 10-20 mg (preferably 20 mg) of Ag-PDA and 15-25 mg (preferably 20 mg) of Cu3P2O8 powder were dispersed in 50 mL of water, and continuously stirred and ultrasonicated for 24-26 h (preferably 24 h). The above solution was quickly frozen with liquid nitrogen, and freeze-dried for 36-40 h to obtain Ag-PDA / Cu3P2O8 nanomaterials (preferably 36 h).

[0013] As an optional embodiment, the specific preparation method of the Ag-PDA comprises the following steps:

[0014] Ultrasonic dispersion of 90-110 mg of PDA nanoparticles in 20 mL of water (preferably 100 mg), continuous stirring and ultrasonic treatment for 2-3 h (preferably 2 h) to make it uniformly dispersed. Then add 4-6 mL of AgNO3 solution as Ag precursor (preferably 5 mL), and ultrasonic treatment for 10-15 min (preferably 10 min) in a dark environment. Quickly freeze the solution with liquid nitrogen, then irradiate it under ultraviolet light for 30-40 min (preferably 30 min), and add liquid nitrogen to maintain the freezing effect. Thaw the solution, centrifuge, wash with water and repeat 3-4 times, and finally freeze-dry to obtain Ag-PDA powder.

[0015] As an optional embodiment, a solid phase reaction method is used to synthesize Cu3P2O8 powder. The precursor materials are CuO and (NH4)2HPO4. CuO and (NH4)2HPO4 are mixed and homogenized in a molar ratio of 2:1-1:1 by ball milling in EtOH with ZrO2 grinding media for 24 to 36 hours (preferably 36 hours). The ball-milled mixture is then quickly dried at 85 to 95°C (preferably 90°C), and then annealed in air at a heating rate of 4.5 to 5.5°C / min at 1000 to 1100°C for 4 to 5 hours (preferably annealed at 1000°C for 4 hours at a heating rate of 5°C / min) to obtain pure phase crystalline Cu3P2O8.

[0016] The second object of the present invention is to provide a method for realizing light-controlled multiple loop-mediated isothermal amplification and detection of macrolide resistance genes lin B, erm B, and ere D by utilizing the photothermal effect of sensitizer Ag-PDA / Cu3P2O8 nanomaterials.

[0017] Specifically, the present invention provides a method for simultaneously detecting lin B, erm B, and ere D, comprising the following steps:

[0018] S1. Light-regulated multiple LAMP

[0019] Under the irradiation of a 532nm external coaxial reflective LED light source, the sample to be tested is subjected to an amplification reaction by a light-regulated multiple LAMP system to obtain a light-regulated multiple LAMP product, wherein the light-regulated multiple LAMP system comprises the following components: a primer set, a sensitizer Ag-PDA / Cu3P2O8, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer, wherein the DNA template is resistance genes lin B, erm B, and ere D;

[0020] S2. Photothermal test paper detection

[0021] Capture probes were fixed on the T and C regions of the test strip to obtain a multi-target photothermal test strip targeting the target genes lin B, erm B, and ere D light-regulated multiple LAMP products;

[0022] The photothermal signal probe was obtained by modifying the sensitizer Ag-PDA / Cu3P2O8 with lin B, erm B, and ere D nucleic acid chains respectively;

[0023] The photothermal signal probe and the photothermal test paper are used to detect the above-mentioned light-regulated multiple LAMP products. The detection area of ​​the photothermal test paper captures the light-regulated multiple LAMP products and the photothermal signal probe in a sandwich form, and gives a photothermal temperature change signal positively correlated with the content of the light-regulated multiple LAMP products under the irradiation of a 532nm external coaxial reflection LED light source. According to the temperature change signal, lin B, erm B, and ere D in the sample to be tested are qualitatively and / or quantitatively determined.

[0024] As an optional implementation, in the light-regulated multiple LAMP system, the concentration of each group of DNA templates is 3% to 6% v / v.

[0025] 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.

[0026] As an optional embodiment, in the light-regulated multiple LAMP system, the concentration of the two external primers F3 and B3 in each set of primers is 2% to 4% v / v, the concentration of the two internal primers FIP and BIP is 2% to 6% v / v, and the concentration of the two loop primers loop F and loop B is 0 to 4% v / v.

[0027] As an optional embodiment, the light-regulated multiple LAMP system further includes the following components: BSA, dNTPs, betaine and ddH2O.

[0028] As an optional embodiment, the light-regulated multiple LAMP buffer includes Tris-HCl, (NH4)2SO4, KCl, MgSO4, and Tween-20.

[0029] As an optional embodiment, the photothermal signal probe is used independently of the photothermal test paper body, or is dropped and dried on the conjugate pad and the conjugate pad is integrated on the photothermal test paper.

[0030] As an optional implementation, the irradiation parameters of the 532nm external coaxial reflection LED light source are power 4 to 8W and irradiation time 10 to 25 minutes.

[0031] Beneficial effects: The photosensitive material for lin B, erm B, and ere D provided by the present invention uses the photoactive sensitizer Ag-PDA / Cu3P2O8 to generate photoelectrons and photothermal effects, which are in direct contact with the light-regulated multiple LAMP system at the molecular level, can quickly control the temperature, and is significantly more efficient than traditional water baths and metal baths. At the same time, the sensitizer is used to synthesize the signal probe of the photothermal test paper to provide sensitive and quantifiable signals. The whole set of methods can quickly achieve efficient amplification and detection of lin B, erm B, and ere D target DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the working process of the present invention;

[0033] Figure 2 The scanning electron microscope and X-ray element energy spectrum of the Ag-PDA / Cu3P2O8 nanomaterial of the present invention;

[0034] Figure 3 is the photothermal temperature rise of the sensitizer of the present invention;

[0035] Figure 4 To verify the light-regulated multiple LAMP primers of the present invention;

[0036] Figure 5 The electrophoresis photos, color development and photothermal test paper photos of the light-regulated single and multiple LAMP products of the present invention are shown;

[0037] Figure 6 The light-regulated LAMP of the present invention is combined with lateral flow chromatography test paper for photothermal quantification;

[0038] Figure 7 The electrophoresis diagram of the products of the optical regulation of multiple LAMP of erm B using an external coaxial reflection LED light source and a scattered light LED light source respectively;

[0039] Figure 8 The present invention uses gel electrophoresis of light-regulated multiplex LAMP and water-bath LAMP amplification products. DETAILED DESCRIPTION

[0040] 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.

[0041] The present invention discloses a photosensitive material for macrolide resistance genes lin B, erm B, ere D, and a method for applying the same to light-regulated loop-mediated isothermal amplification (LAMP) combined with photothermal test paper analysis. Specifically, the method includes two steps: light-regulated LAMP and photothermal test paper detection:

[0042] (1) The samples were simultaneously irradiated with a 532nm external coaxial reflection LED light source. The sensitizer Ag-PDA / Cu3P2O8 provided photoelectrons and heat for LAMP through light regulation. The target gene in the positive sample was used as a template to trigger the amplification reaction and obtain a large amount of double-stranded DNA amplification products.

[0043] (2) Ag-PDA / Cu3P2O8 is modified with three nucleic acid chains of lin B, erm B, and ere D to obtain three signal probes. The detection area of ​​the photothermal 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 light-regulated LAMP product. According to the temperature change signal, lin B, erm B, and ere D in the original sample are further quantified. The present invention uses the photoactivity of the sensitizer Ag-PDA / Cu3P2O8 to generate photoelectrons and photothermal effects, quickly controls the temperature and directly contacts the light-regulated LAMP system at the molecular level, and then performs color development detection with the photothermal test paper, and the photothermal efficiency is significantly improved.

[0044] The following examples use lin B, erm B and ere D as examples of resistance genes to fully illustrate the entire amplification method.

[0045] Embodiment 1:

[0046] The principle of the present invention is as follows Figure 1 As shown, a method for driving multiple LAMPs and performing specific detection using the photothermal effect of sensitizer Ag-PDA / Cu3P2O8 comprises the following steps:

[0047] (1) Preparation of sensitizer Ag-PDA / Cu3P2O8:

[0048] 100 mg of PDA nanoparticles were ultrasonically dispersed in 20 mL of deionized water, stirred continuously and ultrasonically treated for 2 h to make them uniformly dispersed. Then 5 ml of AgNO3 solution was added as an Ag precursor and ultrasonically treated for 10 min in a dark environment. The solution was quickly frozen with liquid nitrogen, then irradiated under ultraviolet light for 30 min, and liquid nitrogen was added to maintain the freezing effect. The solution was thawed, centrifuged, washed with water and repeated 3 times, and finally freeze-dried to obtain Ag-PDA powder.

[0049] Among them, the preparation of Cu3P2O8 powder adopts the solid phase reaction method: the precursor materials are CuO and (NH4)2HPO4. CuO and (NH4)2HPO4 are mixed and homogenized in a molar ratio of 3:2 by ball milling in EtOH with ZrO2 grinding media for 24 hours. The ball-milled mixture is then quickly dried at 90°C and then annealed at 1000°C for 4h in air at a heating rate of 5°C / min to obtain pure phase crystalline Cu3P2O8.

[0050] Ag-PDA / Cu3P2O8 nanomaterials were prepared by co-modifying Cu3P2O8 powder with Ag / PDA nanoparticles: 20 mg of Ag-PDA and 20 mg of Cu3P2O8 powder were redispersed in 50 mL of water, stirred and ultrasonicated for 24 h, and the solution was quickly frozen with liquid nitrogen and freeze-dried for 36 h to obtain Ag-PDA / Cu3P2O8 nanomaterials.

[0051] Determination of Ag-PDA / Cu3P2O8 nanomaterials by scanning electron microscopy and X-ray elemental spectrum mapping Figure 2 shown.

[0052] The sensitizer Ag-PDA / Cu3P2O8 solution was irradiated with a 532nm external coaxial reflection LED light source and compared with the solutions of Ag-PDA and Cu3P2O8 with the same concentration. The photothermal heating curves of the three are as follows: Figure 3 As shown, it can be seen that the heating efficiency of the Ag-PDA / Cu3P2O8 composite is significantly improved compared with that of a single substance, which is beneficial to improving its efficiency as a sensitizer.

[0053] (2) Primer sequence design:

[0054] According to the principle of loop-mediated isothermal amplification, amplification primer sets were designed for six different regions of the target gene sequence. 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). The primer sequences are shown in Table 1:

[0055] Table 1 Primer sequences of the light-regulated multiple loop-mediated isothermal amplification system of the present invention

[0056]

[0057] (3) Plasmid DNA extraction:

[0058] Bacillus subtilis containing the target genes (lin B, erm B, ere D) were cultured for 24 hours respectively, and the plasmid DNA containing the target genes was extracted. The specific method was: plasmid DNA was extracted using a DNA extraction kit, and the kit included RNaseA, Buffer S, Solution I, Solution II, Solution III, Wash Solution, Elution Buffer, EZ-10 Column, and Collection Tube.

[0059] (4) Construction of light-regulated multiple loop-mediated isothermal amplification system:

[0060] The light-regulated multiple LAMP system includes the following components: a primer set, a sensitizer Ag-PDA / Cu3P2O8 (referring to the Ag-PDA / Cu3P2O8 nanomaterial 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.

[0061] Under the premise of maintaining the final volume at 25 μL, the concentrations of the components and the reaction volumes of the light-regulated multiple LAMP system are shown in Table 2 below.

[0062] Table 2 Concentrations of components and reaction volumes of the light-regulated multiple LAMP system in this example

[0063]

[0064] (5)LED light source excitation:

[0065] The multiple LAMP system was regulated by irradiating a 532nm external coaxial reflection LED light source to excite the sensitizer in the system to provide the energy required for multiple LAMP. The excitation power was 7W and the excitation time was 20min.

[0066] (6) Light-regulated multiple LAMP and analysis of detection results:

[0067] Three photothermal signal probes were obtained by modifying the sensitizer Ag-PDA / Cu3P2O8 with three nucleic acid chains. The specific method was as follows: 20 μL of three nucleic acid chains were added to 2 mL of sensitizer Ag-PDA / Cu3P2O8 respectively, oscillated and mixed, then 50 μL of sodium citrate buffer was added, incubated at 37°C for 30 min, then 100 μL of HEPES buffer was added, incubated at 37°C for 30 min, centrifuged, and 150 μL of photothermal reconstitution solution was added to dissolve, thus obtaining three photothermal signal probes modified with the sensitizer Ag-PDA / Cu3P2O8 with three nucleic acid chains.

[0068] Capture probes were fixed in the T and C regions of the test paper to obtain a multi-target photothermal test paper for the light-regulated LAMP products of the target genes lin B, erm B, and ere D. The probe sequences of the photothermal test paper for detecting light-regulated LAMP products are shown in Table 3 below.

[0069] Table 3 Probe sequences of the photothermal test paper for detecting light-regulated LAMP products in this embodiment

[0070]

[0071] The constructed multi-target photothermal test paper is used to detect light-regulated LAMP products and provide them with photothermal heat, thereby stimulating the photothermal effect of the sensitizer CsYF4@Yb2O3 in the photothermal signal probe, and realizing the detection efficacy of the photothermal test paper; and the photothermal signal of the T zone on the photothermal 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; quantitative testing can also be further performed according to the intensity of the temperature change signal, and the standard curve used in the quantitative test can be characterized and drawn according to the concentration gradient method.

[0072] (7) Specificity verification

[0073] After adding all reagents except primers and templates to 6 light-regulated LAMP test tubes, add

[0074] ①lin B template, erm B primer, ere D primer;

[0075] ②lin B template, lin B primer, erm B primer, ere D primer;

[0076] ③erm B template, ere D primer;

[0077] ④erm B template, erm B primer, ere D primer;

[0078] ⑤ere D template, erm B primer;

[0079] ⑥ere D template, erm B primer, ere D primer.

[0080] 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 lin B, erm B, and ere D.

[0081] Figure 5The photo of the test paper shows that the photothermal test paper can specifically detect the amplification products of specific target genes in the light-regulated multiple LAMP system in colorimetric and photothermal modes respectively.

[0082] Figure 6 This is a concentration gradient detection experiment for the three target genes lin B, erm B and ere D, 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 lin B, erm B and ere D. After experimental verification, the detection limit of the three target genes lin B, erm B and ere D reached 10 copies / μL.

[0083] In addition, other LED light sources and external coaxial reflection LED light sources are used as light-regulated LAMP light sources to compare their amplification effects. Figure 7 As shown in the figure, only the external coaxial reflection LED light source can give a bright diffuse strip, that is, a high-efficiency amplification, while other LED light sources do not induce effective amplification. The reason may be that other LEDs generate light energy and heat energy when working, and 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.

[0084] Comparative Example 1: Water bath ring-mediated isothermal amplification

[0085] This example compares the photothermal effect of Ag-PDA / Cu3P2O8 nanomaterials to achieve photoregulated multiple ring-mediated isothermal amplification with the traditional water bath ring-mediated isothermal amplification to verify the advancement of the method of the present invention in terms of amplification effect.

[0086] The water bath ring-mediated isothermal amplification method comprises the following steps:

[0087] (1) Primer sequence design:

[0088] Same as Example 1

[0089] (2) Plasmid DNA extraction:

[0090] Same as Example 1

[0091] (3) Construction of isothermal amplification system:

[0092] The water bath ring-mediated isothermal amplification system was constructed. The water bath ring-mediated amplification system included the following components: primer set, MgSO4, isothermal amplification buffer (Tris-HCl, (NH4)2SO4, KCl, MgSO4, Tween-20), dNTPs, betaine, Bst3.0 DNA polymerase, ddH2O and DNA template.

[0093] Under the premise of maintaining the final volume at 25 μL, the concentrations of the components and the reaction volumes of the water bath ring-mediated isothermal amplification system are shown in Table 4 below:

[0094] Table 4 Concentrations and reaction volumes of components in water bath ring-mediated isothermal amplification system

[0095]

[0096] (4) Amplification process:

[0097] Add the components of the water bath ring-mediated isothermal amplification system into a 1.5 mL centrifuge tube and place it in a 65°C water bath for 60 min.

[0098] (5) Amplification product analysis:

[0099] The amplified products were analyzed by agarose gel electrophoresis.

[0100] Gel electrophoresis images such as Figure 8 As shown, the negative group image without template DNA only shows the primer group, without diffuse bands, while the image of the positive amplification group containing template DNA that has been successfully amplified will show diffuse bands. At the same time, the water bath group of comparative example 1 has amplification bands for 20 minutes, and the light-regulated amplification group has clear and bright diffuse bands for 20 minutes, indicating that the amplification efficiency of the light-regulated nucleic acid amplification group is better than that of the water bath group.

[0101] 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 sensitizer for lin B, erm B, ere D, characterized in that: The sensitizer for lin B, erm B, and ere D genes is Ag-PDA / Cu3P2O8, which is prepared by co-modifying Cu3P2O8 powder with Ag / PDA nanoparticles. The preparation method of the sensitizer Ag-PDA / Cu3P2O8 comprises the following steps: 10-20 mg of Ag-PDA and 15-25 mg of Cu3P2O8 powder were dispersed in 50 mL of water, continuously stirred and ultrasonically treated for 24-26 h, frozen in liquid nitrogen, and then freeze-dried for 36-40 h to obtain Ag-PDA / Cu3P2O8 nanomaterials; The sensitizer Ag-PDA / Cu3P2O8 is used for the light-regulated multiple loop-mediated isothermal amplification reaction of lin B, erm B, and ere D genes, namely, the light-regulated multiple LAMP reaction, and / or for the photothermal test paper detection of lin B, erm B, and ere D genes.

2. The sensitizer for lin B, erm B, ere D according to claim 1, characterized in that The preparation method of the Ag-PDA comprises the following steps: 90-110 mg of PDA nanoparticles were ultrasonically dispersed in 20 mL of water, and the mixture was continuously stirred and ultrasonically treated for 2-3 h to make it uniformly dispersed; then 4-6 mL of AgNO3 solution was added as an Ag precursor and ultrasonically treated for 10-15 min in a dark environment; the mixture was quickly frozen with liquid nitrogen and then irradiated under ultraviolet light for 30-40 min, while liquid nitrogen was added to maintain the freezing effect; the solution was thawed, centrifuged, washed with water, and finally freeze-dried to obtain Ag-PDA powder.

3. The sensitizer for lin B, erm B, ere D according to claim 1, characterized in that The Cu3P2O8 powder is synthesized by a solid phase reaction method, which specifically includes the following steps: The precursor materials are CuO and (NH4)2HPO4. CuO and (NH4)2HPO4 are mixed and homogenized in a molar ratio of 2:1-1:1 by ball milling in EtOH with ZrO2 grinding media for 24 to 36 hours; the ball-milled mixture is then dried at 85 to 95°C, and then annealed at 1000 to 1100°C in air at a heating rate of 4.5 to 5.5°C / min for 4 to 5 hours to obtain pure phase crystalline Cu3P2O8.

4. A method for simultaneously detecting macrolide resistance genes lin B, erm B, and ere D by light-regulated multiple loop-mediated isothermal amplification combined with photothermal test paper, characterized in that: Using the sensitizer Ag-PDA / Cu3P2O8 for lin B, ermB, ere D as described in any one of claims 1 to 3, the method comprises the following steps: S1. Light-regulated multiple LAMP reactions Under the irradiation of 532nm external coaxial reflection LED light source, the sample to be tested undergoes a light-regulated multiple LAMP amplification reaction through the light-regulated multiple LAMP system to obtain a light-regulated multiple LAMP product, wherein: The light-regulated multiple LAMP system includes the following components: a primer set, a sensitizer Ag-PDA / Cu3P2O8, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer, wherein the DNA template is the macrolide resistance genes lin B, ermB, and ere D; S2. Photothermal test paper detection Capture probes were fixed on the T and C regions of the test strip to obtain a multi-target photothermal test strip targeting the target genes lin B, erm B, and ere D light-regulated multiple LAMP products; The photothermal signal probes were obtained by modifying the nucleic acid chains of lin B, erm B, and ere D with the sensitizer Ag-PDA / Cu3P2O8; The photothermal signal probe and the photothermal test paper are used to detect the light-regulated multiple LAMP products. The detection area of ​​the photothermal test paper captures the light-regulated multiple LAMP products and the photothermal signal probe in a sandwich form, and gives a photothermal temperature change signal positively correlated with the content of the light-regulated multiple LAMP products under the irradiation of a 532nm external coaxial reflection LED light source. According to the temperature change signal, lin B, erm B, and ere D in the sample to be tested are qualitatively and / or quantitatively determined.

5. The method according to claim 4, characterized in that The concentration of the Ag-PDA / Cu3P2O8 nanomaterial in the light-regulated multiple LAMP system is 0.1-0.4 mg / mL.

6. The method according to claim 4, characterized in that The DNA template refers to a plasmid DNA containing a target gene, including a DNA template of a macrolide resistance gene. The plasmid DNA extraction method includes any one of a DNA extraction kit extraction method, an adsorption column method, a magnetic bead method, and a hexadecyltrimethylammonium bromide method (CTAB method).

7. The method according to claim 4, characterized in that In the light-regulated multiple LAMPLAMP system, the concentration of each group of DNA templates is 3% to 6% v / v.

8. The method according to claim 4, characterized in that The primer set is a primer sequence corresponding to the DNA template, and may include three sets of primers, each set of primers including two upstream and downstream external primers F3 and B3, two upstream and downstream internal primers FIP and BIP, and two loop primers loop F and loop B, so as to improve the detection efficiency; Preferably, in the light-regulated multiple LAMP system, the concentration of the two external primers F3 and B3 in each set of primers is 2% to 4% v / v, the concentration of the two internal primers FIP and BIP is 2% to 6% v / v, and the concentration of the two loop primers loop F and loop B is 0 to 4% v / v; Preferably, the light-regulated multiple LAMPLAMP system further comprises the following components: BSA, dNTPs, betaine and ddH2O; Preferably, the isothermal amplification buffer comprises Tris-HCl, (NH4)2SO4, KCl, MgSO4, and Tween-20.

9. The method according to claim 4, characterized in that The irradiation parameters of the 532nm external coaxial reflection LED light source are power of 4 to 8W and irradiation time of 10 to 25 minutes.

10. The method according to claim 4, characterized in that The photothermal signal probe is used independently of the photothermal test paper body, or is dropped and dried on a conjugate pad and the conjugate pad is integrated on the photothermal test paper.

Images