A photothermal lateral flow analysis device and application for simultaneous detection of multiple β-lactam antibiotic resistance genes

Abstract

The present invention discloses a photothermal lateral flow analysis device and its application for simultaneously detecting multiple β-lactam antibiotic resistance genes. The device includes n photothermal signal probes, a test strip, an 808 nm surface laser light source, and a thermal detection device. The n photothermal signal probes are complexes of the first complementary fragment of the resistance gene to be detected—the complementary fragment of the quality control probe—CuS@SeS2-Au, where n≥2. The second complementary fragments of n resistance genes to be detected are sequentially fixed in the detection area of the test strip, and the quality control probe is fixed in the quality control area. The n photothermal signal probes, the sample solution to be detected, and the buffer are mixed, and the sample pad end of the test strip is inserted into the mixed solution for chromatography. The entire detection area is irradiated with an 808 nm surface laser, and the photothermal temperature change in the detection area is collected by the thermal detection device. Standard curves are established respectively according to the photothermal temperature change and the concentrations of the n resistance genes to be detected, and quantitative analysis of the n β-lactam antibiotic resistance genes to be detected is carried out.

Description

Technical Field

[0001] The present invention relates to a photothermal lateral flow chromatography device for simultaneously detecting multiple β-lactam antibiotic resistance genes and its application, belonging to the technical field of food analysis and detection. Background Art

[0002] Antibiotic resistance genes are the "culprits" leading to bacterial antibiotic resistance. They are naturally occurring gene fragments encoding functions to resist antibiotics. Due to their biological activity, complex migration and transformation pathways, and diverse types, they are regarded as a new type of environmental pollutant. Affected by human activities, the selection pressure of antibiotics in the environment persists, and the abundance of resistance genes in the environment is continuously increasing, resulting in the "rampant spread" of drug-resistant microorganisms. Among them, β-lactam antibiotic resistance genes are a type of resistance genes that can enable bacteria to produce resistance to β-lactam antibiotics. They spread across hosts and regions with the environment and food, causing food safety risks and at the same time limiting the selection of antibacterial drugs in clinical treatment, seriously threatening human health. Therefore, there is an urgent need to develop a multi-flux rapid detection method for β-lactam antibiotic resistance genes.

[0003] Lateral flow chromatography analysis method has been widely used in the field of rapid detection due to its fast detection performance, low cost, and good sensitivity. In the past 5 - 10 years, a signal amplification strategy based on current lateral flow analysis technology has emerged. This strategy mainly relies on laser excitation of plasmonic nanomaterials. When the excitation light matches the surface plasmon resonance of the nanoparticles, after the particles absorb light energy, they release energy in the form of heat, achieving an effect of signal amplification. Small changes in the analyte concentration can be monitored through changes in temperature, with a low background signal and high sensing sensitivity, and it has been successfully applied to the detection of various targets.

[0004] In recent years, photothermal lateral flow analysis technology has received increasing attention. Zhou et al. developed a photothermal paper-based device for detecting pathogen DNA using oxidized TMB as a photothermal reagent (Analytical Chemistray 2020, 92, 14830 - 14837), and Zhang et al. developed a highly sensitive lateral flow immunoassay strip for the quantitative detection of ochratoxin A (OTA) based on multifunctional photothermal Fe3O4@Au nanoparticles (Talanta 2021, 222, 121478).

[0005] However, existing photothermal lateral flow assays cannot achieve simultaneous detection of multiple analytes. The reason is that all photothermal lateral flow assays use a point laser as the light source, which can only irradiate one detection site on the test strip detection area at a time. Even if there are areas for multiple analytes on the detection area, it can only irradiate each area one by one and measure the temperature of each area one by one. Multiple analytes can only be detected sequentially, and the total analysis time is the sum of the individual analysis times. Therefore, how to achieve simultaneous detection of multiple analytes in photothermal lateral flow analysis is a difficult point and bottleneck in the field. Summary of the Invention

[0006] Technical Problem:

[0007] Existing photothermal lateral flow assays for β-lactam antibiotic resistance genes have the following limitations: First, the light source is point excitation, and the spot area is small (5*8mm 2 ), and only single-target detection can be achieved each time; Second, it takes 10-20 minutes to collect and analyze data for one target, and the analysis time for multiple targets is the sum of the above times, and the detection efficiency needs to be improved.

[0008] Improving the analysis throughput is a key issue in the development of point-of-care testing technology and also the current development trend of analytical chemistry. Exploring from the perspective of technology or device, developing a photothermal lateral flow analysis method for simultaneous detection and evaluation of multiple β-lactam antibiotic resistance genes has important scientific research and practical significance.

[0009] Technical Solution:

[0010] On the one hand, the present application provides a photothermal lateral flow analysis device and application for simultaneous detection of multiple β-lactam antibiotic resistance genes. Using the photothermal lateral flow analysis device to detect multiple β-lactam antibiotic resistance genes, the method includes the following steps:

[0011] (1) Construct a photothermal lateral flow analysis device for simultaneous detection of multiple β-lactam antibiotic resistance genes, specifically including four parts: preparing a test strip, preparing n photothermal signal probes, building an 808nm surface laser light source and a thermal detection device. Among them, the test strip includes a bottom plate, on which a sample pad, an NC membrane, and an absorption pad are sequentially overlapped and pasted in the horizontal direction. The NC membrane includes a detection area and a quality control area, i.e., the C area; the detection area of the test strip includes n independent T areas, and different second complementary fragments of the β-lactam antibiotic resistance genes to be detected are fixed on each T area, denoted as T n area, n≥2; the quality control area, i.e., the C area, of the test strip is fixed with a quality control probe; the photothermal signal probe is a complex of the first complementary fragment of the resistance gene to be detected - the complementary fragment of the quality control probe - CuS@SeS2-Au; the 808nm surface laser light source is formed by expanding a 808nm point laser through a surface divergence laser beam expander, and the spot size can be adjusted to cover all T simultaneouslyn Zone;

[0012] (2) Mix n kinds of photothermal signal probes, the sample solution to be tested, and the buffer solution to form a mixed solution. The resistance genes present in the sample will bind to the first complementary fragments of the resistance genes to be tested on the corresponding photothermal signal probes. Then, insert the sample pad end of the test strip into the mixed solution for chromatography;

[0013] (3) Irradiate the detection zone with an 808nm surface laser to simultaneously excite all n kinds of photothermal signal probes in the T n zone, and use a thermal imaging or temperature measurement device to collect the photothermal temperature changes of the n kinds of photothermal signal probes in the T n zone for qualitative and / or quantitative analysis.

[0014] As an alternative embodiment, the qualitative analysis refers to determining whether the sample solution to be tested contains β-lactam antibiotic resistance genes based on whether there are photothermal temperature changes; the quantitative analysis refers to respectively establishing working curves based on the positive correlation between the photothermal temperature changes and the concentrations of n kinds of resistance genes to be tested, and performing quantitative analysis of the n kinds of resistance genes to be tested.

[0015] As an alternative embodiment, in step (3), the photothermal temperature changes in each detection zone, i.e., the T n zone, are positively correlated with the content of the corresponding β-lactam antibiotic resistance genes to be tested. Specifically:

[0016] When the sample does not contain a certain β-lactam antibiotic resistance gene, the corresponding T zone in the T n zone does not show color, and the photothermal temperature change is close to zero;

[0017] When the sample contains a certain β-lactam antibiotic resistance gene, the corresponding T zone in the T n zone shows a dark purple color, and the photothermal temperature change increases;

[0018] The quality control zone serves as a reference for verifying the effectiveness of the test strip results and always shows a dark purple color.

[0019] As an alternative embodiment, the β-lactam antibiotic resistance genes are gene fragments that can make bacteria resistant when free in the environment or present in cells, including but not limited to at least two of mecA, ampC, blaTEM, blaCTX-M, fmtC.

[0020] As an alternative embodiment, the first complementary fragment of the resistance gene to be detected bound to the photothermal signal probe is partially complementary to the nucleic acid sequence of the resistance gene to be detected. The second complementary fragment of the resistance gene to be detected fixed in the T region is also partially complementary to the nucleic acid sequence of the resistance gene to be detected and is different or not completely the same as the first complementary fragment. The first complementary fragment of the resistance gene to be detected, the resistance gene to be detected, and the second complementary fragment of the resistance gene to be detected can form a sandwich structure; the quality control probe is partially or completely complementary to the complementary fragment of the quality control probe on the photothermal signal probe.

[0021] As an alternative embodiment, the preparation method of the device includes:

[0022] (1) Preparation of the photothermal signal probe: The CuS@SeS2-Au composite material is respectively combined with n kinds of mercapto-labeled first complementary fragments of the resistance gene to be detected, and then combined with the complementary fragment of the quality control probe. Finally, salmon sperm DNA is used to block non-specific binding sites to form a complex, that is, a complex of the first complementary fragment of the resistance gene to be detected - the complementary fragment of the quality control probe - CuS@SeS2-Au is prepared to obtain the photothermal signal probe. The photothermal signal probe is stored in a sealed container in the form of a solution or freeze-dried powder for later use.

[0023] (2) Construction of the test strip: The second complementary fragments of n kinds of β-lactam antibiotic resistance genes to be detected in the T n region are sequentially fixed in the T region to be detected, and the quality control probe is dropped or sprayed in the C region. The quality control probe is partially or completely complementary to the complementary fragment of the quality control probe on the photothermal signal probe. After drying, it is stored in a vacuum bag for later use;

[0024] As an alternative embodiment, the specific method for preparing the CuS@SeS2-Au composite material is: Dilute the CuS@SeS2 nanocrystal dispersion, take the diluted CuS@SeS2 solution in a centrifuge tube, add a trisodium citrate solution, mix well and then add a HAuCl4 solution, add ultrapure water, and then oscillate and react at room temperature to obtain the CuS@SeS2-Au composite material, which is refrigerated for later use.

[0025] As an alternative embodiment, for the preparation of the photothermal signal probe, the specific method is: Take the CuS@SeS2-Au composite material in a centrifuge tube, add a weak alkaline solution to adjust the pH of the system to 6-7.5, oscillate and mix well, and then add the mercapto-labeled first complementary fragment of the resistance gene to be detected and the complementary fragment of the quality control probe respectively. After oscillating and reacting at room temperature for 30-60 min, add salmon sperm DNA to block, and oscillate and react at room temperature for 1-1.5 h. After centrifugation, remove the supernatant, and then redissolve the remaining substance in a buffer solution to obtain n kinds of photothermal signal probes, which are refrigerated for later use.

[0026] As an alternative embodiment, the buffer solution includes any one of phosphate buffer solution, borate buffer solution or carbonate buffer solution.

[0027] As an alternative embodiment, the n testing areas of the T zone are sequentially fixed with n second complementary fragments of the tested β-lactam antibiotic resistance genes to be detected, specifically: in the n T zone, 5-20 mM PBS solution containing 0.5-10 mg / mL of the second complementary fragment of the tested resistance gene is respectively dropped or sprayed;

[0028] As an alternative embodiment, the C zone is fixed with a quality control probe, specifically: in the C zone, 5-20 mM PBS solution containing 0.05-10 mg / mL of a nucleic acid fragment that is partially or completely complementary to the complementary fragment of the quality control probe on the photothermal signal probe is dropped or sprayed.

[0029] As an alternative embodiment, the running buffer solution is a phosphate or citrate solution containing sucrose, salmon sperm DNA, and Tween.

[0030] On the other hand, the present invention provides a photothermal lateral flow analysis device for simultaneously detecting multiple β-lactam antibiotic resistance genes. The photothermal lateral flow analysis device includes four parts: n photothermal signal probes, a test strip, an 808 nm surface laser light source, and a thermal detection device:

[0031] The main body of the test strip includes a bottom plate, on which a sample pad, an NC membrane, and an absorption pad are sequentially overlapped and pasted in the horizontal direction. Among them, the nitrocellulose membrane is used to separate and detect the analyte in the sample from other substances. The sample pad is used for loading samples, and the absorption pad is used to absorb excess liquid. The PVC bottom plate provides physical support for the test strip; the NC membrane, that is, the nitrocellulose membrane, includes a detection area, namely the n T zone, and a quality control area, namely the C zone, where n≥2; among them, the n T zone includes n independent T areas, with a T area set every 3-5 mm. Different second complementary fragments of the tested resistance genes, that is, capture probes, are respectively fixed on each T area; a quality control probe is fixed on the C zone, and the quality control probe is partially or completely complementary to the complementary fragment of the quality control probe on the photothermal signal probe;

[0032] The 808 nm surface laser is formed by expanding a 808 nm point laser through a surface diverging laser beam expander; the surface diverging laser beam expander is used as an optical accessory for the 808 nm point laser. Through the surface diverging laser beam expander, a small area point light spot of 5*8 mm 2 can be expanded by 1.5-30 times. The surface laser can simultaneously irradiate n T areas, where n≥2. When the expansion multiple is 2.5 times, the original light spot of 5*8 mm 2 can be expanded to 12.5*20 mm2 , the laser can irradiate 3 T regions simultaneously;

[0033] The thermal detection device refers to a thermal imaging or temperature measurement device.

[0034] As an alternative embodiment, the optothermal signal probe is a complex formed by CuS@SeS2-Au composite material binding to the first complementary fragments of n kinds of β-lactam antibiotic genes to be detected respectively, then binding to the complementary fragments of the quality control probe, and finally blocking the non-specific binding sites with salmon sperm DNA; in this application, the optothermal signal probe is used in a form independent of the main structure of the optothermal test strip. After chromatographic migration, the optothermal signal probe flows through the detection region, and the optothermal signal probe that has bound to the antibiotic resistance gene to be detected in the sample will be captured in a sandwich form by the second complementary fragment of the antibiotic resistance gene in the corresponding T n region, while the optothermal signal probe that has not bound to the antibiotic resistance gene to be detected in the sample will directly flow through the detection region and then be captured in the quality control region.

[0035] As an alternative embodiment, the thermal imaging or temperature measurement device includes, but is not limited to, a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imager, a handheld infrared thermal imaging analyzer, or an infrared thermal imaging temperature gun. The thermal imaging or temperature measurement device collects optothermal imaging photos and outputs and displays them through a connected intelligent display terminal.

[0036] As an alternative embodiment, the intelligent display terminal includes a computer or a smart phone.

[0037] The detection principle of the optothermal lateral flow analysis device for detecting β-lactam antibiotic resistance genes of the present invention is explained as follows by taking three β-lactam antibiotic resistance genes mecA, ampC, and blaTEM as examples: When there are no three β-lactam antibiotic resistance genes in the sample, the optothermal signal probe that has not bound to the sample after passing through the three detection regions (T n region) cannot be captured. Therefore, there is no accumulation in the T n region, so it does not show color, and when irradiating the three T regions with an 808 nm surface laser simultaneously, the optothermal temperature change is close to zero. When there is one or more of the three β-lactam antibiotic resistance genes in the sample, when the sample is incubated with the optothermal signal probe, the resistance genes in the sample bind to some of the optothermal signal probes and swim forward. When passing through the corresponding T n region on the NC membrane, they are sequentially captured to form a sandwich complex, showing a deep purple color. When irradiating the three T regions with an 808 nm surface laser simultaneously, the optothermal temperature change increases significantly. The degree of temperature change in the T n region is proportional to the content of the resistance gene in the sample. The excessive free optothermal signal probe will bind to the quality control probe on the quality control region (C region) and always show a deep purple color as a reference for verifying the effectiveness of the test strip result.

[0038] Beneficial effects:

[0039] The detection method provided by the present invention can simultaneously complete the photothermal quantitative detection of multiple β-lactam antibiotic resistance genes within 20 minutes by one chromatography, with higher sensitivity, and broadens the detection throughput compared with the existing photothermal lateral flow analysis method without increasing the detection time. Brief description of the drawings

[0040] Figure 1 It is a schematic structural diagram of the device of the present invention;

[0041] Figure 2 It is the corresponding situation of the test strip of the present invention for negative and positive samples and the working curve in an embodiment;

[0042] Figure 3 It is a schematic diagram of the sample test result in an embodiment of the present invention. Detailed implementation manners

[0043] According to the following embodiments, the present invention can be better understood. However, the specific material ratios, process conditions and their results described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0044] The following embodiments of the present application disclose the preparation and application of a photothermal lateral flow analysis device for simultaneously detecting multiple β-lactam antibiotic resistance genes, including the following steps:

[0045] (1) Prepare a photothermal signal probe: The CuS@SeS2-Au composite material is respectively combined with the first complementary fragments of n kinds of mercapto-labeled β-lactam antibiotic resistance genes to be detected, then combined with the complementary fragment of the quality control probe, and finally the non-specific binding sites are blocked with salmon sperm DNA to prepare a photothermal signal probe;

[0046] (2) Assemble the test strip: Fix n kinds of streptavidin-conjugated biotin-labeled second complementary fragments of the resistance genes to be detected in sequence in the detection area (T n zone), and fix the streptavidin-conjugated biotin-labeled quality control probe in the quality control area (C zone). The quality control probe is partially or completely complementary to the complementary fragment of the quality control probe on the photothermal signal probe;

[0047] (3) After mixing n kinds of photothermal signal probes, the sample solution to be detected and the buffer solution, insert the sample pad end of the test strip into the mixed solution for chromatography;

[0048] (4) Irradiate the detection area with an 808nm surface laser, collect the temperature with a thermal imaging or temperature measuring device, establish a standard curve according to the temperature and the concentrations of n kinds of resistance genes to be detected respectively, and perform quantitative analysis of n kinds of target substances.

[0049] The present invention can achieve simultaneous photothermal quantitative analysis of multiple β-lactam antibiotic resistance genes. According to the number of T regions, the magnification of the surface divergence laser beam expander in the surface laser can be adjusted to adjust the surface laser irradiation area, so that the spot area meets the coverage requirements of n T regions. In the following examples, mecA, ampC, and blaTEM are used as examples of β-lactam antibiotic resistance genes to fully and detailedly illustrate the preparation and application methods of the entire device.

[0050] Example 1:

[0051] Preparation of a photothermal lateral flow analysis device for simultaneous detection of multiple β-lactam antibiotic resistance genes and its detection of negative and positive samples, the specific steps are as follows

[0052] 1. Preparation of test strip materials

[0053] 1.1 Preparation of CuS@SeS2-Au composite material

[0054] Dilute the CuS@SeS2 dispersion with deionized water to a volume fraction of 1%. Take the diluted CuS@SeS2 solution in a centrifuge tube, add ultrapure water, trisodium citrate solution (0.3 mol / L), and HAuCl4 solution (m / v = 1%), then immediately place it on a vortex oscillator and oscillate at room temperature for 7 min to obtain the composite nanomaterial CuS@SeS2-Au of CuS@SeS2 nanocrystals and AuNPs, and store it in a 4°C refrigerator for later use.

[0055] 1.2 Preparation of photothermal signal probe

[0056] Take 1 mL of CuS@SeS2-Au in three centrifuge tubes respectively, add 0.2 mol / L K2CO3 solution to adjust the pH values of the systems to 6.5, 6.5, and 6 respectively, oscillate and mix well, add 5 μL of 1 mg / mL mercapto-labeled mecA first complementary fragment (sequence: 5’-SH-CATATGAGATAGGCATCGTTCCAA-3’), 3 μL of mercapto-labeled ampC first complementary fragment (5’-SH-ACCATGGGGTATCAGGGAGATG-3’), and 1 μL of mercapto-labeled blaTEM first complementary fragment (sequence: 5’-SH-GTGCGCGGAACCCCTATT-3’) respectively. Then add 1 mg / mL quality control probe complementary fragment (sequence: 5’-SH-TTTTTTTTTTTTTTT-3’) to the three centrifuge tubes respectively. After mixing, react at room temperature with oscillation (150 rpm) for 45 min. After the reaction, add 100 μL of salmon sperm DNA (m / m = 10%) to block for 1 h (150 rpm), centrifuge at 12,000 r / min for 30 min, remove the supernatant, and redissolve in 100 μL of buffer (20 mmol / L Na3PO4, 5% salmon sperm DNA, 0.25% Tween-20, 10% sucrose) to obtain the complexes of the first complementary fragments of the three target resistance genes - quality control probe complementary fragment - CuS@SeS2-Au, that is, the photothermal signal probes of the three targets, and store them in a 4 °C refrigerator for standby.

[0057] 1.3 Preparation of solutions in the detection area (T n area)

[0058] Dilute streptavidin-conjugated biotin-labeled mecA second complementary fragment (sequence: 5’-TGCGAAATCACTTAAATATTCATCCAT-biotin-3’), ampC second complementary fragment (sequence: 5’-CAAAGCGCGTAACCGGATTGG-biotin-3’), and blaTEM second complementary fragment (5’-TTACCAATGCTTAATCAGTGAGGC-biotin-3’) to 0.4 mg / mL, 0.6 mg / mL, and 0.2 mg / mL respectively with 10 mM PBS solution, corresponding to the solutions in areas T1, T2, and T3 respectively.

[0059] 1.4 Preparation of solutions in the quality control area (C area)

[0060] Dilute the quality control probe to 0.2 mg / mL with 10 mM PBS solution.

[0061] The nucleic acid fragment sequence of the quality control probe is as follows:

[0062] 5’-AAAAAAAAAAAAAAA-biotin-3’

[0063] 2. Preparation of test strip

[0064] According to Figure 1 the membrane combination method, paste the NC membrane in the middle of the PVC bottom plate. The sample pad and the absorbent pad are respectively lapped on the left and right ends of the NC membrane, so that they cover and press the NC membrane by about 2 mm. Cut the assembled large card into strips with a width of 3 mm to obtain blank test strips. Respectively in Figure 1 the T n area and the C area, drop 0.5 μL of the T n area solution and 0.5 μL of the C area solution at intervals of 5 mm in turn. After spotting, place the test strip in an oven and dry it at 37 °C for 60 min, and store it in a vacuum bag for later use.

[0065] 3. Working curve drawing

[0066] Use PBS solution (pH 7.4) to prepare 1 mg / mL mecA gene, ampC gene, and blaTEM gene standard solutions respectively, and dilute the standard solutions of the antibiotic resistance genes to be measured with 10 mM PBS solution (pH 7.4) to concentrations of 1 ng / mL, 5 ng / mL, 25 ng / mL, 125 ng / mL, and 625 ng / mL.

[0067] Take 20 μL of each of the above gradient dilution solutions of the antibiotic resistance genes to be measured, a total of 60 μL, and place them in a centrifuge tube. Then take 5 μL of each of the three photothermal probes, a total of 15 μL, and add them to the centrifuge tube. Then add 15 μL of the running buffer to the centrifuge tube and mix for 10 min. Then insert the test strip into the centrifuge tube for chromatography. After chromatography, irradiate it with an 808 nm surface laser (power: 1.96 W / cm 2 ) for 5 min, and use the infrared thermal imaging accessory to monitor the temperature changes in the T1, T2, and T3 detection areas.

[0068] The results are shown as Figure 2 , the T n area of the positive test strip shows obvious dark purple, and the photothermal temperature change is relatively high. The T n area of the negative test strip does not show color, and the photothermal temperature change is close to zero.

[0069] The working curves of mecA, ampC, and blaTEM are shown as Figure 2 (C), (D), and (E) respectively.

[0070] Example 2:

[0071] Specific verification of a photothermal lateral flow analysis device for simultaneously detecting multiple β-lactam antibiotic resistance genes against multiple β-lactam antibiotic resistance genes includes the following steps:

[0072] 1. Preparation of test strip material

[0073] Same as Example 1

[0074] 2. Preparation of test strip

[0075] Same as Example 1

[0076] 3. Sample pretreatment

[0077] Prepare 1 mg / mL mecA gene, ampC gene, and blaTEM gene as positive test solutions for standby with 10 mM PBS solution (pH 7.4), and use 10 mM PBS solution (pH 7.4) as the negative test solution for standby.

[0078] 4. Sample detection

[0079] Add 24 μL of mecA gene test solution and 48 μL of negative test solution to centrifuge tube 1, add ampC gene test solution and 48 μL of negative test solution to centrifuge tube 2, add blaTEM gene test solution and 48 μL of negative test solution to centrifuge tube 3. Take 3 μL of each of the three photothermal probes, a total of 9 μL, and add them to the three centrifuge tubes. Then add 9 μL of running buffer (10 mM PBS solution, containing 5% sucrose, 1% salmon sperm DNA, 1% Tween-20, pH 7.4) to mix and incubate for 10 min. Then insert the test strip into the centrifuge tube for chromatography. After the test strip chromatography is completed, irradiate with an 808 nm laser (power 1.96 W / cm 2 ) for 5 min, and use an infrared thermal imaging accessory to monitor the temperature change of T n in the detection area. The results are shown as Figure 3 and Table 1.

[0080] As shown in Table 1, only the mecA detection area of test strip 1 shows deep purple and has a relatively high photothermal temperature change; only the ampC detection area of test strip 2 shows deep purple and has a relatively high photothermal temperature change; only the blaTEM detection area of test strip 3 shows deep purple and has a relatively high photothermal temperature change; the specific recognition results between the probes are good.

[0081] Table 1 Specific verification results of the photothermal lateral flow analysis device for multiple β-lactam antibiotic resistance genes

[0082]

[0083] Example 3:

[0084] An optothermal lateral flow analysis device for simultaneously detecting multiple β-lactam antibiotic resistance genes is applied to the analysis of actual samples, including the following steps:

[0085] 1. Preparation of test strip material

[0086] Same as Example 1

[0087] 2. Preparation of test strip

[0088] Same as Example 1

[0089] 3. Sample pretreatment

[0090] Respectively take 1 - 5 ml of methicillin-resistant Staphylococcus aureus (containing mecA, ampC, blaTEM genes) and ordinary Staphylococcus aureus culture solution (less than 1.0×10 9 bacteria), centrifuge at 10000 rpm (11500×g) for 1 min, and discard the culture solution. Use a commercial DNA extraction kit to extract the total genomic DNA of the colonies as the test solution for standby.

[0091] 4. Sample detection

[0092] Add 72 μL of the actual sample test solution into three centrifuge tubes respectively, then add 9 μL of CuS@SeS2-Au-DNA1, 9 μL of CuS@SeS2-Au-DNA2, and 9 μL of CuS@SeS2-Au-DNA3 (denoted as T1, T2, T3 respectively), and then add 9 μL of running buffer (10 mM PBS solution, containing 5% sucrose, 1% salmon sperm DNA, 1% Tween-20, pH 7.4) respectively, mix and incubate for 10 min, and then insert the test strip into the centrifuge tube for chromatography. After the test strip chromatography is completed, irradiate with an 808 nm laser (power 1.96 W / cm 2 ) equipped with a surface divergence laser beam expander for 5 min, and use an infrared thermal imaging accessory to monitor the temperature change in the detection area T n .

[0093] For the test sample without β-lactam antibiotic resistance genes, the detection area corresponding to the test strip shows lighter color and lower optothermal temperature change close to zero; for the test samples containing mecA, ampC, and blaTEM respectively, the detection areas of the test strips all show dark purple and high optothermal temperature change; the optothermal lateral flow analysis device has good accuracy in detecting actual samples.

[0094] Table 2 Detection results of the optothermal lateral flow analysis device for multiple β-lactam antibiotic resistance genes applied to the analysis of actual samples

[0095]

[0096]

[0097] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A photothermal lateral flow analysis method for simultaneously detecting multiple β-lactam antibiotic resistance genes, characterized in that, Simultaneously detecting multiple β-lactam antibiotic resistance genes by using a photothermal lateral flow analysis device, the method comprising the following steps: (1)Construct a photothermal lateral flow analysis device for simultaneously detecting multiple β-lactam antibiotic resistance genes, specifically including four parts: preparing a test strip, preparing n photothermal signal probes, building an 808 nm surface laser light source and a thermal detection device. Among them, the test strip includes a bottom plate, on which a sample pad, an NC membrane, and an absorption pad are sequentially overlapped and pasted in the horizontal direction. The NC membrane includes a detection area and a quality control area, namely the C area; the detection area of the test strip includes n independent T areas, and different second complementary fragments of the β-lactam antibiotic resistance genes to be detected are respectively fixed on each T area, denoted as T n zone, n≥2; A quality control probe is immobilized on the quality control region, i.e., the C region, of the test strip; The photothermal signal probe is a complex of a first complementary fragment of the resistance gene to be detected - a complementary fragment of the quality control probe - CuS@SeS2-Au; when preparing n kinds of photothermal signal probes, take CuS@SeS2-Au in a centrifuge tube, add the first complementary fragment of the resistance gene to be detected labeled with a thiol group and the complementary fragment of the quality control probe to obtain a photothermal signal probe composed of a complex of a first complementary fragment of the resistance gene to be detected - a complementary fragment of the quality control probe - CuS@SeS2-Au; The second complementary fragment in the detection region is labeled with streptavidin-conjugated biotin; The 808 nm surface laser light source is formed by expanding the 808 nm dot laser through a surface-divergent laser beam expander, and the spot size can be adjusted to cover all T areas simultaneously. n areas; (2) Mix n kinds of photothermal signal probes, the sample solution to be tested, and a buffer solution to form a mixed solution. The resistance genes present in the sample will bind to the first complementary fragments of the resistance genes to be detected on the corresponding photothermal signal probes, and then insert the sample pad end of the test strip into the mixed solution for chromatography; (3) n kinds of photothermal signal probes migrate through the detection area due to chromatography. The photothermal signal probes that have already bound to the target resistance gene in the sample will be captured by the corresponding second complementary fragments in the T n region in a sandwich form, while the photothermal signal probes that have not bound to the target resistance gene in the sample will directly flow through the detection area and then be captured in the quality control area; irradiate the detection area with an 808 nm surface laser to simultaneously excite all n n kinds of photothermal signal probes in the T n region, and collect the photothermal temperature changes of the n kinds of photothermal signal probes in the T region with a thermal imaging or temperature measurement device for qualitative and / or quantitative analysis.

2. The application method according to claim 1, characterized in that, In the step (3), the photothermal temperature change in each detection region, i.e., the T n region, is positively correlated with the content of the corresponding β-lactam antibiotic resistance gene to be detected, specifically: When the sample does not contain a certain β-lactam antibiotic resistance gene, the corresponding T n region does not show color, and the photothermal temperature change is close to zero; When a certain β-lactam antibiotic resistance gene is present in the sample, its corresponding T n region shows dark purple and the photo-thermal temperature change increases; The quality control region serves as a reference for verifying the validity of the test strip results and always shows a dark purple color.

3. The method according to claim 1, characterized in that, The β-lactam antibiotic resistance gene is a gene fragment that can confer drug resistance to bacteria when it is free in the environment or present in cells, including but not limited to at least two of mecA, ampC, blaTEM, blaCTX-M, fmtC.

4. The method according to claim 1, wherein The first complementary fragment of the resistance gene to be detected bound to the photothermal signal probe is partially complementary to the nucleic acid sequence of the resistance gene to be detected. The second complementary fragment of the resistance gene to be detected immobilized in the T region is also partially complementary to the nucleic acid sequence of the resistance gene to be detected and is different from the first complementary fragment. The first complementary fragment of the resistance gene to be detected, the resistance gene to be detected, and the second complementary fragment of the resistance gene to be detected can form a sandwich structure; the quality control probe is partially or completely complementary to the complementary fragment of the quality control probe on the photothermal signal probe.

5. The method according to claim 4, wherein The preparation method of CuS@SeS2-Au includes: diluting the CuS@SeS2 nanocrystal dispersion, taking the diluted CuS@SeS2 solution in a centrifuge tube, adding a trisodium citrate solution, mixing evenly and then adding a HAuCl4 solution, adding ultrapure water, and then oscillating and reacting at room temperature to obtain a CuS@SeS2-Au composite material, which is stored in the refrigerator for later use.

6. The method according to claim 4, wherein The preparation method of the photothermal signal probe includes: taking CuS@SeS2-Au in a centrifuge tube, adding a weak alkaline solution to adjust the pH of the system to 6-7.5, oscillating and mixing evenly, and then adding the first complementary fragment of the resistance gene to be detected and the complementary fragment of the quality control probe respectively. After oscillating and reacting at room temperature for 30-60 min, adding salmon sperm DNA, and oscillating and reacting at room temperature for 1-1.5 h. After centrifugation, the supernatant is removed, and the remaining substance is redissolved in a buffer solution to obtain n kinds of photothermal signal probes respectively; the photothermal signal probes are stored in a sealed container in the form of a solution or freeze-dried powder and stored in the refrigerator for later use.

7. The method according to claim 1, characterized in that, The test strip detection area is respectively dropped or sprayed with a PBS solution containing 0.5 - 10 mg / mL of the second complementary fragment of the resistance gene to be detected; the C area is dropped or sprayed with a PBS solution containing 0.05 - 10 mg / mL of the quality control probe.

8. A photothermal lateral flow analysis device for detecting multiple β-lactam antibiotic resistance genes, characterized in that, The device includes four parts: a test strip, n kinds of photothermal signal probes, an 808 nm surface laser light source, and a thermal detection device; The test strip includes a bottom plate, on which a sample pad, an NC membrane, and an absorption pad are sequentially overlapped and pasted in the horizontal direction. The NC membrane includes a detection area, i.e., the T n zone, and a quality control area, i.e., the C zone, where n≥2. Among them, the T n zone includes n independent T zones, and different second complementary fragments of the β-lactam antibiotic resistance genes to be detected are respectively fixed on each T zone, and a quality control probe is fixed on the C zone; The photothermal signal probe is a complex of the first complementary fragment of the resistance gene to be detected - the complementary fragment of the quality control probe - CuS@SeS2-Au; when preparing n kinds of photothermal signal probes, take CuS@SeS2-Au in a centrifuge tube, add the first complementary fragment of the resistance gene to be detected labeled with a mercapto group and the complementary fragment of the quality control probe to obtain a photothermal signal probe composed of a complex of the first complementary fragment of the resistance gene to be detected - the complementary fragment of the quality control probe - CuS@SeS2-Au; The second complementary fragment in the detection area is labeled with streptavidin-conjugated biotin; The 808 nm surface laser light source is formed by expanding the 808 nm point laser through a surface divergence laser beam expander, and the spot size can be adjusted to cover all T areas simultaneously. n area; The thermal detection device refers to a thermal imaging or temperature measurement device.

9. The method according to claim 1 and the device according to claim 8, characterized in that, The photothermal signal probe is a complex formed by CuS@SeS2-Au binding to n kinds of the first complementary fragments of the resistance gene to be detected respectively, then binding to the complementary fragment of the quality control probe, and finally blocking non-specific binding sites with salmon sperm DNA.

10. The device according to claim 8, wherein The thermal imaging or temperature measurement device includes but is not limited to any one of a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imager, a handheld infrared thermal imaging analyzer, or an infrared thermal imaging temperature gun. The thermal imaging or temperature measurement device collects photothermal imaging photos and outputs and displays them through a connected intelligent display terminal, and the intelligent display terminal includes a computer or a smart phone.

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