Long-persistence immunochromatographic test strip for detecting foodborne pathogenic bacteria and preparation method thereof

Abstract

The application relates to a long-afterglow immunochromatographic test paper for detecting foodborne pathogenic bacteria and a preparation method thereof. The adsorption layer of the test paper comprises an adsorption fiber layer, a long-afterglow antibody fiber layer, a cellulose membrane layer and a water-absorbing material layer. The detection line on the cellulose membrane layer is composed of anti-foodborne pathogenic bacteria monoclonal antibodies or polyclonal antibodies. The long-afterglow antibody fiber layer is adsorbed with long-afterglow antibodies, which are composed of long-afterglow nano-materials and anti-foodborne pathogenic bacteria monoclonal antibodies or polyclonal antibodies. The test paper combines long-afterglow nano-materials with immunochromatographic technology, does not need any label, has low manufacturing cost and strong light stability. After long-afterglow antibodies are introduced into the test paper, the antigen-antibody double signal amplification effect is achieved, and higher sensitivity than that of ordinary colloidal gold test paper can be obtained.

Description

Technical Field

[0001] This invention relates to a long-afterglow immunochromatographic test strip for detecting foodborne pathogens and its preparation method, belonging to the field of bioengineering. Background Technology

[0002] In recent years, diseases caused by foodborne microorganisms have become the leading cause of death among humans. Traditional plate assays require bacterial culture, isolation and identification, biochemical analysis, and serological identification. While the results are relatively accurate, the process is cumbersome and time-consuming (4-7 days), making it difficult to effectively prevent and control outbreaks of foodborne pathogens. Currently, many rapid detection methods have been established, but in practical applications, most methods still require improvement in both sensitivity and specificity. Immunological detection techniques are rapid and easy to operate, but their sensitivity needs improvement. Molecular biology techniques have relatively high sensitivity and specificity, but they require skilled operators and specific environmental conditions, as well as specialized instruments. Biosensor detection techniques can be used to detect pathogens and their toxins, without requiring pre-enrichment of samples, but the equipment is complex and difficult to promote widely. Metabolic detection techniques are rapid and simple, but they require a large amount of data to establish corresponding mathematical models and are susceptible to environmental factors, leading to poor consistency of results.

[0003] Immunochromatographic test strips offer advantages such as rapid detection, simple operation, low cost, and easy storage, making them suitable for on-site screening. Given the large number of food enterprises in my country, coupled with limited testing resources, massive testing volumes, and difficulties in implementing other molecular and immunological tests, immunochromatographic test strip technology is currently one of the most valuable rapid testing technologies for widespread adoption in my country.

[0004] Long-afterglow nanoparticles (PLNPs) are special nanomaterials that, when excited by energy (gamma rays, X-rays, ultraviolet light, visible light, and electron beams, etc.), emit visible or near-infrared light for a prolonged period. Their spectral emission region can be modulated by changing the synthesis conditions and elemental composition, allowing them to emit different colors of light. In recent years, the applications of PLNPs have expanded from initial civilian uses such as decoration, safety displays, and dials to various advanced scientific fields, including biomedicine, clinical medicine, life sciences, and energy and environmental engineering. PLNPs possess advantages not found in other fluorescent labeling materials, such as long luminescence lifetime, high luminescence intensity, and low toxicity. After surface modification, they are more suitable for in vivo studies in living organisms. They also exhibit good photochemical stability, avoiding photobleaching, and can be excited in vitro for optical imaging, effectively avoiding the influence of the organism's self-emission and the background light of the excitation source on the imaging effect. In recent years, they have also begun to be used as novel fluorescent probes in the field of biological fluorescent labeling. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a long-afterglow immunochromatographic test strip for detecting foodborne pathogens and its preparation method, thereby solving the technical problems of low sensitivity, long detection time, and complex detection process in methods for detecting foodborne pathogens in food.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The long-afterglow immunochromatographic test strip for detecting foodborne pathogens includes a support layer with an adsorption layer on top. From the test end, the adsorption layer consists of an adsorption fiber layer, a long-afterglow antibody fiber layer, a cellulose membrane layer, and an absorbent material layer at the handle end. Adjacent layers of the adsorption fiber layer, long-afterglow antibody fiber layer, cellulose membrane layer, and absorbent material layer are interconnected. A detection line and a control line are provided on the cellulose membrane layer. The detection line is composed of monoclonal or polyclonal antibodies against foodborne pathogens, and the control line is composed of goat anti- or rabbit anti-mouse IgG antibodies, or goat anti-rabbit IgG antibodies. Long-afterglow antibodies are adsorbed on the long-afterglow antibody fiber layer, and these antibodies are monoclonal or polyclonal antibodies against foodborne pathogens labeled with long-afterglow nanomaterials.

[0008] The long afterglow nanomaterial is Sr2MgSi2O7:Eu 2+ ,Dy 3+ The material has an excitation peak of 254 nm or 350 nm, an emission peak of 470 nm, an afterglow duration of 10 h, and particle size of nanoparticles with a diameter of 50-200 nm.

[0009] The long afterglow nanomaterial Sr2MgSi2O7:Eu 2+ ,Dy 3+ The marking method is as follows:

[0010] Sr2MgSi2O7:Eu with surface aminated modification 2+ ,Dy 3+ Nanomaterials were dispersed in PBS buffer solution, and 25% glutaraldehyde solution was added. The mixture was reacted at room temperature for 3 hours. Excess glutaraldehyde was washed away by centrifugation. The reaction solution was then dispersed in PBS buffer solution, and monoclonal or polyclonal antibodies against foodborne pathogens were added. The mixture was reacted at 4°C for 3 hours. After centrifugation and washing, labeled long-afterglow antibodies were obtained and stored in PBS buffer solution at 4°C.

[0011] The Sr2MgSi2O7:Eu 2+ ,Dy 3+ The amination modification method for nanomaterials is as follows:

[0012] Grinding Sr2MgSi2O7:Eu 2+ ,Dy 3+ While producing nanomaterials, NaOH solution is continuously added to Sr2MgSi2O7:Eu 2+ ,Dy 3+ Nanomaterials were ultrasonically dispersed in NaOH solution, and separated after repeated washing to obtain HO-PLNPS nanoparticles with hydroxyl groups on their surface.

[0013] HO-PLNPS was ultrasonically dispersed in a PVP solution, and after standing, the supernatant was separated and extracted. The supernatant was then centrifuged to obtain PVP-PLNPS nanoparticles coated with PVP.

[0014] PVP-PLNPS was dispersed in a mixed solution of ethanol and ultrapure water, ammonia was added, and the mixture was ultrasonically dispersed. After the solution was homogeneous, it was placed in an ice-water bath, and tetraethyl orthosilicate was slowly added dropwise. The mixture was continuously stirred, and SiO2-PLNPS was obtained through repeated centrifugation and washing. Finally, the modifier APTES was added to modify the solution, generating aminated Sr2MgSi2O7:Eu. 2+ ,Dy 3+ Nanomaterials.

[0015] The preparation method of the long-afterglow immunochromatographic test strip for detecting foodborne pathogens includes the following steps:

[0016] (1) Prepare monoclonal or polyclonal antibodies against foodborne pathogens;

[0017] (2) Preparation of long-afterglow antibodies;

[0018] (3) Prepare an adsorption fiber layer, wherein the adsorption fiber layer is made of glass fiber cotton, nylon membrane, polyvinylidene fluoride membrane or polyester membrane;

[0019] (4) Preparation of long-afterglow antibody fiber layer;

[0020] (5) Prepare a cellulose membrane layer, wherein the cellulose membrane layer is a nitrocellulose membrane, a pure cellulose membrane or a carboxylated cellulose membrane, and the imprint is detected by spraying a spot on the cellulose membrane with a spotting instrument, and then dried for later use.

[0021] (6) Assemble the test strip by sequentially attaching the adsorbent fiber layer, long afterglow antibody fiber layer, cellulose membrane layer and water-absorbing material layer to the support layer with adhesive, and assembling the support layer, adsorbent layer and protective layer into a test strip.

[0022] The method for preparing the adsorbent fiber layer is as follows: glass fiber cotton, nylon membrane, polyvinylidene fluoride membrane or polyester membrane are immersed in TBS buffer solution until wet, then freeze-dried for later use; the concentration of the TBS buffer solution is 0.01M and the pH is 7.8; by mass percentage concentration, the TBS buffer solution also contains 0.5% PVP, 1% sucrose, 0.5% Tween-20 and 1% BSA.

[0023] The detection method for the long-afterglow immunochromatographic test strip for detecting foodborne pathogens includes the following steps:

[0024] (1) Pre-enrichment of samples: Add liposome solution containing long afterglow nanomaterials to enrichment medium, and then add the sample for enrichment culture;

[0025] (2) Test strip detection: Insert the test strip into the sample solution, take it out and lay it flat after 10-20 seconds, and after 5-10 minutes, observe whether there is afterglow on the test line of the test strip to determine whether the sample contains foodborne pathogens, or perform semi-quantitative analysis by using a fluorescence strip reader.

[0026] Beneficial effects of this invention:

[0027] (1) The long afterglow nanomaterial of the present invention has a long afterglow time after being excited, so there is no need to use a fluorescent lamp to continuously irradiate it during the detection. It can be observed with the naked eye. At the same time, it avoids the problems of photobleaching and matrix autofluorescence that are common in other fluorescent test strips, and greatly improves the accuracy of the test strip.

[0028] The silicate long afterglow luminescent material used in this invention not only has good chemical and thermal stability, but its main raw material (tetraethoxysilane) is abundant, readily available, and inexpensive. Its sintering temperature is lower than that of the aluminate system, and the preparation method is simple and the production cost is low.

[0029] (2) The test strip of this invention has high specificity and high sensitivity. This invention combines long-afterglow nanomaterials with immunochromatography. Target foodborne pathogens in the sample will emit a long afterglow after being cultured in an enrichment medium containing liposomes encapsulating the long-afterglow nanomaterials. No labeling process is required, resulting in low production cost and strong photostability. Introducing the long-afterglow antibody into the test strip achieves a dual signal amplification effect of antigen and antibody, achieving higher sensitivity than ordinary colloidal gold test strips. This test strip has the advantages of high sensitivity and good specificity, and can detect trace contamination as low as 1 CFU / mL, meaning that a trace contamination sample of 1 CFU / mL can be detected after pre-enrichment culture.

[0030] (3) Simple and fast. This test strip can be used to directly detect food samples after enrichment treatment. The results can be observed directly after excitation under ultraviolet light for 2-10 seconds. It can also be read directly with the help of a long-persistence reader to achieve semi-quantitative detection. During the test, simply insert the test strip into the sample for 10-20 seconds, and the test result can be determined within 5-10 minutes. It saves time and effort, is easy to operate, and can be completed in one step.

[0031] (4) The results are visually clear, intuitive, and accurate. The test strip uses blue “|” and “||” marks as negative and positive indicators, respectively. One blue “|” mark on the cellulose membrane indicates that the sample does not contain foodborne pathogens; two blue “||” marks indicate that the sample contains foodborne pathogens. This result can be directly observed with the naked eye, making the judgment visually clear, intuitive, accurate, simple, and easy to understand, and reducing the likelihood of false positives and false negatives due to human error.

[0032] (5) Cost-saving. This test strip requires no additional instruments or reagents, allowing for testing anytime, anywhere. The low cost saves significant investment in expensive instruments and equipment. It enables qualitative and semi-quantitative detection, detecting trace contamination as low as 1 CFU / mL, resulting in low overall cost.

[0033] (6) Wide applicability and easy to promote and apply. This test strip can meet the needs of different levels of personnel, including professional testing, customs quarantine, health quarantine, quality monitoring, livestock product processing, farmers, and individual consumers. It is suitable for both single sample testing and large-scale sample screening, and can be applied to fields such as disease diagnosis, bacterial detection, and environmental monitoring. This invention is of great significance in ensuring food safety and protecting consumer health, and has significant economic and social benefits. Attached Figure Description

[0034] Figure 1 A schematic diagram of the test strip structure of the present invention.

[0035] Figure 2 A top view of the test strip structure of the present invention.

[0036] Among them, 1 is the support layer, 2 is the adsorbent fiber layer, 3 is the long afterglow antibody fiber layer, 4 is the cellulose membrane layer, 5 is the water-absorbing material layer, 6 is the invisible detection imprint, 7 is the invisible control imprint, 8-1 is the sample end protective film, 8-2 is the handle end protective film, and 9 is the sample marking line.

[0037] Figure 3 The sensitivity test results of the test strip.

[0038] Wherein, the horizontal axis Pos (mm) represents the detection position of the test strip; the vertical axis Int (mv) represents the fluorescence intensity. Detailed Implementation

[0039] The specific embodiments of the present invention will be further described in detail below with reference to examples. Unless otherwise specified, the instruments and equipment involved in the examples are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the experimental methods involved are all conventional methods.

[0040] Example 1: Preparation process of the test strip of the present invention

[0041] The method for preparing the test strip of the present invention includes the following steps: preparation of monoclonal or polyclonal antibodies against foodborne pathogens, preparation of an adsorbent fiber layer, preparation of a cellulose membrane layer, and assembly of the test strip.

[0042] (1) Preparation of monoclonal or polyclonal antibodies against foodborne pathogens

[0043] Monoclonal antibody preparation: using an inactivated concentration of 10 8 Immunize 6-8 week old Balb / C mice 3-4 times with whole-cell (ATCC standard strain) foodborne pathogens at CFU / mL intervals, with each immunization spaced 3-5 weeks apart, until the antibody titer meets the requirements (titer reaching 1:10). 5 After a super immunization, blood was collected from the infraorbital sinus of the immunized mice 3-4 days later, and positive serum was separated (to be used as a positive control later).

[0044] Immunized mice were euthanized by dislocation of the neck. The mice were disinfected by soaking them in 75% (v / v) alcohol for 5-10 minutes. The spleens were aseptically removed, cut into pieces, ground, filtered through 120-mesh nylon gauze, and centrifuged at 1000 rpm for 10 minutes to collect the spleen cells.

[0045] 1×10 8 Spleen cells and NSO myeloma cells were mixed at a ratio of 10:1 (v / v), centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. 0.7–1.0 mL of 50% (v / v) PEG4000 solution was slowly added to the cell pellet in a 37°C water bath and allowed to react for 1 min. Then, 15 mL of serum-free 1640 medium was slowly added to terminate the PEG reaction. The mixture was incubated at 37°C for 5–10 min, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The cell pellet was resuspended in HAT selective medium and added to the wells of a 96-well cell culture plate (100–200 μL / well). The plate was incubated at 37°C in a 5% CO2 incubator for 10–14 days. Positive wells were screened using an indirect ELISA method. Wells with strong positive results, high inhibition rates, and vigorous cell growth were selected for three limiting dilutions for cloning, followed by large-scale culture to establish hybridoma cell lines. The monoclonal antibody secreted in this embodiment can specifically react with foodborne pathogens, with an affinity constant reaching 10. 10 ~10 12Monoclonal antibodies targeting specific antigenic determinants of foodborne pathogens are used to detect the printing of imprinted T-lines.

[0046] Preparation of polyclonal antibodies: New Zealand white rabbits were immunized with inactivated foodborne pathogens at an immunogen concentration of 10. 8 CFU / mL, subcutaneously injected at 4-6 points on the back. For the initial immunization, an equal volume of Freund's complete adjuvant is added to the immunogen and thoroughly emulsified. For the booster immunization, Freund's incomplete adjuvant is added to the immunogen and thoroughly emulsified. 4-5 consecutive immunizations are administered 2-3 weeks after the initial immunization, with each immunization spaced 2-3 weeks apart. Ten to fifteen days after the last immunization, the titer is measured by indirect ELISA to reach 10. 5 At the above time, blood is collected and hyperimmune serum is separated and collected.

[0047] IgG antibodies were extracted using the saturated ammonium sulfate precipitation method. One part of hyperimmune serum was mixed with two parts of PBS (pH 7.2), and an equal volume of saturated ammonium sulfate solution was added. The mixture was incubated at 4°C for 12 hours, centrifuged at 4°C and 2500 rpm for 15 minutes, and the supernatant was discarded. The precipitate was then dissolved in an appropriate amount of PBS (pH 7.2), and saturated ammonium sulfate solution was added to a final concentration of 33% (v / v). The mixture was incubated at 4°C for 2 hours, centrifuged at 4°C and 2500 rpm for 15 minutes, and the supernatant was discarded. The precipitate was then dissolved in an appropriate amount of PBS (pH 7.2), and dialyzed against PBS (pH 7.2) at 4°C for 48–72 hours, with several changes of the medium. The mixture was centrifuged at 4°C and 12000 rpm for 15 minutes, and the supernatant was collected to obtain purified polyclonal antibodies against foodborne pathogens. The purified antibodies were stored at -20°C for use in T-line printing.

[0048] (2) Preparation of long-afterglow antibody fiber layer (binding pad)

[0049] The preparation of long-afterglow antibody fiber layers first requires the preparation of long-afterglow nanomaterials, specifically long-afterglow antibodies. The specific steps are as follows:

[0050] 1) Preparation of long afterglow nanomaterials (PLNPS)

[0051] The long afterglow nanomaterial is Sr2MgSi2O7:Eu 2+ ,Dy 3+ Nanoparticles with an excitation peak at 254 or 350 nm, an emission peak at 470 nm, an afterglow duration of 10 h, and a diameter of 50-200 nm can be prepared using hydrothermal or sol-gel methods.

[0052] Hydrothermal preparation: Using Sr(NO3)2, Mg(NO3)2·6H2O, Eu(NO3)3·6H2O, Dy(NO3)3·5H2O, and TEOS (tetraethoxysilane) as raw materials, the raw materials were dissolved and mixed according to stoichiometric ratios. Surfactant CTAB (molar ratio to silicon source: 0.16:1) and metal complexing agent EDTA (molar ratio to metal ions in the mixture: 1:1) were added. The pH of the mixture was adjusted to 7 with ammonia water, and the mixture was poured into a hydrothermal reactor and reacted at 110℃ for 24 h. After washing and drying, the precursor was obtained. In a reducing atmosphere produced by carbon powder, the mixture was held at 550℃ for 1 h, then heated to 900℃ and held at this temperature for 2 h to obtain Sr2MgSi2O7:Eu 2+ ,Dy 3+ Nanoluminescent materials.

[0053] Sol-gel method preparation: Using SrCO3, Mg(NO3)2·6H2O, Eu(NO3)3·6H2O, Dy(NO3)3·5H2O and TEOS as raw materials, a certain amount of SrCO3 powder was weighed and dissolved in concentrated nitric acid by gentle heating to prepare a 0.1M Sr(NO3)2 solution. All materials were dissolved in deionized water according to stoichiometric ratio to prepare a 10mL solution, and the pH was adjusted to 2.0 with concentrated nitric acid. 2mL of TEOS was quickly added to the system, and the mixture was stirred at room temperature until the system was homogeneous and transparent. The solution was then heated to 70℃ to hydrolyze the TEOS and induce a sol-gel reaction. The prepared wet gel was dried at 110℃ and aged overnight to obtain an opaque solid. The obtained opaque solid product was transferred to a crucible and calcined at 1000℃ for 10h under a weak reducing atmosphere (volume ratio: 10% H2, 90% Ar) to obtain white solid particles, namely Sr2MgSi2O7:Eu 2+ ,Dy 3+ Nanoluminescent materials.

[0054] 2)Sr2MgSi2O7:Eu 2+ ,Dy 3+ Surface silicification and amination

[0055] The prepared PLNPS was ground while NaOH solution was continuously added, and then the powder was dispersed in NaOH solution. The powder was ultrasonically dispersed and stirred to make the surface of the nanoparticles contain hydroxyl groups. After repeated washing and separation, HO-PLNPS nanoparticles with hydroxyl groups on the surface were obtained.

[0056] Next, HO-PLNPS was dispersed in a PVP (polyvinylpyrrolidone) solution, ultrasonically dispersed, and after standing, the supernatant was separated and extracted. The supernatant was then centrifuged to obtain PVP-PLNPS nanoparticles encapsulated in PVP.

[0057] PVP-PLNPS was then dispersed in an equal-volume mixture of ethanol and ultrapure water, and 1-3% (v / v) ammonia was added. The mixture was then sonicated. After the solution was homogeneous, it was placed in an ice-water bath, and tetraethyl orthosilicate was slowly added dropwise. The mixture was continuously stirred, and SiO2-PLNPS was obtained through repeated centrifugation and washing. The modifier APTES was then added to generate aminated PLNPS.

[0058] 3) Labeling of long-afterglow antibodies

[0059] Glutaraldehyde method: Glutaraldehyde is a homologous bifunctional crosslinking agent with two aldehyde groups that can form Schiff bases with the amino groups of modified fluorescent nanoparticles and antibody proteins, respectively.

[0060] Sr2MgSi2O7:Eu surface modified with amination 2+ ,Dy 3+ Nanoparticles were added to PBS buffer solution until completely dispersed. 25% glutaraldehyde solution was added to bring the final concentration of nanoparticles to 1.25% (wt). The reaction was carried out at room temperature for 3 hours. Excess glutaraldehyde was washed away by centrifugation. The reaction solution was then dispersed in PBS buffer solution, and monoclonal or polyclonal antibodies against foodborne pathogens were added. The reaction was carried out at 4°C for 3 hours. After centrifugation and washing, long-afterglow antibodies were obtained and stored in PBS buffer solution at 4°C.

[0061] Next, long-afterglow antibody (concentration of 1-2 mg / mL) is sprayed onto the fiber membrane using a spotting instrument to create a long-afterglow trace, which is then dried at 37°C in the dark for later use.

[0062] (3) Preparation of adsorption fiber layer (sample pad)

[0063] The test end adsorption fiber layer is prepared using glass fiber cotton, nylon membrane, polyvinylidene fluoride (PVDF) membrane, or polyester membrane. The fiber material is cut into strips 1.5 cm wide, immersed in the sample pad treatment solution for 30 min, and dried at 37 °C for later use.

[0064] Preparation method of the adsorbent fiber layer: Glass fiber cotton, nylon membrane, polyvinylidene fluoride membrane, or polyester membrane is immersed in a TBS buffer solution, wherein the concentration of the buffer solution is 0.01M and the pH is 7.8. The buffer solution also contains PVP, sucrose, Tween-20, and BSA; wherein the mass percentage concentration of PVP is 0.5%, the mass percentage concentration of sucrose is 1%, the mass percentage concentration of Tween-20 is 0.5%, and the mass percentage concentration of BSA is 1%. The solution is lyophilized to remove moisture and then stored for later use.

[0065] (4) Preparation of cellulose membrane (analytical pad)

[0066] The cellulose membrane layer is made of nitrocellulose membrane, pure cellulose membrane or carboxycellulose membrane, cut into strips with a width of 1.5 cm. Using a spotting instrument, monoclonal or polyclonal antibodies against foodborne pathogens are sprayed onto the cellulose membrane to make an invisible detection blot. Goat anti or rabbit anti-mouse IgG antibodies, or goat anti-rabbit IgG antibodies are sprayed onto the membrane to make an invisible control blot. The membrane is then dried at 37°C for later use.

[0067] (5) Assembly of long afterglow test paper

[0068] The absorbent fiber layer (sample pad), long-afterglow antibody fiber layer (binding pad), cellulose membrane layer (analysis pad), and absorbent material layer are then attached from right to left onto the support layer (base plate) with adhesive, and cut into test strips 3-4 cm wide.

[0069] (6) The detection reaction principle of the test strip of the present invention

[0070] When the test end of the foodborne pathogen detection strip is inserted into the sample solution, the sample solution carries the luminescent foodborne pathogen to diffuse into the cellulose membrane layer through a siphon effect, and finally penetrates into the absorbent material layer at the handle end. During diffusion, the foodborne pathogens labeled with long afterglow in the pre-enriched bacteria can form a complex of long afterglow antibody 1 and long afterglow bacteria with the long afterglow antibody on the conjugate pad. When it flows to the detection T line, it will be captured by the foodborne pathogen antibody 2 on it, thus showing a detection imprint. After ultraviolet excitation (or direct reading using a fluorescence reader), a blue detection imprint band "|" is formed, which indicates a positive result. Conversely, if there are no foodborne pathogens in the sample solution, they cannot bind to the long afterglow antibody on the conjugate pad, nor can they bind to the foodborne pathogen antibody detection imprint on the cellulose membrane, and no blue detection imprint band "|" is displayed, which indicates a negative result. Regardless of whether the sample contains foodborne pathogens, the long afterglow antibody of the long afterglow nanomaterial will capture the goat anti or rabbit anti-mouse IgG (or goat anti-rabbit IgG) antibody on the cellulose membrane, forming a blue control imprint band "|".

[0071] The detection method of the long afterglow immunochromatographic test strip of the present invention is as follows:

[0072] (1) Pre-enrichment of samples: Samples should be tested as soon as possible after collection. If immediate testing is not possible, they can be stored at 2-4℃ for 18 hours.

[0073] 1) Long afterglow Sr2MgSi2O7:Eu 2+ ,Dy 3+ Preparation of liposomes

[0074] Add long-afterglow nanomaterial stock solution to 20 mL of physiological saline to achieve a final concentration of 10. -6mol / L. Add 100 mg of lecithin to obtain a lecithin mixture. Sonicate at 4°C three times for 10 min each time. During sonication, place the lecithin mixture in an ice-water mixture and cool for 10 min every 10 min until the solution is clear. Centrifuge at 3000 r / min to remove large liposomes and insoluble matter. Dialyze the supernatant in 5 L PBS for 24 h, changing the PBS three times during this period. Store at 4°C for later use.

[0075] 2) Live markers of pathogenic bacteria

[0076] Taking advantage of the fact that liposomes can fuse with cell membranes, long-afterglow liposomes were co-cultured with foodborne pathogens, thereby enabling the foodborne pathogens to be labeled during detection.

[0077] Aseptically, take 25g (or 25mL) of sample and add it to EC or NB broth containing 1% long-afterglow liposome solution. Place the solution in a homogenizer and homogenize continuously for 1-2 minutes on a tapping homogenizer, or place it in a homogenizing cup containing 225mL of EC or NB broth and homogenize at 8000-10000r / min for 1-2 minutes. Incubate at 37℃ for several hours, and prepare positive and negative controls at the same time.

[0078] (2) Test strip detection: Insert the test strip into the sample solution, take it out and lay it flat after 10-20 seconds, and after 5-10 minutes, observe whether there is a long afterglow on the test line of the test strip to determine whether the sample contains foodborne pathogens, or perform semi-quantitative analysis by using a fluorescence strip reader.

[0079] Example 2: Test strip of the present invention

[0080] The test strip was obtained according to the method in Example 1, as shown in the schematic diagram. Figure 1 , Figure 2 ,

[0081] In the diagram, 1 represents the support layer, made of thin plastic strips; 2 represents the absorbent fiber layer, made of glass fiber cotton; 3 represents the long-afterglow antibody fiber layer, on which long-afterglow monoclonal antibodies are adsorbed; 4 represents the cellulose membrane layer, made of nitrocellulose membrane; and 5 represents the absorbent material layer at the handle end, made of absorbent filter paper. The absorbent fiber layer 2, long-afterglow antibody fiber layer 3, cellulose membrane layer 4, and absorbent material layer 5 are sequentially adhered and fixed onto the support layer 1, with the fibers interpenetrating at their junctions. An invisible detection imprint (detection line) 6, made of monoclonal antibodies against foodborne pathogens, is located on the cellulose membrane layer 4; an invisible control imprint (control line) 7, made by imprinting a goat anti-mouse IgG antibody solution onto the cellulose membrane to form a "|", with two parallel imprint bands forming a combined imprint band "||".

[0082] 8-1 is the sample end protective film, a white protective film covering the adsorbent fiber layer and the long-afterglow antibody fiber layer. 8-2 is the handle end protective film, covering the absorbent material layer; other colored protective films (such as yellow) can be used. 9 is the sample marker line, located approximately 0.5 cm off-center from the adsorbent fiber layer on the white protective film corresponding to the junction of the adsorbent fiber layer and the long-afterglow antibody fiber layer. An arrow and the word "max" are printed on the protective film to the right of the marker line. Figure 2 ).

[0083] Example 3

[0084] The test strip structure is the same as in Example 2. The difference is that the long-afterglow antibody fiber layer adsorbs polyclonal antibodies against foodborne pathogens labeled with long-afterglow nanomaterials, the adsorption fiber layer is made of nylon membrane, the cellulose membrane layer is made of pure cellulose membrane, and the invisible imprint is goat anti-rabbit IgG antibody.

[0085] Sample preparation and testing procedures:

[0086] Bread testing: Aseptically sample 25g of bread and add it to EC or NB broth containing 1% long-afterglow liposomes. Place the solution in a homogenizer and homogenize continuously for 1-2 minutes on a tapping homogenizer. Incubate at 37°C for several hours.

[0087] Operating procedure: Insert the test strip into the sample solution, remove it after 10-20 seconds and lay it flat. Irradiate the test strip with a UV lamp for 2-10 seconds. After 5-10 minutes, observe whether there is a long afterglow on the test line of the test strip to determine whether the sample contains foodborne pathogens. Alternatively, a semi-quantitative analysis can be performed using a fluorescence strip reader.

[0088] Result Interpretation: (a) Positive: If two blue "+" bands are displayed on the cellulose membrane, the test result is positive, indicating that the sample contains foodborne pathogens; (b) Negative: If one blue "+" band is displayed on the cellulose membrane, the test result is negative, indicating that the sample does not contain foodborne pathogens; (c) Invalid: If no blue band is displayed on the cellulose membrane, the test strip is invalid.

[0089] Example 4

[0090] The test strip structure is basically the same as that in Example 2, except that: the cellulose membrane layer adsorbs polyclonal antibodies against foodborne pathogens, the adsorbent fiber layer is made of nylon membrane, and the cellulose membrane layer is made of pure cellulose membrane.

[0091] For testing milk samples: Aseptically take 25 mL of the sample and add it to EC or NB broth containing 1% long afterglow liposomes. Place the solution in a homogenizer and then into a homogenizing cup containing 225 mL of EC or NB broth. Homogenize at 8000-10000 r / min for 1-2 min and incubate at 37℃ for several hours.

[0092] Operating procedure: Insert the test strip into the sample solution, remove it after 10-20 seconds and lay it flat. Irradiate the test strip with a UV lamp for 2-10 seconds. After 5-10 minutes, read the value directly through a fluorescence reader. The value is the long afterglow intensity of the detection T line. Plot a standard curve based on the peak value or peak area and calculate the actual content.

[0093] Example 5

[0094] The structure of the test paper is basically the same as that in Example 2, except that the adsorption fiber layer is made of polyvinylidene fluoride (PVDF) membrane and the cellulose membrane layer is made of carboxylated cellulose membrane.

[0095] For testing jelly: the pre-enrichment of samples, result determination and operation methods are the same as in Example 2, and will not be repeated.

[0096] Example 6

[0097] The test strip structure is basically the same as that in Example 2, except that the adsorption fiber layer is made of polyester film, the cellulose membrane layer is made of carboxylated cellulose membrane, and the invisible detection imprint band is “├”.

[0098] For testing meat samples, the pre-sample enrichment, result determination, and operation methods are the same as in Example 2, and will not be repeated.

[0099] Example 7 Performance of the test strip of the present invention

[0100] (1) Test strip sensitivity detection: Taking Escherichia coli O157:H7 as an example, a single colony of Escherichia coli was inoculated into a culture medium containing long-afterglow liposomes and cultured. The bacterial count was performed using the plate count method. The bacterial solution was diluted to 10⁻⁶. 9 10 8 10 7 10 6 10 5 10 4 10 3 The CFU / mL concentration was measured using the test strip from Example 2. After detection, the test strip was excited with a UV lamp, and the visually determined limit of detection (LOD) was the lowest bacterial concentration at which the T-line was visible. Semi-quantitative analysis of the test strip was performed using a fluorescence strip reader, and the results are as follows: Figure 3 As shown, Figure 3 The bacterial concentrations corresponding to the T-line in the test, from top to bottom, are 10. 9 10 8 107 10 6 10 5 10 4 10 3 CFU / mL, test strip at bacterial concentration of 10 4 -10 8 A linear correlation was observed at CFU / mL, and the LOD was 10. 4 CFU / mL.

[0101] (2) Test strip specificity detection: The specificity was determined using a cross-reactivity test. A concentration of 10... 8 Table 1 shows the common foodborne pathogens at CFU / mL. When tested with long-afterglow test strips, a positive result ("+") indicates cross-contamination. The results showed that the long-afterglow test strips could only detect three strains of Escherichia coli, while other common foodborne pathogens could not be detected, indicating that the test strips have good specificity.

[0102] Table 1. Common foodborne pathogens and their sources used in cross-reactivity studies.

[0103]

[0104] (3) Determination of the stability of the test strip of the present invention: Taking the detection of Escherichia coli O157:H7 as an example, the same batch of long afterglow test strips were sealed with desiccant and placed at 4°C in the dark. The sensitivity was tested after 1 day, 1, 5, 10, 15, and 20 weeks of storage. The results showed that the sensitivity of the test strip after 20 weeks of storage was 10. 4 The CFU / mL value remained unchanged after 1 day of storage, indicating that the test strip can maintain stable performance for at least 20 weeks at 4°C.

[0105] (4) Simulated bacterial contamination experiment: Cake, biscuit, and beverage samples were purchased from a local supermarket. PCR was used to identify that none of the three samples were contaminated with E. coli O157:H7. 25g of each sample was aseptically placed in a culture medium containing long-afterglow liposomes and cultured. A live bacteria solution was added to the culture medium to achieve a bacterial concentration of 1 CFU / mL. The culture flasks were placed in a shaker at 37℃, and samples were taken for testing every 2 hours. The results showed that the bacteria could be detected in cake, biscuit, and beverage samples after 10 hours of culture; the long-afterglow test strip could detect samples with an initial bacterial concentration of 1 CFU / mL.

Claims

1. A long-afterglow immunochromatographic test strip for detecting foodborne pathogens, comprising a support layer, wherein an adsorption layer is provided on the support layer, characterized in that: The adsorption layer consists of, from the test end, an adsorption fiber layer, a long-afterglow antibody fiber layer, a cellulose membrane layer, and a water-absorbing material layer at the handle end. Adjacent layers of the adsorption fiber layer, long-afterglow antibody fiber layer, cellulose membrane layer, and water-absorbing material layer are interconnected. A detection line and a control line are provided on the cellulose membrane layer. The detection line is composed of monoclonal or polyclonal antibodies against foodborne pathogens. The control line is composed of goat anti- or rabbit anti-mouse IgG antibodies, or goat anti-rabbit IgG antibodies. Long-afterglow antibodies are adsorbed on the long-afterglow antibody fiber layer. The long-afterglow antibodies are composed of monoclonal or polyclonal antibodies against foodborne pathogens labeled with long-afterglow nanomaterials. The long afterglow nanomaterial is Sr2MgSi2O7:Eu 2+ ,Dy 3+ The material has an excitation peak of 254 nm or 350 nm, an emission peak of 470 nm, an afterglow duration of 10 h, and particle size of nanoparticles with a diameter of 50-200 nm. The long afterglow nanomaterial Sr2MgSi2O7:Eu 2+ ,Dy 3+ The marking method is as follows: Sr2MgSi2O7:Eu with surface aminated modification 2+ ,Dy 3+ Nanomaterials were dispersed in PBS buffer solution, and 25% glutaraldehyde solution was added. The mixture was reacted at room temperature for 3 hours. Excess glutaraldehyde was washed away by centrifugation. The reaction solution was then dispersed in PBS buffer solution, and monoclonal or polyclonal antibodies against foodborne pathogens were added. The mixture was reacted at 4°C for 3 hours. After centrifugation and washing, labeled long-afterglow antibodies were obtained and stored in PBS buffer solution at 4°C.

2. The long afterglow immunochromatographic test strip as described in claim 1, characterized in that, The Sr2MgSi2O7:Eu 2+ ,Dy 3+ The amination modification method for nanomaterials is as follows: Grinding Sr2MgSi2O7:Eu 2+ ,Dy 3+ While producing nanomaterials, NaOH solution is continuously added to Sr2MgSi2O7:Eu 2+ ,Dy 3+ Nanomaterials were ultrasonically dispersed in NaOH solution, and separated after repeated washing to obtain HO-PLNPS nanoparticles with hydroxyl groups on their surface. HO-PLNPS was ultrasonically dispersed in a PVP solution, and after standing, the supernatant was separated and extracted. The supernatant was then centrifuged to obtain PVP-PLNPS nanoparticles coated with PVP. PVP-PLNPS was dispersed in a mixed solution of ethanol and ultrapure water, ammonia was added, and the mixture was then ultrasonically dispersed. After the solution is thoroughly mixed, place it in an ice-water bath and slowly add tetraethyl orthosilicate; stir continuously, and obtain SiO2-PLNPS through repeated centrifugation and washing; finally, add the modifier APTES to modify it, generating aminated Sr2MgSi2O7:Eu 2+ ,Dy 3+ Nanomaterials.

3. A method for preparing a long-afterglow immunochromatographic test strip for detecting foodborne pathogens as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Prepare monoclonal or polyclonal antibodies against foodborne pathogens; (2) Preparation of long-afterglow antibodies; (3) Prepare an adsorption fiber layer, wherein the adsorption fiber layer is made of glass fiber cotton, nylon membrane, polyvinylidene fluoride membrane or polyester membrane; (4) Preparation of long-afterglow antibody fiber layer; (5) Prepare a cellulose membrane layer, wherein the cellulose membrane layer is made of nitrocellulose membrane, pure cellulose membrane or carboxylated cellulose membrane, and the imprint is sprayed on the cellulose membrane with a spotting instrument, and then dried for later use; (6) Assemble the test strip by attaching the adsorbent fiber layer, long afterglow antibody fiber layer, cellulose membrane layer and water-absorbing material layer to the support layer with adhesive in sequence, and assemble the support layer, adsorbent layer and protective layer into a test strip.

4. The preparation method according to claim 3, characterized in that, The method for preparing the adsorbent fiber layer is as follows: glass fiber cotton, nylon membrane, polyvinylidene fluoride membrane or polyester membrane are immersed in TBS buffer solution until wet, then freeze-dried for later use; the concentration of the TBS buffer solution is 0.01M and the pH is 7.8; by mass percentage concentration, the TBS buffer solution also contains 0.5% PVP, 1% sucrose, 0.5% Tween-20 and 1% BSA.

5. A method for detecting foodborne pathogens using a long-afterglow immunochromatographic test strip as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Pre-enrichment of samples: Add liposome solution containing long afterglow nanomaterials to enrichment medium, and then add the sample for enrichment culture; (2) Test strip detection: Insert the test strip into the sample solution, take it out and lay it flat after 10-20 seconds, and after 5-10 minutes, observe whether there is afterglow on the test line of the test strip to determine whether the sample contains foodborne pathogens, or perform semi-quantitative analysis by using a fluorescence strip reader.

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