CN108362872A - A kind of quantitative detecting method of the white diarrhea and avian infectious bronchitis nephritis virus of non-diagnostic purpose - Google Patents

Abstract

The invention discloses a kind of rapid detection methods of the white diarrhea and avian infectious bronchitis nephritis virus of non-diagnostic purpose.The detection method of the present invention prepares the coupled to Nano magnetic bead (IgG MNPs) for being marked with S. pullonum and avian infectious bronchitis nephritis virus antibody and antibody and the silicon dioxide nanosphere (GOx IgG SiNPs) of enzyme double labelling first, when there are purpose bacterium to be measured, form the immune complex IgG MNPs/S.pullorum and S.gallinarum GOx IgG SiNPs of sandwich " sandwich " structure, then pass through enzymic catalytic reaction, reaction product forms complexing product with color developing agent, the concentration of white diarrhea and avian infectious bronchitis nephritis virus is obtained finally by the absorbance value of detection architecture, to realize the quantitative detection of S. pullonum and avian infectious bronchitis nephritis virus.The cascade reaction combination Fe that the present invention is triggered with glucose oxidase (GOx)³⁺‑SCN^‑Color Appearance System realizes the quantitative detection of S. pullonum and avian infectious bronchitis nephritis virus, and the detection method is convenient and efficient, easy to operate, has good sensitivity, specificity and stability.

Description

A method for quantitative detection of pullorum and Salmonella gallinarum for non-diagnostic purposes

technical field

The invention belongs to the technical field of food and food raw material safety detection, in particular to a non-diagnostic non-diagnostic non-diagnostic pullorum and salmonella gallinarum typhi quantitative detection method.

Background technique

Salmonella pullorum (S.pullorum) and Salmonella gallinarum (S.gallinarum) are the pathogens of pullorum (Pullorum Disease, PD) and chicken typhoid (Fowl typhoid, FT), causing huge damage to the poultry industry. losses and seriously threaten food safety. Since Salmonella pullorum and Salmonella gallinarum have the same O1, O9, O12 antigens, it is difficult to strictly distinguish the two and it is not necessary in actual testing, so the two are usually tested together. The detection of Salmonella pullorum and Salmonella gallinarum is very meaningful for the prevention and control of food safety risks.

The detection methods for pathogenic bacteria include standard methods and a variety of rapid detection methods, such as digital classification and identification method, immunolabeling technology and multiple detection methods based on molecular biology. Existing methods have their own advantages and disadvantages. Therefore, it is an inevitable trend of development and application to use multi-disciplinary new knowledge, new materials and new technologies to construct better rapid detection technology.

Chen et al. reported a method of using glucose oxidase to catalyze glucose solution to produce hydrogen peroxide, using PSS-GN-Pt nanocomposite as a peroxidase mimic to catalyze the color development of tetramethylbiphenyl, and then realize the Colorimetric determination of glucose. This provides a new idea for us to design and construct a new colorimetric system. Glucose oxidase catalyzes glucose to produce H ₂ O ₂ , and H ₂ O ₂ can be catalyzed and oxidized with the assistance of horseradish peroxidase (HRP). color reaction. The classic reaction of ferric ions (Fe ³⁺ ) is the color reaction with thiocyanate anion (SCN ^- ), resulting in a red color visible to the naked eye. Therefore, this reaction is often applied to the determination of Fe ³⁺ . In addition, Fe ²⁺ can be oxidized to Fe ³⁺ in an acidic environment. Therefore, the cascade reaction triggered by glucose oxidase combined with the Fe ^{3+ -SCN} -chromogenic system can be used to establish a new immunoassay method.

In summary, the present invention aims at the defects of the existing detection technology of Salmonella pullorum and Salmonella gallinarum typhi, and develops a magnetic control immunocolorimetric method based on glucose oxidase-triggered cascade reaction system combined with Fe ³⁺ -SCN ^- color development system Using IgG-MNPs to isolate and enrich the target bacteria Salmonella pullorum and Salmonella gallinarum typhi, GOx-IgG-SiNPs as signal indicators; construct IgG-MNPs-S. pullorum and S. gallinarum-GOx-IgG- SiNPs sandwich structure, GOx can catalyze glucose to produce H ₂ O ₂ , H ₂ O ₂ oxidizes Fe ²⁺ into Fe ³⁺ , Fe ³⁺ can quickly complex with SCN ^- to generate a red product, by measuring the red product Absorbance, in order to achieve quantitative determination of the concentration of Salmonella pullorum and Salmonella gallinarum typhi in food.

Contents of the invention

The purpose of the present invention is to provide a non-diagnostic purpose of pullorum and Salmonella gallinarum quantitative detection method. The invention realizes the quantitative detection of Salmonella pullorum and Salmonella gallinarum in food by combining the cascade reaction triggered by GOx and the Fe ³⁺ -SCN ^- color development system. The detection method is convenient, quick, easy to operate, and has good sensitivity and specificity. sex and stability.

In order to achieve the above-mentioned purpose, the present invention takes the following technical solutions:

A quantitative detection method for pullorum pullorum and Salmonella gallinarum typhi for non-diagnostic purposes, comprising the following steps:

(1) Covalently bind pullorum and Salmonella typhi antibodies (Anti-S.pullorum and S.gallinarum) and glucose oxidase (GOx) to the surface of silica nanospheres (SiNPs) to form enzyme labels ( GOx-IgG-SiNPs), standby;

(2) The surface of magnetic nanoparticles with Fe ₃ O ₄ /SiO ₂ core-shell structure was modified with pullorum and Salmonella typhi antibodies (Anti-S.pullorum and S.gallinarum) to obtain antibody-coupled magnetic nanoparticles (IgG -MNPs), spare;

(3) Add the antibody-coupled nano-magnetic beads (IgG-MNPs) prepared in step (2) to the sample suspension to be tested and incubate at 37°C for a certain period of time, wash and magnetically separate to obtain a magnetic immune complex A (IgG-MNPs-S. pullorum and S. gallinarum);

(4) Add the enzyme marker (GOx-IgG-SiNPs) prepared in step (1) to the immune complex (IgG-MNPs-S.pullorum and S.gallinarum) prepared in step (3) and incubate at 37°C After a certain period of time, after washing and magnetic separation, magnetic immune complex B (IgG-MNPs-S.pullorum and S.gallinarum-GOx-IgG-SiNPs) is obtained;

(5) adding glucose to the immune complex B obtained in step (4) to carry out the enzyme-catalyzed reaction;

(6) After the enzyme catalyzed reaction is completed, add the chromogenic reagent KSCN solution, and utilize a microplate reader to measure the absorbance value of the sample at a wavelength of 466nm. The standard curve can be calculated to obtain pullorum and Salmonella gallinarum typhi content.

In the above method, the silica nanospheres in the step (1) are prepared by the following method:

Mix and stir Triton X-100, n-hexanol, and cyclohexane evenly, then add ultrapure water and stir evenly to form a W/O microemulsion, then add tetraethyl orthosilicate, after stirring for reaction, add ammonia water as a catalyst for hydrolysis Condensation reaction, and then through the steps of demulsification, centrifugation and washing to obtain the product silicon dioxide nano microspheres.

In the above method, the specific preparation steps of the enzyme marker in the step (1) are as follows:

(1) Ultrasonic disperse silica nano-microspheres in absolute ethanol, then add silane coupling agent 3-aminopropyltriethoxysilane (APTES), react at room temperature for 12h, use absolute ethanol and Phosphate buffered saline (PBS) was washed at least three times respectively to obtain amino chemically modified silica nanospheres;

(2) Ultrasonically disperse amino-chemically modified silica nanospheres into phosphate buffer, add glutaraldehyde solution, stir for 3 hours in the dark, and wash with phosphate buffer several times to obtain aldehyde-modified Silica nanospheres; add pullorum and Salmonella gallinarum antibodies, stir and react at room temperature for 3 hours, centrifuge and wash at least 3 times with phosphate buffer, resuspend in phosphate buffer, add glucose oxidase (GOx ) to stir the reaction to obtain the enzyme-labeled substance (GOx-IgG-SiNPs) precipitate, add bovine serum albumin (BSA) to the GOx-IgG-SiNPs precipitate and stir the reaction at room temperature to block the remaining surface of the enzyme-labeled substance (GOx-IgG-SiNPs) Non-specific binding sites, resulting in enzyme labels.

In the above method, the magnetic nanoparticles of Fe ₃ O ₄ /SiO ₂ core-shell structure in the step (2) are prepared by the following method:

(1) Add the solution containing Fe ³⁺ to the deionized water that has been deoxygenated in advance, add FeSO ₄ 7H ₂ O, after the above-mentioned FeSO ₄ 7H ₂ O solid dissolves, quickly add ammonia water and stir vigorously, N ₂ protection Under the conditions, 80°C vigorously stirred and reacted for 60 minutes. After the liquid was cooled, it was fully washed with deionized water to obtain Fe ₃ O ₄ magnetic nanoparticles;

(2) Take Fe ₃ O ₄ magnetic nanoparticles and add them to the mixed solvent of absolute ethanol, deionized water and ammonia water. After stirring vigorously, add tetraethyl orthosilicate, and stir and react in a constant temperature water bath at 40°C for 24 hours to obtain magnetic nanoparticles. particles (MNPs).

In the above method, the antibody-coupled nano-magnetic beads (IgG-MNPs) in the step (2) are prepared by the following method:

(1) Disperse magnetic nanoparticles (MNPs) in absolute ethanol, then add silane coupling agent 3-aminopropyltriethoxysilane, react at room temperature for 12 hours, buffer with absolute ethanol and phosphate solution were washed three times or more to obtain amino-modified MNPs;

(2) Resuspend the amino-modified magnetic nanoparticles in phosphate buffer, add glutaraldehyde solution, and stir for 6 hours in the dark, then fully wash with phosphate buffer several times to obtain aldehyde-modified MNPs; Then add Salmonella pullorum and Salmonella typhi antibodies, stir and react at room temperature for 3h, wash, then add bovine serum albumin (BSA) to precipitate with antibody-coupled nano-magnetic beads and react with stirring at room temperature to block non-specific binding sites on the surface of IgG-MiNPs point to obtain antibody-coupled nanomagnetic beads (IgG-MNPs).

In the above method, the concentration of the antibody-coupled nano-magnetic beads in the step (3) is preferably 6 mg/mL, and the incubation time is preferably 45 min.

In the above method, the concentration of the enzyme label in the step (4) is preferably 20 mg/mL, and the incubation time is preferably 30 min.

In the above method, the glucose in the step (5) is added in the form of a solution with a concentration of 50 mM dissolved in tris-HCl with a pH of 7.0.

In the above method, the chromogenic reagent in the step (6) is a solution containing 4.5 mM FeSO ₄ and 0.3 mM KSCN prepared with 0.2 M HCl.

In the above method, the linear equation of the standard curve in the step (6) is y=0.1992x-0.31102, x is the logarithm value of Salmonella pullorum and Salmonella gallinarum concentration with 10 as the base, and the unit is CFU/mL, y is the absorbance value.

The invention realizes quantitative detection of Salmonella pullorum and Salmonella gallinarum by combining cascade reaction triggered by glucose oxidase (GOx) and Fe ³⁺ -SCN ^- color development system. To construct this detection platform, two kinds of nanoprobes including antibody-coupled magnetic nanoparticles (IgG-MNPs) labeled with S. , where IgG-MNPs were used as capture probes for rapid capture and enrichment of the target bacteria Salmonella pullorum and Salmonella gallinarum, and GOx-IgG-SiNPs were used as detection probes for signal transduction. When the target bacteria to be tested are present, the IgG-MNPs S.pullorum S.gallinarum-GOx-IgG-SiNPs sandwich structure immune complex B is formed, and the GOx carried by the GOx-IgG-SiNPs in the sandwich structure can catalyze the glucose solution to produce glucose Acid and H ₂ O ₂ , the generated H ₂ O ₂ can further oxidize Fe ²⁺ to Fe ³⁺ , and Fe ³⁺ can quickly undergo a complex reaction with SCN ^- to form a red complex product. The absorbance value of the colored product is linearly correlated with the logarithm of the concentration of the target bacteria Salmonella pullorum and Salmonella gallinarum, whereby the concentration of Salmonella pullorum and Salmonella gallinarum in the food can be quantitatively obtained by measuring the absorbance value.

The present invention has the following technical characteristics:

1) The present invention realizes the quantitative detection of Salmonella pullorum and Salmonella gallinarum in food by using the cascade reaction triggered by glucose oxidase combined with the Fe ³⁺ -SCN ^- color development system. The detection method is convenient, fast, economical and easy to operate.

2) The present invention has good sensitivity, specificity and stability.

3) The immunosensing system based on the Fe ^{3+ -SCN} -chromogenic system of the present invention can be further expanded and applied to detect other pathogenic bacteria by changing the target antibody.

Description of drawings

Figure 1a is a schematic diagram of the preparation process of antibody-coupled nano-magnetic beads;

The schematic diagram of the preparation process of the enzyme marker in Fig. 1b;

Figure 1c Schematic diagram of the principle of the method for the quantitative determination of pullorum pullorum Salmonella gallinarum by combining the cascade reaction triggered by GOx with the Fe ³⁺ -SCN ^- chromogenic system;

The relationship diagram between the absorbance value of Fig. 2 embodiment 2 and the concentration of antibody-coupled nano-magnetic beads;

Fig. 3 embodiment 2 absorbance value and the relation figure of enzyme marker concentration;

The relation figure of Figure 4 embodiment 3 absorbance value and pullorum and Salmonella gallinarum concentration;

Fig. 5 embodiment 4 specificity experiment: the relation figure of absorbance value and bacterial species;

Fig. 6 Stability experiment of Example 5: a graph of the relationship between absorbance value and storage time.

Detailed ways

The following specific examples are further descriptions of the methods and technical solutions provided by the present invention, but should not be construed as limiting the present invention.

Example 1:

(1) Synthesis of silica nanoparticles (SiNPs):

SiNPs were synthesized by the inverse microemulsion method. The specific synthesis process is as follows: 2mL Triton X-100 (surfactant), 2mL n-hexanol (cosurfactant), 8mL cyclohexane (organic solvent) were stirred for 20min until clear and transparent, and 480μL ultrapure water was added as the dispersed phase , stirred for 20 min to make the water droplets completely dispersed in the above oil phase to form a uniform and stable W/O microemulsion, and then added 100 μL tetraethyl orthosilicate (TEOS) as a precursor for the synthesis of SiNPs, and stirred for 30 min. Add 100 μL of ammonia water (catalyst) to the system solution to promote TEOS to form nano-microspheres through hydrolysis and condensation reaction faster. After 48 hours of magnetic stirring reaction, 10 mL of acetone was added to demulsify the microemulsion, and the precipitate was collected after centrifugation at 10,000 rpm for 5 minutes, and then centrifuged and washed 3 times with acetone, absolute ethanol and ultrapure water, respectively. The obtained SiNPs were dispersed in ultrapure water and stored at room temperature for future use.

(2) Synthesis of enzyme markers (GOx-IgG-SiNPs):

The preparation principle of GOx-IgG-SiNPs is shown in Fig. 1b. Take 30 mg of SiNPs prepared by the above inverse microemulsion method and centrifuge and wash them three times with absolute ethanol, then sonicate the precipitate for 30 min to fully disperse it in 10 mL of absolute ethanol, then add 200 μL of silane coupling agent 3-ammonia Propyltriethoxysilane (APTES), after reacting at room temperature for 12h, APTES made the amino group be introduced to the surface of SiNPs through hydrolysis and condensation reaction, with absolute ethanol and 0.01M phosphate buffer (PBS, pH7.4) Centrifuge and wash three times respectively to obtain amino chemically modified SiNPs (SiNPs-NH ₂ ).

Ultrasonic disperse SiNPs-NH ₂ into 10mL PBS, add 5mL 2.5% glutaraldehyde solution, and stir for 3 hours in the dark, centrifuge and wash with PBS for 6 times to obtain aldehyde-modified SiNPs, then resuspend in 4mL PBS, add 40 μL pullorum and Salmonella typhi antibodies (anti-S. pullorum and S. gallinarum), stirred at room temperature for 3 hours, centrifuged with PBS 10,000 rpm and washed 3 times, resuspended in 4 mL PBS, added 500 μL 6mg/mL GOx at 4 °C The reaction was stirred for 6 h, and the unbound GOx molecules were washed several times with PBS to obtain GOx-IgG-SiNPs. The GOx-IgG-SiNPs pellet was resuspended in 4 mL of PBS, and 1 mL of 1% BSA was added to react at room temperature for 1.5 h to block the remaining non-specific binding sites on the surface of GOx-IgG-SiNPs. Centrifuge and wash with PBS for 3 times, resuspend in 2 mL of PBS, and store in a 4°C refrigerator for later use.

(3) Synthesis of Fe ₃ O ₄ magnetic nanoparticles:

With slight modification according to the method reported in _the literature, _Fe3O4 magnetic nanoparticles were synthesized by chemical co-precipitation method ^. Add 2.7mL of 1M Fe ³⁺ solution to 70mL of deionized water that has been deoxygenated in advance, add 0.375g of FeSO ₄ 7H ₂ O, and after the above-mentioned FeSO ₄ 7H ₂ O solid dissolves, quickly add 5mL of ammonia water and stir vigorously to Vacuum pump to remove oxygen and introduce high-purity nitrogen to create an oxygen-free environment. Under N ₂ protection conditions, stir vigorously at 80°C for 60 minutes. After the liquid is cooled, it is fully washed with deionized water to obtain Fe ₃ O ₄ magnetic nanoparticles. Suspend in 60 mL of deoxygenated deionized water and store in a 4°C refrigerator for later use.

(4) Synthesis of Magnetic Nanoparticles (MNPs):

The process of coating SiO ₂ onto the surface of Fe ₃ O ₄ magnetic nanoparticles refers to the classic , with minor improvements. The specific coating process is as follows: Take 1 mL of the above-mentioned Fe ₃ O ₄ magnetic nanoparticles and add them to 60 mL of absolute ethanol, 10 mL of deionized water and 9 mL of ammonia water, stir vigorously for 20 min, add 1 mL of TEOS, and stir in a constant temperature water bath at 40 °C for 24 h. Wash several times with deionized water to obtain MNPs, resuspend in 500 μL deionized water, and store in a 4°C refrigerator for later use.

(5) Synthesis of antibody-coupled nanomagnetic beads (IgG-MNPs):

The preparation principle of IgG-MNPs is shown in Figure 1a. Take 500 μL of the above-synthesized MNPs and wash them three times with absolute ethanol, and then fully disperse them in 10 mL of absolute ethanol, then add 200 μL of silane coupling agent APTES, and react at room temperature for 12 hours, APTES makes amino groups through hydrolysis and condensation reaction. It was introduced onto the surface of MNPs, and washed three times with absolute ethanol and 0.01M phosphate buffer (PBS, pH 7.4) to obtain amino-modified MNPs (MNPs-NH ₂ ).

Resuspend MNPs-NH ₂ into 10mL PBS, add 5mL 2.5% glutaraldehyde solution, and stir for 6 hours in the dark, wash with PBS for 6 times to obtain aldehyde-modified MNPs, and then resuspend the precipitate with 4mL PBS , adding 40 μL of anti-S.pullorum and S.gallinarum, stirred at room temperature for 3h, washed 3 times with PBS, and obtained IgG-MNPs. IgG-MNPs were resuspended in 4 mL of PBS, and 1 mL of 1% BSA was added to react at room temperature for 1.5 h to block non-specific binding sites on the surface of IgG-MNPs. After washing 3 times with PBS, resuspend in 500 μL PBS, and store in 4°C refrigerator for use.

(6) GOx-triggered enzymatic cascade reaction combined with Fe ³⁺ -SCN ^- chromogenic system to detect pullorum and Salmonella gallinarum typhi:

The detection principle of Pullorum pullorum and Salmonella gallinarum (S.pullorum and S.gallinarum) using Fe ^{3+ -SCN} -chromogenic system is shown in Figure 1c. The specific detection steps are as follows: 20 μL of a solution of antibody-coupled nanomagnetic beads IgG-MNPs with a concentration of 6 mg/mL was added to 1 mL of pullorum and Salmonella gallinarum suspension and incubated at 37 °C for 45 min, then washed 3 times with PBS to remove untreated bacteria. Combined pullorum and Salmonella gallinarum and other substances were magnetically separated to obtain IgG-MNPs-S.pullorum-and-S.gallinarum immune complex A. After the immune complex A was resuspended in 20 μL of PBS, 20 μL of enzyme-labeled GOx-IgG-SiNPs at a concentration of 20 mg/mL was added and incubated at 37°C for 30 min, and then thoroughly washed with PBS to remove unbound GOx-IgG-SiNPs. Magnetic separation to obtain IgG-MNPs-S. pullorum-and-S.gallinarum-GOx-IgG-SiNPs sandwich "sandwich" structure immune complex B, add 50μL 50mM glucose (dissolved in pH7.0tris-HCl) at 37℃ After performing the enzyme-catalyzed reaction for 1 min, an equal volume of chromogenic reagent (4.5 mM FeSO ₄ , 3% KSCN solution prepared with 0.2 M HCl) was added successively, and its absorbance value at a wavelength of 466 nm was measured on a microplate reader within 5 min. All measurements were performed at room temperature (25±1.0°C).

Example 2: Establishment of GOx-triggered enzymatic cascade reaction combined with Fe ³⁺ -SCN ^- chromogenic system for the quantitative detection of pullorum and Salmonella gallinarum typhi

(1) Optimization of the concentration of antibody-coupled nano-magnetic beads

Change the concentration of antibody-coupled nanomagnetic beads IgG-MNPs: 2.4mg/mL, 3mg/mL, 4mg/mL, 6mg/mL, 12mg/mL, 24mg/mL, 48mg/mL, other experimental conditions are the same as the implementation Same as Example 1, quantitatively detect 1 mL of 10 ⁷ CFU·mL ^-1 samples of S. pullorum and S. gallinarum to be tested. It can be seen from Figure 2 that as the concentration of IgG-MNPs increases, the absorbance value gradually reaches the maximum value and then maintains a plateau period and then shows a downward trend. Therefore, the optimal concentration of IgG-MNPs for analysis and testing is 6 mg/mL.

(2) Optimization of enzyme marker concentration

Change the concentration of the enzyme marker GOx-IgG-SiNPs as follows: 2mg/mL, 2.5mg/mL, 3.3 mg/mL, 5mg/mL, 10mg/mL, 20mg/mL, 40mg/mL, other experimental conditions are the same as the implementation Same as Example 1, quantitatively detect 1 mL of 10 ⁷ CFU·mL ^-1 samples of S. pullorum and S. gallinarum to be tested. It can be seen from Figure 3 that as the concentration of the enzyme label increases, the absorbance value remains stable after gradually reaching the maximum value, so the optimal concentration of GOx-IgG-SiNPs selected for analysis and testing is 20 mg/mL.

Embodiment 3: Sensitivity detection

Sensitivity was determined by detecting 8.4×10 ⁰ CFU·mL ^-1 to 8.4×10 ⁷ CFU·mL ^-1 pullorum and Salmonella gallinarum typhi (S. pullorum and S. gallinarum ) under the conditions of Example 1. The relationship between the absorbance value and the concentration of pullorum and Salmonella gallinarum is shown in Figure 4, when the concentration of S. pullorum and S. gallinarum changes from 8.4×10 ³ CFU·mL ^-1 to 8.4×10 ⁷ CFU·mL ^-1 , the absorbance value has a good linear relationship with the logarithmic value of the concentration of S.pullorum and S.gallinarum. The linear equation is y=0.1992x-0.31102, and the coefficient of variation R ² =0.98706. The detection limit of this method for the target bacteria S.pullorum and S.gallinarum was 2.36×10 ³ CFU·mL ^-1 .

Embodiment 4: specific detection

Good specificity is particularly important in immunoassays, so the specific effect of this newly established method for the analysis and detection of pullorum and Salmonella gallinarum (S. pullorum and S. gallinarum) was investigated. Under the conditions of Example 1, 10 ⁷ CFU·mL ^-1 of S.pullorum and S.gallinarum), Shigella sonnei (Sh.sonnei), Escherichia coli (E.coli), Salmonella typhimurium (S. typhimurium), Lactobacillus freundii (C.freundii) and PBS were used as blank controls. The experimental results are shown in Figure 5, only S.pullorum and S.gallinarum caused obvious color changes and the absorbance value was much higher than that of other control groups, which indicated that this method can specifically and effectively detect S.pullorum and S. gallinarum .

Embodiment 5: Stability experiment

GOx-IgG-SiNPs were stored in a refrigerator at 4°C for 1, 7, 30, 60, 90, and 120 days, respectively, and then the probe was used to detect 10 ⁶ CFU·mL ^-1 of S. pullorum and S. gallinarum. The experimental results showed that after GOx-IgG-SiNPs were stored for 7, 30, 60, 90, and 120 d, their signal intensity values changed to 94.29%, 97.77%, 99.98%, 62.32%, and 61.37% of the initial signal value, respectively, which indicated that the enzyme The biological activity of the marker has not decreased significantly, and still has a high reactivity, and it can still be used for analysis and detection of S. pullorum and S. gallinarum within 60 days of storage without obvious signal weakening.

Example 6: Example of detection of Pullorum pullorum and Salmonella gallinarum typhi in food

Chicken livers were used to simulate actual samples. Chicken livers were purchased from supermarkets and homogenized into a homogenate with a sterile homogenizer. S.pullorum and S.gallinarum were added to the prepared chicken liver suspension, and the final concentrations of S.pullorum and S.gallinarum were 8.4×10 ³ CFU·mL ^-1 , 8.4×10 ⁵ CFU·mL ^-1 and For 8.4×10 ⁷ CFU·mL ^-1 actual samples, three parallel groups were set up for each sample. The test results are shown in Table 1. The relative error between the newly established method and the national standard method for detecting S. pullorum and S. gallinarum is not more than 17.14%. Therefore, the method established in this study is stable and accurate, and can be used to detect actual samples. S. pullorum and S. gallinarum.

Table 1 The method of the present invention detects the result of adding the standard chicken liver sample and the national standard method contrast

a The average value of three detections

The descriptions of the above embodiments are only used to help understand the method of the present invention and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (10)

1. a quantitative detection method of pullorum pullorum and Salmonella gallinarum typhi of non-diagnostic purpose, is characterized in that, comprises the following steps: (1) Covalently binding the pullorum and Salmonella gallinarum antibodies and glucose oxidase to the surface of the silica nanospheres to form an enzyme marker for subsequent use; (2) modifying the surface of magnetic nanoparticles with Fe ₃ O ₄ /SiO ₂ core-shell structure with pullorum pullorum and Salmonella gallinarum antibodies to obtain antibody-coupled nano-magnetic beads for future use; (3) adding the antibody-coupled nano-magnetic beads prepared in step (2) to the sample suspension to be tested and incubating at 37°C for a certain period of time, washing and magnetic separation to obtain magnetic immune complex A; (4) adding the enzyme marker prepared in step (1) to the immune complex prepared in step (3) and incubating at 37°C for a certain period of time, washing and magnetic separation to obtain magnetic immune complex B; (5) adding glucose to the immune complex B obtained in step (4) to carry out the enzyme-catalyzed reaction; (6) After the enzyme catalyzed reaction is completed, add the chromogenic reagent KSCN solution, and utilize a microplate reader to measure the absorbance value of the sample at a wavelength of 466nm. The standard curve can be calculated to obtain pullorum and Salmonella gallinarum typhi content. 2. quantitative detection method as claimed in claim 1, is characterized in that, the silica nano-microsphere in described step (1) is prepared by following method: Mix and stir Triton X-100, n-hexanol, and cyclohexane evenly, then add ultrapure water and stir evenly to form a W/O microemulsion, then add tetraethyl orthosilicate, after stirring for reaction, add ammonia water as a catalyst for hydrolysis Condensation reaction, and then through the steps of demulsification, centrifugation and washing to obtain the product silicon dioxide nano microspheres. 3. quantitative detection method as claimed in claim 1, is characterized in that, the concrete preparation step of enzyme marker in described step (1) is as follows: (1) Ultrasonic disperse silica nanospheres into absolute ethanol, then add silane coupling agent 3-aminopropyltriethoxysilane, react at room temperature for 12h, buffer with absolute ethanol and phosphate Washing with liquid for at least three times respectively to obtain amino chemically modified silica nanospheres; (2) Ultrasonically disperse amino-chemically modified silica nanospheres into phosphate buffer, add glutaraldehyde solution, stir for 3 hours in the dark, and wash with phosphate buffer several times to obtain aldehyde-modified Silica nanospheres; then add pullorum and Salmonella gallinarum antibodies, stir and react at room temperature for 3 hours, centrifuge and wash at least 3 times with phosphate buffer, then resuspend in phosphate buffer, add glucose oxidase and stir for reaction , to obtain the enzyme-labeled precipitate, add bovine serum albumin and the enzyme-labeled precipitate to react with stirring at room temperature to block the remaining non-specific binding sites on the surface of the enzyme-labeled substance, and obtain the enzyme-labeled substance. 4. The quantitative detection method according to claim 1, characterized in that, the magnetic nanoparticles of the _Fe3O4 / _SiO2 core-shell structure in _the step (2) are prepared by the following method: (1) Add the solution containing Fe ³⁺ to the deionized water that has been deoxygenated in advance, add FeSO ₄ 7H ₂ O, after the above-mentioned FeSO ₄ 7H ₂ O solid dissolves, quickly add ammonia water and stir vigorously, N ₂ protection Under the conditions, 80°C vigorously stirred and reacted for 60 minutes. After the liquid was cooled, it was fully washed with deionized water to obtain Fe ₃ O ₄ magnetic nanoparticles; (2) Take Fe ₃ O ₄ magnetic nanoparticles and add them to the mixed solvent of absolute ethanol, deionized water and ammonia water. After stirring vigorously, add tetraethyl orthosilicate, and stir and react in a constant temperature water bath at 40°C for 24 hours to obtain magnetic nanoparticles. particles. 5. quantitative detection method as claimed in claim 1, is characterized in that, the antibody-coupled magnetic nano-bead in described step (2) is prepared by following method: (1) Disperse the magnetic nanoparticles in absolute ethanol, then add the silane coupling agent 3-aminopropyltriethoxysilane, react at room temperature for 12 hours, wash with absolute ethanol and phosphate buffer More than three times to obtain amino-modified magnetic nanoparticles; (2) Resuspend the amino-modified magnetic nanoparticles in phosphate buffer, add glutaraldehyde solution, and stir for 6 hours in the dark, then fully wash with phosphate buffer several times to obtain aldehyde-modified magnetic nanoparticles Particles; then add Salmonella pullorum and Salmonella typhi antibodies, stir and react at room temperature for 3 hours, wash, then add bovine serum albumin to precipitate antibody-coupled nano-magnetic beads and react at room temperature to block the non-specificity of the surface of antibody-coupled nano-magnetic beads Combining sites to obtain antibody-coupled magnetic nanobeads. 6. The quantitative detection method according to claim 1, wherein the concentration of the antibody-coupled nano-magnetic beads in the step (3) is preferably 6 mg/mL, and the incubation time is preferably 45 min. 7. The quantitative detection method according to claim 1, wherein the concentration of the enzyme marker in the step (4) is preferably 20 mg/mL, and the incubation time is preferably 30 min. 8. The quantitative detection method according to claim 1, characterized in that, the glucose in the step (5) is added in the form of a solution having a concentration of 50 mM dissolved in tris-HCl at pH=7.0. 9. The quantitative detection method according to claim 1, characterized in that, the chromogenic reagent in the step (6) is a solution containing 4.5mM FeSO ₄ and 3wt% KSCN prepared with 0.2M HCl. 10. quantitative detection method as claimed in claim 1 is characterized in that, the linear equation of standard curve is y=0.1992x-0.31102 in the described step (6), and x is Salmonella pullorum and Salmonella gallinarum concentration with 10 as The logarithmic value of the bottom, the unit is CFU/mL, and y is the absorbance value.