CN114636817B - Kit and method for detecting escherichia coli O157H 7 based on polyacrylamide hydrochloride mediated copper growth enhancement - Google Patents
Kit and method for detecting escherichia coli O157H 7 based on polyacrylamide hydrochloride mediated copper growth enhancement Download PDFInfo
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- CN114636817B CN114636817B CN202210118304.2A CN202210118304A CN114636817B CN 114636817 B CN114636817 B CN 114636817B CN 202210118304 A CN202210118304 A CN 202210118304A CN 114636817 B CN114636817 B CN 114636817B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
Abstract
The invention discloses a kit and a detection method for detecting Escherichia coli O157: H7 based on polyacrylamide hydrochloride mediated copper growth enhancement. The kit comprises an immunochromatography test strip, a gold-labeled antibody, a polyacrylamide hydrochloride solution, a copper ion solution and an ascorbic acid solution; the immunochromatographic test strip comprises a bottom plate, and a sample pad, a nitrocellulose membrane and absorbent paper which are sequentially lapped and fixed on the bottom plate; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is sprayed with an anti-Escherichia coli O157H 7 capture antibody, and the quality control line is sprayed with a goat anti-mouse antibody. By adopting the kit and the detection method, the non-specificity between the nitrocellulose membrane and the copper metal ions can be eliminated, so that the detection stability is improved, the maximized and effective signal-to-noise ratio is provided, and the high-sensitivity detection of Escherichia coli O157: H7 is realized.
Description
Technical Field
The invention relates to the technical field of immunochromatography detection, in particular to a kit and a detection method for detecting Escherichia coli O157: H7 based on polyacrylamide hydrochloride mediated copper growth enhancement.
Background
Escherichia coli O157H 7 causes severe infectious diseases such as diarrhea, hemolytic uremic syndrome, epilepsy, and respiratory failure at low concentrations (700 CFU/mL). However, the monitoring and detection of E.coli O157: H7 based on conventional culture methods is time consuming and does not provide on-site detection. The immunochromatographic test strip has been widely used for immediate detection of environmental and food safety due to its advantages of being portable, user-friendly and low-cost. However, the traditional colorimetric immunochromatographic test strip based on colloidal gold nanoparticles has the inherent defect of insufficient sensitivity, and the detection application of low-abundance targets is hindered. The signal intensity is very important for the detection performance of the test strip. In order to improve the detection level of the immunochromatographic test strip, a proper signal amplification strategy is selected on the basis of gold nanoparticles so as to improve the performance of the test strip and ensure repeatability and reliability.
Amplification strategies based on gold nanoparticles mainly include enzyme-catalyzed mediated deposition of coloured substrates and metal growth methods. The peroxidase activity of gold nanoparticles is often susceptible to the immunological properties of the probe and the sample protein, resulting in poor methodological stability. The commonly used metal growth method is a research hotspot of a signal amplification technology of a test strip based on colloidal gold as a signal probe. According to the metal growth strategy, the color development test strip is soaked in the enhancing solution, metal ions gather on the surface of the colloidal gold to be reduced into a metal simple substance by taking the colloidal gold as a core under the action of a reducing agent, and then the metal simple substance is deposited on the surface of the colloidal gold probe to form a nano shell layer, so that the signal intensity of the colloidal gold probe is obviously enhanced, and the detection sensitivity of the test strip is greatly improved. Gold, silver and copper growth are the most common strategies for enlarging the size of gold nanoparticles. Among them, copper is a cheap metal, and has been receiving attention because of its advantages in the field of catalysis. However, the lattice constants of gold and copper are very different, causing lattice mismatch. The growth process and shape control synthesis of the metal nanoshell with large lattice mismatch is not controllable. In addition, due to the metal growth method of the test strip soaking, copper can be deposited on the surface of the existing gold nanoparticles or the substrate, and particularly the specific treatment process of the nitrocellulose membrane enables the additionally added copper ions to be easily adsorbed during incubation, so that a false positive result or an obvious background is caused. The mode is easily influenced by the environment and operators, and the operation process needs to be accurately grasped, so that the popularization and application of the method in practice are greatly limited. Therefore, new controllable models need to be introduced to further improve accuracy, sensitivity and detection efficiency.
Disclosure of Invention
The invention aims to provide a method for sensitively detecting Escherichia coli O157: H7 aiming at the technical defects of the prior art, so as to solve the technical problem that the detection method for Escherichia coli O157: H7 in the prior art is low in sensitivity.
The invention also aims to solve the technical problem that the double-antibody sandwich immunochromatographic test strip method aiming at Escherichia coli O157: H7 in the prior art has low detection sensitivity due to poor color rendering property of colloidal gold.
The invention aims to solve the technical problem of improving the luminous signal and the detection sensitivity in the method for detecting the content of the Escherichia coli O157: H7 by executing the double-antibody sandwich immunochromatographic test strip through a polyacrylamide hydrochloride mediated amplification system.
In order to solve the problems, the invention introduces the polyacrylamide hydrochloride positive polyelectrolyte, and can realize uniform deposition on a non-planar substrate with any shape and size, wherein the substrate comprises gold nanoparticles and related probes. In addition, polyacrylamide hydrochloride with abundant amine groups is chelated with copper ions. The polyacrylamide hydrochloride can reduce the gold-copper interfacial energy, so that copper ions can be easily adsorbed on the surface of the target shell without any additional treatment. Thus, polyallylamine hydrochloride may serve a particular function as a versatile intermediate linkage. The strong adhesion of polyacrylamide hydrochloride eliminates non-specificity between copper ions and the nitrocellulose membrane, providing a maximized and effective signal-to-noise ratio. According to the invention, the polyacrylamide hydrochloride mediated amplification system is applied to the test strip, and naked eye semi-quantitative and colloidal gold reader quantitative detection of Escherichia coli O157: H7 in a food sample is realized based on the color intensity of the detection line.
On one hand, the invention provides a kit for detecting Escherichia coli O157: H7 based on polyacrylamide hydrochloride mediated copper growth enhancement, which comprises an immunochromatography test strip, a gold-labeled antibody, a polyacrylamide hydrochloride solution, a copper ion solution and an ascorbic acid solution; the immunochromatographic test strip comprises a bottom plate, and a sample pad, a nitrocellulose membrane and absorbent paper which are sequentially lapped and fixed on the bottom plate; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is sprayed with an anti-Escherichia coli O157H 7 capture antibody, and the quality control line is sprayed with a goat anti-mouse antibody.
Preferably, the preparation method of the gold-labeled antibody comprises the following steps: mixing an anti-Escherichia coli O157: H7 detection antibody and colloidal gold nanoparticles, adding a potassium carbonate solution, reacting under stirring, then adding a bovine serum albumin solution, continuing to react under stirring, centrifuging after the reaction is finished, and re-dissolving the precipitate in a phosphate solution to obtain the gold-labeled antibody.
Preferably, the preparation method of the gold-labeled antibody comprises the following steps: mixing 40 mu L and 1mg/mL anti-Escherichia coli O157H 7 detection antibody with 1mL and 40nm gold nanoparticle solution, adding 20 mu L and 0.2mol/L potassium carbonate solution to adjust pH, stirring and reacting for 30min under the condition of 100rpm, then adding 100 mu L10% bovine serum albumin solution, stirring and reacting for 30min under the condition of 100rpm, centrifuging for 10min under the condition of 5000g, and re-dissolving the precipitate in 100 mu L0.01 mol/L, pH 7.4.4 phosphate buffer solution to prepare the colloidal gold labeled antibody.
Preferably, the phosphate buffer solution (0.01M, pH 7.4) is prepared by adding 16g of sodium chloride, 0.2g of potassium dihydrogen phosphate and 2.9g of potassium dihydrogen phosphate to 1L of ultrapure water, and dissolving the mixture by stirring.
Preferably, the concentration of the capture antibody of the anti-Escherichia coli O157H 7 is 1mg/mL, and the concentration of the goat anti-mouse antibody is 1mg/mL; the spraying density of the detection line and the quality control line is 1 mu L/cm, and the distance is 6mm. And (5) placing the sprayed nitrocellulose membrane in an air drying oven for drying for 8h.
Preferably, the concentration of the polyacrylamide hydrochloride solution is 20mg/mL; the concentration of the copper ion solution is 10mg/mL; the concentration of the ascorbic acid solution is 50mg/mL. The molecular weight of the polyallylamine hydrochloride is 50kDa.
Preferably, the solvents used for preparing the polyacrylamide hydrochloride solution, the copper ion solution and the ascorbic acid solution are ultrapure water.
Preferably, the copper ion solution is a copper sulfate solution or a copper chloride solution.
Preferably, the immunochromatographic test strip is assembled by the following steps: firstly, sticking the nitrocellulose membrane on a bottom plate, then sticking the sample pad and the absorbent paper on the upper part and the lower part of the bottom plate, overlapping the sample pad and the absorbent paper on the nitrocellulose membrane for 1.5mm, finally cutting the assembled test paper strip into 4mm, sealing the test paper strip in a plastic bag, and storing the test paper at room temperature.
On the other hand, the invention also provides a method for detecting Escherichia coli O157H 7 based on polyacrylamide hydrochloride mediated copper growth enhancement, the detection method is to use the kit for detection, and the method specifically comprises the following steps:
(1) Mixing and incubating a gold-labeled antibody and a sample solution, adding the mixture into a sample hole of an immunochromatography test strip, and recording the reading of a first reaction of the test strip after reaction;
(2) Mixing the polyacrylamide hydrochloride solution and the copper ion solution, standing for reaction, soaking the test strip in the first round in the mixed solution, transferring the test strip into the ascorbic acid solution for reduction reaction, and recording the reading of the test strip after the second round of reaction.
Preferably, in the step (1), the amount of the gold-labeled antibody is 2 μ L, the amount of the sample solution is 70 μ L, the two are mixed and reacted for 10min, then the mixture is added into a sample hole, and after the reaction on a test strip for 15min, a colloidal gold reader is used for reading.
Preferably, in the step (2), the mixing volume ratio of the polyacrylamide hydrochloride solution and the copper ion solution is 1:1, after mixing and reacting for 10min, the first round of test strips are soaked in the mixed solution for 5min, and then the test strips are transferred to the ascorbic acid solution for reduction reaction for 10min.
The beneficial effects of the invention are as follows:
(1) The polyacrylamide hydrochloride mediated signal amplification system obtained by the invention is suitable for detecting pathogenic bacteria in food samples, and has the advantages of simple method, low cost, high stability and high sensitivity.
(2) According to the invention, after a capture antibody-antigen-antibody-colloidal gold nanoparticle compound is formed on a detection line by using a traditional sample adding process of an immunochromatographic test strip, copper ions can be directly adsorbed on the surface of gold nanoparticles coated with polyacrylamide hydrochloride, and then a stable metal copper shell layer is formed on the gold nanoparticles originally combined on the detection line in the presence of an ascorbic acid solution serving as a reducing agent.
(3) Based on good physicochemical and biological properties of the polyacrylamide hydrochloride, an analysis method combining a polyacrylamide hydrochloride-based high-efficiency signal amplification system and a colloidal gold immunochromatography test strip platform is designed by utilizing the adhesion capability and the chelating effect of the polyacrylamide hydrochloride, the method improves the detection stability by eliminating the non-specificity between a nitrocellulose membrane and copper ions, provides a maximized and effective signal-to-noise ratio, and realizes the high-sensitivity detection of Escherichia coli O157: H7.
Drawings
FIG. 1 is a schematic diagram of the method of the present invention.
FIG. 2 shows the results of naked eye detection of the presence or absence of PAH hydrochloride-mediated signal amplification in the enhancement system at colloidal gold concentrations of 0, 8, and 150 pM.
FIG. 3 is a graph showing the amplification effect of a polyacrylamide hydrochloride based signal amplification system on different concentrations of colloidal gold on nitrocellulose membranes.
FIG. 4 is a calibration curve for detecting Escherichia coli O157: H7 with different concentrations by processing a traditional colloidal gold immunochromatographic test strip based on a polyacrylamide hydrochloride signal amplification system.
FIG. 5 is a detection object diagram for detecting Escherichia coli O157H 7 with different concentrations by processing a traditional colloidal gold immunochromatographic test strip based on a polyacrylamide hydrochloride signal amplification system.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The principle schematic diagram of the method for detecting Escherichia coli O157: H7 based on polyacrylamide hydrochloride mediated copper growth enhancement is shown in figure 1.
Example 1: preparation of colloidal gold-labeled antibody
Mixing 40 μ L of anti-Escherichia coli O157: H7 detection antibody (1 mg/mL) and 1mL of colloidal gold nanoparticle (40 nm) solution, adding 20 μ L of 0.2mol/L potassium carbonate solution to adjust pH, stirring at 100rpm for reaction for 30min, adding 100 μ L of 10% bovine serum albumin solution, stirring at 100rpm for reaction for 30min, centrifuging at 5000g for 10min, and re-dissolving the precipitate in 100 μ L of phosphate buffer solution (0.01M, pH 7.4) to obtain colloidal gold labeled antibody.
Example 2: exploration on amplification stability of colloidal gold signals with different concentrations
A colloidal gold solution was sprayed onto a nitrocellulose membrane at a concentration of 0 to 200pmol/L using a dot-film applicator, and dried overnight at 37 ℃. And recording the reading value of the test strip detection line by using a colloidal gold reader. Mixing 20mg/mL polyacrylamide hydrochloride solution and 10mg/mL copper ion solution according to a volume ratio of 1:1, standing for reaction for 10min, soaking the test strip in the mixed solution for 5min, transferring the test strip into 50mg/mL sodium ascorbate solution, soaking for 10min for reduction reaction, and recording the reading after the second round of reaction. Each concentration was determined in five replicates on the same day.
FIG. 2 shows the results of naked eye detection of the presence or absence of polyacrylamide hydrochloride-mediated signal amplification in the enhancement system at colloidal gold concentrations of 0, 8 and 150 pM. The test paper strip of number 1 is sprayed with colloidal gold of different concentrations and is not subjected to signal amplification treatment (i.e., is not subjected to polyacrylamide hydrochloride solution, copper ion solution or sodium ascorbate solution treatment). The test paper strip numbered 2 is sprayed with colloidal gold of different concentrations and is treated with a copper ion solution and a sodium ascorbate solution. The test paper strip of number 3 is sprayed with colloidal gold with different concentrations and is treated by polyacrylamide hydrochloride solution, copper ion solution and sodium ascorbate solution. The results show no significant signal enhancement in the absence of polyallylamine hydrochloride and a clear yellow background, and it can be seen that there is no mediated amplification of polyallylamine hydrochloride at different concentrations of colloidal gold, with a significantly different background (test strip No. 2). The experimental result well indicates the necessity of the existence of the polyacrylamide hydrochloride as an intermediate connector, and can effectively reduce the adsorption of metal ions on the nitrocellulose membrane. In contrast, the polyallylamine hydrochloride-mediated copper enhancement was good, still remained a high dark red color, and the background color was unchanged, further indicating that the polyallylamine hydrochloride-mediated system had excellent signal enhancement (test strip No. 3).
The data analysis of FIG. 3 shows that the color development signal is enhanced with the increase of the concentration of the colloidal gold, and the signal difference and the concentration before and after enhancement have a certain linear trend, which indicates that the enhanced color development intensity is restricted by the in-situ growth of the gold core. Before amplification, the concentration of the colloidal gold needs to reach 50pmol/L to have an obvious signal, and the concentration of the colloidal gold only needs to be 1.5625pmol/L after the treatment of a polyacrylamide hydrochloride system, thereby illustrating the superiority of the colloidal gold treated by the polyacrylamide hydrochloride in the aspect of color development. Moreover, the relative standard deviation (CV) of the color intensity after the polyacrylamide hydrochloride treatment was in the range of 2.31% to 8.36% for samples having the same sample concentration. These results indicate that the model has high reproducibility and signal accuracy.
Example 3: preparation of Escherichia coli O157H 7 double-antibody sandwich immunochromatographic test strip
Escherichia coli O157: H7 capture antibody (1 mg/mL) and goat anti-mouse antibody (1 mg/mL) were sprayed onto nitrocellulose membranes using a spotting apparatus, respectively, as test strip detection lines and control lines, and dried overnight at 37 ℃. The two lines were sprayed at a density of 1. Mu.L/cm and a distance of 6mm. The test strip assembly sequence is as follows: firstly, sticking the nitrocellulose membrane on a bottom plate, then sticking the sample pad and the absorbent paper on the upper part and the lower part of the bottom plate, overlapping the sample pad and the absorbent paper on the nitrocellulose membrane by about 1.5mm, finally cutting the assembled test paper strip into 4mm, sealing the test paper strip in a plastic bag, and storing the test paper at room temperature.
Example 4: escherichia coli O157H 7 double-antibody sandwich immunochromatographic test strip with colloidal gold labeled antibody as signal probe
When the traditional immunochromatographic test strip is used for detecting residues of Escherichia coli O157H 7 in milk, the method is implemented by the following steps: sample pretreatment, detection by using an immunochromatographic test strip based on colloidal gold, and analysis of results.
1) Sample pretreatment
The pretreatment of the sample is carried out according to national standards.
2) Detection of Escherichia coli O157H 7 residue in milk by using colloidal gold-based immunochromatographic test strip
Firstly, mixing and incubating Escherichia coli O157: H7 standard substance or sample solution (70 mu L) diluted to different concentrations and a colloidal gold probe (2 mu L) for 10min, adding the mixture into a test strip sample hole, measuring three concentrations in parallel, reacting on a test strip for 15min, and recording the test strip detection line reading value by using a colloidal gold reader. Mixing polyacrylamide hydrochloride solution (20 mg/mL) and copper ion solution (10 mg/mL) according to a volume ratio of 1:1, standing for reaction for 10min, soaking the test strip in the mixed solution for 5min, transferring the test strip into sodium ascorbate solution (50 mg/mL), soaking for 10min for reduction reaction, and recording the reading of the test strip after the second round of reaction. And (3) plotting the signal value of the detection line as a vertical coordinate and the concentration of the Escherichia coli O157H 7 as a horizontal coordinate, drawing a standard curve, calculating and comparing the sensitivity of the test strip for detecting the Escherichia coli O157H 7 and determining a linear quantitative range.
3) Analysis results
The standard curve was plotted with the concentration of E.coli O157H 7 standard or sample solution as the X-axis and the average value of absorbance obtained for each concentration of the standard or sample solution as the Y-axis, as shown in FIGS. 4A and 4B. The picture of the detected object is shown in figure 5.
As a result, as shown in FIGS. 4A and 4B, it can be seen that the concentration of E.coli O157: H7 and the absorbance showed a good exponential relationship. The traditional immunochromatography test strip using colloidal gold as a probe is 5 multiplied by 10 3 CFU/mL-1×10 5 The detection limit is 3.5 multiplied by 10 and is linearly related under the concentration of CFU/mL 3 CFU/mL. After the polyacrylamide hydrochloride mediated system is used for enhancement, more complex is formed on a detection line, and the detection line has darker brown color. Standard curve analysis based on signal intensity showed that the lowest detection limit of this system was 9.8CFU/mL. In contrast, the LOD detected by naked eyes of the conventional colloidal gold immunochromatographic test strip in FIG. 5 is 2.5X 10 3 CFU/mL, 100 times (2.5X 10) that of a PAH hydrochloride-based mediated signal enhancement system 1 CFU/mL)。
Example 5: detection experiment procedure of actual sample
The accuracy of the test strip for enhancing the immunochromatography based on the polyacrylamide hydrochloride mediated copper growth is evaluated by adopting an Escherichia coli O157H 7 standard addition recovery experiment. Adding 5 × 10 final concentration into milk sample 3 ，1×10 3 ，5×10 2 ，1×10 2 ，5×10 1 CFU/mL of E.coli O157H 7 standard, passed through the front of the sampleAfter the test, the sample extract is detected by using the double-antibody sandwich immunochromatographic test strip method provided by the research. Each concentration was measured three times in the same day, every three days, and three batches were repeated to obtain the spiked recovery rate for each concentration and the batch-to-batch variation.
The results are shown in Table 1. The recovery rate (recovery) of Escherichia coli O157: H7 in the milk sample is between 84.92% -120.32%, and the coefficient of variation is less than 13%, which indicates that the method established by the research has good accuracy and precision and can meet the practical application.
TABLE 1 detection of recovery of E.coli O157H 7 in milk based on polyacrylamide hydrochloride mediated copper growth enhancement
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (6)
1. A kit for detecting Escherichia coli O157H 7 based on polyacrylamide hydrochloride mediated copper growth enhancement is characterized by comprising an immunochromatography test strip, a gold-labeled antibody, polyacrylamide hydrochloride solution, copper ion solution and ascorbic acid solution; the immunochromatographic test strip comprises a bottom plate, and a sample pad, a nitrocellulose membrane and absorbent paper which are sequentially lapped and fixed on the bottom plate; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is sprayed with an anti-Escherichia coli O157: H7 capture antibody, and the quality control line is sprayed with a goat anti-mouse antibody;
the preparation steps of the gold-labeled antibody are as follows: mixing 40 mu L of 1mg/mL anti-Escherichia coli O157H 7 detection antibody with 1mL of 40nm gold nanoparticle solution, adding 20 mu L of 0.2mol/L potassium carbonate solution to adjust pH, stirring and reacting for 30min under the condition of 100rpm, then adding 100 mu L of 10% bovine serum albumin solution, stirring and reacting for 30min under the condition of 100rpm, centrifuging for 10min at 5000g, and re-dissolving the precipitate in 100 mu L of 0.01mol/L, pH 7.4.4 phosphate buffer solution to prepare colloidal gold labeled antibody;
the concentration of the anti-Escherichia coli O157H 7 capture antibody is 1mg/mL, and the concentration of the goat anti-mouse antibody is 1mg/mL; the spraying density of the detection line and the quality control line is 1 mu L/cm, and the distance is 6mm;
the concentration of the polyacrylamide hydrochloride solution is 20mg/mL; the concentration of the copper ion solution is 10mg/mL; the concentration of the ascorbic acid solution is 50mg/mL.
2. The kit for detecting Escherichia coli O157H 7 based on polyacrylamide hydrochloride-mediated copper growth enhancement as claimed in claim 1, wherein the copper ion solution is copper sulfate solution or copper chloride solution.
3. The kit for detecting Escherichia coli O157H 7 based on polyacrylamide hydrochloride-mediated copper growth enhancement according to claim 1, wherein the immunochromatographic test strip is assembled by the following steps: sticking the nitrocellulose membrane on a bottom plate, sticking the sample pad and the absorbent paper on the upper part and the lower part of the bottom plate, overlapping the sample pad and the absorbent paper on the nitrocellulose membrane by 1.5mm, and finally cutting the assembled test paper strip into 4 mm.
4. A method for detecting Escherichia coli O157H 7 based on polyacrylamide hydrochloride mediated copper growth enhancement, which is characterized in that the kit of claim 1 is used for detection, and the method comprises the following steps:
(1) Mixing and incubating a gold-labeled antibody and a sample solution, adding the mixture into a sample hole of an immunochromatography test strip, and recording the reading of a first reaction of the test strip after reaction;
(2) Mixing the polyacrylamide hydrochloride solution and the copper ion solution, standing for reaction, soaking the test strip in the first round in the mixed solution, transferring the test strip into the ascorbic acid solution for reduction reaction, and recording the reading of the test strip after the second round of reaction.
5. The method for detecting Escherichia coli O157: H7 based on the polyacrylamide hydrochloride-mediated copper growth enhancement is characterized in that in the step (1), the amount of the gold-labeled antibody is 2 mu L, the amount of the sample solution is 70 mu L, the gold-labeled antibody and the sample solution are mixed and react for 10min, then the mixture is added into a sample hole, and after the mixture reacts on a test strip for 15min, a colloidal gold reader is used for reading.
6. The method for detecting the Escherichia coli O157: H7 based on the polyacrylamide hydrochloride-mediated copper growth enhancement is characterized in that in the step (2), the mixing volume ratio of the polyacrylamide hydrochloride solution to the copper ion solution is 1:1, after the mixing reaction is carried out for 10min, the first round of test strips are soaked in the mixed solution for 5min, and then the test strips are transferred to an ascorbic acid solution for carrying out the reduction reaction for 10min.
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