CN110133262B - Bacterium detection kit - Google Patents
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- CN110133262B CN110133262B CN201910337253.0A CN201910337253A CN110133262B CN 110133262 B CN110133262 B CN 110133262B CN 201910337253 A CN201910337253 A CN 201910337253A CN 110133262 B CN110133262 B CN 110133262B
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- 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
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
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- 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
- G01N33/56938—Staphylococcus
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Abstract
The invention discloses a double-labeling gold nanorod probe, which is formed by dropwise adding urease solution into a gold nanorod suspension, carrying out mixed spinning, and dropwise adding anti-staphylococcus aureus antibody solution for mixed spinning; centrifuging and discarding the supernatant; the double-labeled probe converts the number signal of SA into the change of the pH value of the solution through the catalysis of urease and the specific recognition of IgY; meanwhile, the staphylococcus aureus detection kit is provided, and comprises: the kit comprises a double-labeled gold nanorod probe, non-specific magnetic beads, saturated urea and phenolphthalein test paper; the double-labeled gold nanorod probe is labeled by urease and an anti-staphylococcus aureus antibody; quickly detecting the result by phenolphthalein test paper, wherein the color is changed from white to carmine; the invention has the advantages that: the kit is simple and convenient to operate; the detection result can be interpreted by naked eyes under natural light without any instrument; and can be compared with a standard colorimetric card to obtain a semi-quantitative detection result; the detection is rapid and can be completed within 20 minutes.
Description
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to a bacteria detection kit.
Background
Staphylococcus aureus (1)Staphylococcus aureusSA) is an important zoonosis pathogen, one of the five major pathogens associated with food-borne diseases, and can cause multiple serious infections. The staphylococcus aureus can tolerate a wide range of pH and temperature and humidity, is widely distributed in nature, has a plurality of food contamination opportunities, and is one of the main pathogenic bacteria causing food poisoning. With the widespread use of antibiotics, a number of drug-resistant staphylococcus aureus bacteria have emerged. Diseases caused by staphylococcus aureus become a global public health problem and seriously harm human safety and health. Studies have shown that 30% to 50% of the general population are carriers of SA. Thus, during preparation and transport of the food product, the productAre easily contaminated. Even in the safest food supply united states, 24 million people are reported to be infected with SA each year, resulting in medical costs of $ 167,597,860/year. Meanwhile, staphylococcus aureus is also an important pathogenic bacterium causing mastitis of dairy cows, and causes huge economic loss to the breeding industry. At present, the traditional microbial culture, biochemical identification and the like are mainly used as detection methods for staphylococcus aureus including drug-resistant staphylococcus aureus in China, and an accurate, rapid and sensitive detection method is needed to be established.
The traditional culture method is the most common SA detection method with the most mature technology. However, its process is time consuming and not suitable for handling emergency food safety events. In order to speed up the detection, researchers have developed many alternatives, such as the use of nucleic acid amplification techniques or advanced equipment, with detection times that can be reduced from 2-4 days to several hours, or even tens of minutes. But the reliance on professionals and sophisticated instrumentation of these techniques still limits their widespread use. In addition, complex material processing greatly increases the overall detection cost.
The advent of nanotechnology has made it possible to overcome the above obstacles. In recent years, simpler handling and lower costs with nanoparticles have been the focus of attention. In 2016, Bhaisar et al developed a non-specific magnetic bead (nMB) using carbon-modified iron oxide nanoparticles (Fe)3O4NP). nMB can be used to effectively and non-specifically enrich various gram-positive or gram-negative bacteria due to the carbon dots attached to the surface of the magnetic nanoparticles. Compared with the traditional immunomagnetic separation probe, nMB can be directly used after synthesis without labeling or modifying an antibody. This means that the processing cost and the inspection cost are greatly reduced. Of course, nMB is a convenient, economical and powerful tool for enrichment and detection of bacteria in complex matrices.
The gold nanoparticles (AuNPs) are gold microparticles, have the diameter of 1-100 nm, have high electron density, dielectric property and catalytic action, can be combined with various biological macromolecules, and do not influence the biological activity of the gold nanoparticles. In the biosensor aspect, nanogold is mainly designed as an immunosensor because it can be easily synthesized and functionalized, and is designed by using the change of a detection signal due to antigen-antibody specific binding. In order to enable the nanoparticles to recognize not only the target but also catalyze the substrate, various double-labeled AuNP probes have been developed. Among them, most researchers select gold nanospheres (AuNS) as a carrier. However, it is found that under the same conditions, the protein adsorbed on the surface of the gold nanorod (AuNR) is obviously more than that adsorbed on the surface of the gold nanosphere (AuNS). Similar results were obtained with Hinterwirth et al and Joshi et al. Therefore, we can reasonably conclude that AuNR can bind more antibodies and enzymes simultaneously, and that the performance of the double-labeled AuNR probe is superior to that of the double-labeled AuNS probe. However, to our knowledge, double-labeled AuNR probes have not been applied to the detection of SA at present.
Disclosure of Invention
The invention aims to provide a convenient and economic bacteria detection kit for solving the problems of long time consumption and high cost of the existing bacteria detection method.
The double-labeled gold nanorod probe is prepared by the following method:
1) adding urease solution into the suspension of the gold nanorods drop by drop, and carrying out mixed spinning incubation;
2) dropwise adding an antibacterial antibody solution, and performing mixed spinning incubation;
3) centrifuging and discarding the supernatant to obtain the double-labeled gold nanorod probe;
the step 1) is that 120 muL of urease solution with the concentration of 8-12mg/mL is dropwise added into 1200 muL of gold nanorod suspension, and slowly mixed and rotated for 5 minutes;
step 2) adding 120 mul of antibacterial antibody solution with the concentration of 0.25-1mg/mL drop by drop, and slowly carrying out mixed spinning for 25 minutes;
the gold nanorods are prepared by adopting a seed growth method;
the antibacterial antibody is an antibacterial yolk antibody (IgY). The bacteria are staphylococcus aureus.
A bacteria detection kit comprising: the kit comprises a double-labeled gold nanorod probe, non-specific magnetic beads, saturated urea and phenolphthalein test paper; the double-labeled gold nanorod probe is labeled by urease and an antibacterial antibody;
the concentration of the double-labeled gold nanorod probe is as follows: 0.5-1.5 mg/mL;
the non-specific magnetic bead is a suspension containing the non-specific magnetic bead, and the concentration of the suspension is 0.5-2 mg/mL;
the bacteria are staphylococcus aureus;
the double-labeled gold nanorod probe is prepared by the method.
The invention provides a double-labeled gold nanorod probe, which is prepared by the following method: 1) adding urease solution into the suspension of the gold nanorods drop by drop, and carrying out mixed spinning incubation; 2) dropwise adding an anti-staphylococcus aureus antibody solution for mixed spinning incubation; 3) centrifuging and discarding the supernatant to obtain the double-labeled gold nanorod probe; the double-labeled probe converts the number signal of SA into the change of the pH value of the solution through the catalysis of urease and the specific recognition of IgY; meanwhile, the staphylococcus aureus detection kit is provided, and comprises: the kit comprises a double-labeled gold nanorod probe, non-specific magnetic beads, saturated urea and phenolphthalein test paper; the double-labeled gold nanorod probe is labeled by urease and an anti-staphylococcus aureus antibody; quickly visualizing the detection result by using a phenolphthalein test paper, wherein the color is changed from white to magenta; the invention has the advantages that: (1) the kit is simple and convenient to operate, and is suitable for basic food supervision departments lacking professional technicians; (2) the detection result can be interpreted by naked eyes under natural light without any instrument; and can be compared with a standard colorimetric card to obtain a semi-quantitative detection result; (3) the detection is rapid and can be completed within 20 minutes.
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FIG. 1 is a transmission electron micrograph of gold nanorods;
FIG. 2 is a transmission electron micrograph of a non-specific magnetic bead;
FIG. 3 is a flow chart of detection.
Detailed Description
EXAMPLE 1 preparation of gold nanorods
The gold nanorods are prepared by adopting a seed growth method, and the method comprises the following specific steps:
1) preparing a seed solution: adding 96 mul of 25mM tetrachloroaurate trihydrate aqueous solution to 7.5mL of 0.1M hexadecyltrimethylammonium bromide aqueous solution; then adding 0.5-2mL of 0.02M aqueous solution of sodium borohydride; stirring for 1-3 hours at 30 ℃ after the mixed solution turns to dark brown, wherein the obtained solution is the seed solution;
2) preparing a growth solution: fully mixing 100mL of hexadecyl trimethyl ammonium bromide aqueous solution with the concentration of 0.1M, 2mL of sulfuric acid with the concentration of 0.5M, 1.96mL of tetrachloroauric acid trihydrate aqueous solution with the concentration of 25mM, 0.5-2mL of silver nitrate aqueous solution with the concentration of 0.01M and 0.5-1mL of ascorbic acid aqueous solution with the concentration of 0.1M, and obtaining mixed solution, namely growth solution;
3) adding 0.1-1mL of the seed solution into 50-100mL of the growth solution, and fully mixing; standing at 30 deg.C for 10-15 hr; centrifuging at 9000 rpm for 15 min, and concentrating to 20 mL; regulating the pH value of the solution to 6.0-8.0 by using a potassium carbonate aqueous solution with the concentration of 0.2M to obtain a suspension containing gold nanorods for later use;
the transmission electron microscope image of the gold nanorods is shown in figure 1; the length of the gold nanorod is about 40-50nm, and the diameter of the gold nanorod is about 10-15 nm.
Example 2 preparation of double-labeled gold nanorod probes
Dropwise adding 120 mu L of urease solution with the concentration of 8-12mg/mL into 1200 mu L of the gold nanorod-containing suspension in the embodiment 1, and slowly carrying out mixed spinning for 5 minutes; subsequently, 120 mul of anti-staphylococcus aureus yolk antibody solution with the concentration of 0.25-1mg/mL is added drop by drop, and the slow rotation is carried out for 25 minutes; centrifuging at 8000 rpm for 10 minutes, discarding the supernatant, and obtaining a precipitate part which is the double-labeled gold nanorod probe;
resuspending with 300-plus 500 mul of ultrapure water to obtain suspension containing the double-labeled gold nanorod probe; the absorption spectrum of the double-labeled gold nanorod probe is obviously red-shifted compared with that of the unmarked gold nanorod by using a visible light-ultraviolet spectrophotometer for measurement, and the double-labeled gold nanorod probe is proved to be successfully prepared.
EXAMPLE 3 preparation of non-specific magnetic beads
The nonspecific magnetic bead is Fe with carbon dots wrapped on the surface3O4The nano particles are synthesized by a two-step method;
the method comprises the following steps: preparation of Fe3O4Nanoparticle core: 0.5-1g of ferrous chloride tetrahydrate and 1-2g of ferric chloride hexahydrate are fully dissolved in 30mL of ultrapure water; 35mL of 28% (w/v) ammonia water was added dropwise; stirring for 5 hours at 80 ℃ under the protection of nitrogen; separating black solid matter from the reaction solution with permanent magnet, and washing with ultrapure water to obtain Fe3O4Drying the nanoparticle core for later use;
step two: in Fe3O4Modifying carbon points on the surface of the nanoparticle core: adding 0.05-0.2g of the above Fe3O4Mixing the nanoparticle core and 0.25-1g chitosan in 30mL4% (w/v) glacial acetic acid; transferring the obtained liquid into a reaction kettle, and reacting for 6-24 hours at 180 ℃; separating black solid matters from the reaction solution by using a permanent magnet, cleaning the black solid matters by using ultrapure water to obtain nonspecific magnetic beads, and airing the nonspecific magnetic beads for later use;
FIG. 2 shows a transmission electron micrograph of the nonspecific magnetic beads; the diameter of the nonspecific magnetic bead is about 15-20 nm.
EXAMPLE 4 preparation of phenolphthalein test paper
Immersing Fusion 5 filter paper in 1% (w/v) phenolphthalein solution, air-drying, and cutting into circular paper sheets with diameter of 5mm for use.
EXAMPLE 5 detection of Staphylococcus aureus
The detection flow chart is shown in FIG. 3; mixing 200 mu L of sample liquid to be detected, 200 mu L of suspension containing the double-labeled gold nanorod probe and 200 mu L of suspension (resuspended by ultrapure water) containing non-specific magnetic beads with the concentration of 0.5-2mg/mL, and reacting for 10 minutes at room temperature; performing magnetic separation, and taking 200 muL of supernatant; adding 200 mu L of saturated urea into the obtained supernatant, and reacting for 5 minutes; and immersing the phenolphthalein test paper into the reacted solution, observing the color change of the phenolphthalein test paper, and judging the result.
The method has the advantages of stable detection, short detection time (as low as 1000 CFU/mL), detection completion within 20 minutes, and direct visual interpretation of detection results under natural light. And comparing the result color with a standard color comparison card to obtain a semi-quantitative result. For staphylococcus aureus, escherichia coli O157: h7, Listeria monocytogenes, Salmonella typhimurium, Vibrio parahaemolyticus and other bacteria are detected, only the detection result of staphylococcus aureus is positive, and the rest are negative. As can be seen, the method has strong specificity, and no false positive or false negative results are seen, and the results are shown in tables 1 and 2. All experiments were repeated three times with consistent results.
Example 6 detection of food contamination simulation sample (beef)
Weighing 5g of beef, grinding the beef into minced meat, flatly paving the minced meat on a plane, and irradiating the minced meat for 2 hours under an ultraviolet lamp to remove pathogenic bacteria naturally accumulated in the minced meat; taking 1g of sterilized minced meat, adding 10mL of sterilized normal saline, and fully and uniformly mixing to prepare a food sample matrix; inoculating staphylococcus aureus with different concentrations to the food matrix, and detecting the staphylococcus aureus with the detection kit; the results are shown in Table 3. All experiments were repeated three times with consistent results.
Example 7 detection of food contamination simulation sample (cabbage)
Weighing 5g of Chinese cabbage, grinding into minced cabbage, spreading on a plane, and irradiating under an ultraviolet lamp for 2 hours to remove pathogenic bacteria naturally accumulated in the cabbage; taking 1g of sterilized minced cabbage, adding 10mL of sterilized normal saline, and fully and uniformly mixing to prepare a food sample matrix; inoculating staphylococcus aureus with different concentrations to the food matrix, and detecting the staphylococcus aureus with the detection kit; the detection results are shown in table 4; all experiments were repeated three times with consistent results.
Claims (8)
1. A bacteria detection kit comprising: the kit comprises a double-labeled gold nanorod probe, non-specific magnetic beads, saturated urea and phenolphthalein test paper;
the double-labeled gold nanorod probe is prepared by the following method:
1) adding urease solution into the suspension of the gold nanorods drop by drop, and carrying out mixed spinning incubation;
2) dropwise adding an antibacterial antibody solution, and performing mixed spinning incubation;
3) centrifuging and discarding the supernatant to obtain the double-labeled gold nanorod probe.
2. The bacteria detection kit according to claim 1, wherein: the antibacterial antibody is an antibacterial yolk antibody.
3. The bacteria detection kit according to claim 2, wherein: the bacteria are staphylococcus aureus.
4. A bacteria detection kit according to claim 1, 2 or 3 wherein: the step 1) is that 120 muL of urease solution with the concentration of 8-12mg/mL is added into 1200 muL of gold nanorod suspension drop by drop, and slowly mixed and rotated for 5 minutes.
5. The bacteria detection kit according to claim 4, wherein: and in the step 2), 120 mul of anti-staphylococcus aureus antibody solution with the concentration of 0.25-1mg/mL is added dropwise and slowly mixed for 25 minutes.
6. The bacteria detection kit according to claim 5, wherein: the gold nanorods are prepared by adopting a seed growth method.
7. The bacteria detection kit according to claim 6, wherein: the concentration of the double-labeled gold nanorod probe is as follows: 0.5-1.5 mg/mL.
8. The bacteria detection kit according to claim 7, wherein: the non-specific magnetic bead is a suspension containing the non-specific magnetic bead with the concentration of 0.5-2 mg/mL.
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| CN111323582A (en) * | 2020-03-16 | 2020-06-23 | 西南大学 | ELISA (enzyme-Linked immunosorbent assay) detection kit for staphylococcus aureus SEO and detection method thereof |
| CN111366727B (en) * | 2020-03-19 | 2021-05-25 | 吉林大学 | Kit for detecting salmonella typhimurium and preparation method thereof |
| CN112649602A (en) * | 2020-06-17 | 2021-04-13 | 吉林大学 | Visual kit for detecting staphylococcus aureus based on immunomagnetic beads |
| CN113281507B (en) * | 2021-05-23 | 2022-08-16 | 吉林大学 | Rapid detection method and kit for staphylococcus aureus |
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