CN110658338A - Portable mastitis pathogen MRSA detection method in lactation period - Google Patents
Portable mastitis pathogen MRSA detection method in lactation period Download PDFInfo
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Abstract
The invention discloses a portable method for detecting mastitis pathogenic bacteria MRSA in lactation period. The MRSA is rapidly detected by the combined use of a nano immunomagnetic bead technology and a colloidal gold immunochromatography technology, and the method comprises the following specific steps: the nano magnetic beads are coupled with monoclonal antibodies obtained by a hybridoma technology to form nano immunomagnetic beads; using the other matched monoclonal antibody marked by the colloidal gold as a marked antibody, and respectively using the polyclonal serum and the goat anti-mouse IgG as a detection line T and a quality control line C; the colloidal gold immunochromatographic test paper consists of a binding pad containing a colloidal gold labeled antibody, a chromatographic membrane containing a detection line T and a quality control line C and a water absorption pad; when a sample is detected, the immunomagnetic beads and thalli in the sample are subjected to specific capture enrichment, then the enrichment liquid is added into lysis solution for lysis, then the lysis solution is dropped on a sample pad of the test paper, and interpretation of macroscopic visible color development strips is formed at a detection line T and a quality control line C according to the color of colloidal gold, so that rapid semi-quantitative detection of MRSA is realized.
Description
Technical Field
The invention belongs to the technical field of medical detection, and particularly relates to a portable method for detecting mastitis pathogenic bacteria MRSA in lactation.
Background
MRSA is a stubborn pathogenic bacterium of mastitis in lactation, and the product is developed for early diagnosis and treatment, improving the cure rate of diseases and reducing the damage to breasts in the treatment process. At present, no portable technical product for rapidly detecting MRSA in milk exists clinically.
The immunomagnetic bead enrichment technology takes magnetic microspheres as a solid phase surface, and combines an immunological method to establish a sample concentration method with important application prospect. The magnetic beads are immunomagnetic beads formed by combining antibodies, can be specifically combined with corresponding antigens in a liquid phase, and quickly concentrate a required sample volume by the action of a magnetic field. The immunomagnetic beads can enrich various types of antigens such as nucleic acid with extremely small molecular weight, small molecular toxin, specific protein, cells or pathogenic microorganisms in a short time according to different antibodies coupled to the surfaces of the magnetic beads.
1. Structure of magnetic bead
Structurally, the magnetic microsphere is divided into three parts, wherein a core part is composed of magnetic substances, such as gamma Fe2O3, Fe3O4 and MeFe2O3, so that the magnetic microsphere can be rapidly aggregated under the action of a magnetic field; the outer layer is wrapped by high polymer materials such as polystyrene, polyethyleneimine or polyacrylic acid, so that good magnetic sealing performance is ensured, and the magnetic leakage phenomenon is not easy to occur; the surface of the microsphere is also covered with special activated groups, common chemical groups comprise carboxyl, amino, sulfydryl, tosylate, epoxy and the like, and the groups can be covalently bonded with amino or carboxyl groups on antibody protein.
2. Preparation of magnetic beads
In medical diagnosis and treatment, the nanometer-scale immunomagnetic beads are selected, because the nanometer-scale immunomagnetic beads have larger surface area per unit weight, better dispersibility and extremely quick response to a magnetic field.
The antibody used for preparing the immunomagnetic beads can be a monoclonal antibody or a polyclonal antibody, and the monoclonal antibody has good specificity and is beneficial to being directly used for detecting or sorting cells after enrichment; the affinity of polyclonal antibody is usually higher than that of monoclonal antibody, and it is easy to capture antigen, but because of more epitopes, it is more complex than monoclonal antibody for treatment after enriching antigen.
The system of the buffer solution is mainly selected according to the characteristics of the surface active groups of the magnetic beads, the buffer system with lower ion concentration is generally selected, and 0.5 percent of Tween-20 is added to reduce the aggregation phenomenon of the magnetic beads; the pH of the buffer is usually between 5 and 9.5, and peracid or overbase can affect the activity of the antibody, and is not recommended. Reaction temperature, time and environment are often considered relevant factors. The lower the reaction temperature, the longer the coupling time, and the common reaction conditions are 4 ℃ overnight, room temperature for 3 hours or 37 ℃ for 1 hour.
More sites which are not combined with the antibody still exist on the surface of the prepared immunomagnetic beads, so that small molecules which do not react with the antigen are required to block surface active sites so as to reduce nonspecific adsorption in the enrichment process. Commonly used blocking agents are BSA, Tween-20, gelatin and glycine.
3. Advantages of the invention
The operation steps are simple and time-saving: the magnetic bead enrichment process is high-fidelity (the immunomagnetic bead enrichment method for concentrating samples through the action of a magnetic field can enable the required substances to be highly concentrated, and the biological activity of the substances is not damaged in the enrichment process); high-specificity enrichment; the detection limit is favorably improved (the concentration of a target detection object can be greatly improved in the upstream sample pretreatment step by the immunomagnetic bead enrichment technology, so that the detection limit of the whole detection method is indirectly reduced).
4. Deficiency of
The reduction of the activity, affinity and titer of the antibody after the coupling of the magnetic beads often influences the effect of the application of the downstream technology, and becomes a key factor for restricting the application of the enrichment technology. Therefore, optimizing the preparation method of immunomagnetic beads and ensuring that the antibody keeps good activity and affinity after coupling is an important link for applying the enrichment technology.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a portable method for detecting mastitis pathogenic bacteria MRSA in lactation period, which selects specific antibodies to prepare magnetic beads by improving enrichment conditions and improving antibody activity and taking PB2a (beta lactamase generated by MRSA) and QacA (multidrug efflux protein of MRSA) as antigens to detect the MRSA in milk quickly and conveniently with high specificity.
In order to achieve the purpose, the invention provides a portable method for detecting pathogenic bacteria MRSA of mastitis in lactation period, which is characterized by comprising the following steps: MRSA is rapidly detected by the combined use of an immune nano magnetic bead technology and a colloidal gold immunochromatography technology, and a monoclonal antibody obtained by a hybridoma technology is coupled to the nano magnetic bead to form nano immune magnetic bead; the other paired monoclonal antibody marked by the colloidal gold is used as a marked antibody, and the polyclonal serum and the goat anti-mouse IgG are respectively used as a detection line T and a quality control line C, and the method specifically comprises the following steps:
the method is characterized in that a portable mastitis pathogenic bacterium MRSA detection kit in the lactation period is adopted for detection, and the portable mastitis pathogenic bacterium MRSA detection kit in the lactation period comprises nanometer immunomagnetic beads, strong magnets, staphylococcus aureus lysate and colloidal gold immunochromatography test paper; the colloidal gold immunochromatographic test paper comprises a binding pad of a colloidal gold labeled antibody, a chromatographic membrane containing a detection line T and a quality control line C and a water absorption pad, when a sample is detected, specific capture and enrichment are carried out on nano immunomagnetic beads and thalli in the sample, then an enrichment liquid is added into a lysis solution for lysis, then the lysis solution is dropped on the sample pad of the colloidal gold immunochromatographic test paper, and interpretation of a macroscopic chromogenic strip is formed at the detection line T and the quality control line C according to the color of colloidal gold, so that rapid semi-quantitative detection of MRSA is realized.
As a preferred scheme, the volume ratio of the sample to be detected to the nano magnetic beads is 20:1, 10:1, 5:1 or 2: 1; the method for capturing and enriching the thallus by the nano immunomagnetic beads comprises the following steps: after fully shaking and uniformly mixing the sample and the self-made immunomagnetic beads, standing for reaction for 30-60min, then carrying out magnetic field separation, and washing for 3 times by PBS (phosphate buffer solution) to obtain a thallus enrichment solution;
the volume ratio of the bacterial lysate to the sample to be detected is 1:20, 1:10 or 1: 1; and dripping the obtained lysate into a detection area, and reading T, C line color development after 5-15min to obtain a result.
Further, the portable MRSA detection kit for mastitis pathogenic bacteria in lactation period is adopted for detection, and the method comprises the following steps:
(2.1) adsorbing a sample to be detected with nano immunomagnetic beads: adding the nanometer immunomagnetic beads and the sample to be detected into a container, uniformly mixing, and reacting at 25-40 ℃ for 30-60 min;
(2.2) attracting and gathering the nano magnetic beads by a magnet: placing the container in the previous step on a strong magnet, standing for 3min, and pouring out the liquid;
(2.3) lysis of bacteria: adding 50ul/ml of staphylococcus aureus lysate into the container, and continuously reacting for 30-60min at 25-40 ℃;
(2.4) color development: pouring the lysate onto a sample pad of colloidal gold immunochromatographic test paper, carrying out chromatography for 10-15min to observe color change, and if two dark red strips are present, determining that the test paper is strong positive, thus prompting the infection of drug-resistant staphylococcus aureus; if the color is dark and light, the prompt is weak positive, and the possibility of drug-resistant staphylococcus aureus infection exists; if only one red strip is presented, the drug-resistant staphylococcus aureus infection is not supported; if no red band is present, damage is indicated.
Furthermore, the preparation steps of the nano immunomagnetic beads are as follows:
(3.1) preparation and purification of antibody: using MRSA as antigen to immunize mice by autoclaving, and selecting Balb/c, female mice; wherein the injection amount of the antigen is 100-200ug per mouse, monoclonal antibody cell strains are prepared and screened according to the conventional hybridoma technology and the limiting dilution method, then the MRSA-resistant specific antibody cell strains are proliferated and cultured and injected into the mouse to prepare the ascites, and the obtained ascites is purified by an octanoic acid-sulfuric acid string precipitation method; using MRSA thallus as antigen to immunize a New Zealand white rabbit, purifying the obtained anti-MRSA serum by a sulfuric acid string precipitation method, and storing the obtained polyclonal antibody at-80 ℃ for later use;
(3.2) coupling of nano magnetic beads and antibodies: selecting nano magnetic particles with the particle diameter of 10-800nm, and shaking and uniformly mixing the nano immunomagnetic beads and staphylococcus aureus protein AIgG for 6 hours according to the mass ratio of 15: 1; then adding sodium borohydride according to the mass ratio of the specific antibody to sodium borohydride of 1:1 to obtain the specific antibody coupled nano immunomagnetic beads, purifying by a column method, and storing at 4 ℃.
Further, the diameter of the nano-magnetic particle is 60-300 nm.
The preparation steps of the colloidal gold immunochromatographic test strip are as follows:
the colloidal gold immunochromatographic test paper comprises absorbent paper (a sample pad) (1), a glass fiber membrane (2), a nitrocellulose membrane (3) and absorbent paper (4), wherein the absorbent paper (the sample pad) (1), the glass fiber membrane (2), the nitrocellulose membrane (3) and the absorbent paper (4) are connected end to end; a dried gold-labeled antibody (flow band) is adsorbed on the glass fiber membrane (2); an antigen or antibody strip and a quality control substance strip (a detection strip) capable of directly reacting with a marker are coated on the nitrocellulose membrane (3); the width of the absorbent paper (4) is 20/25 mm;
1. preparing colloidal gold: preparation of colloidal gold by trisodium citrate reduction method-HAuCl4First, 0.01% aqueous solution is prepared. 1ml of 1% HAuCl was added accurately with magnetic stirring4While simultaneously adding 1.5-3ml of 1% aqueous solution of trisodium citrate. Continuing heating after the color is stable, cooling to room temperature, adding pure water to make up to 100ml, and storing at 4 ℃ in dark.
2. Preparing immune colloidal gold: preparing mouse anti-PBP 2a protein (Qac protein) colloidal gold, drying and adsorbing on a colloidal gold binding pad (glass fiber membrane); 5-20ug of mAb was bound per ml of colloidal gold. Wherein, the T line fixes a mouse anti-PBP 2a protein (Qac protein) antibody, and the concentration of the antibody is 3-8 mg/mL; goat anti-mouse IgG was immobilized on line C at an antibody concentration of 1-5 mg/mL.
3. Detecting line fixed antibody: PBP2a protein (Qac protein) antibody was fixed to T-line, and goat anti-mouse IgG was fixed to C-line. 500ul of each immune colloidal gold is respectively taken, and the immune colloidal gold is respectively and uniformly dripped on glass fiber with the width of 1cm, the distance between the two immune colloidal gold and the glass fiber is 1-2cm, and the immune colloidal gold is dried and then adsorbed on a colloidal gold binding pad (glass fiber membrane).
4. All the components are connected end to end and fixed on a PVC rubber plate and sealed.
The invention has the following beneficial effects and advantages:
in recent years, along with the opening of a two-fetus policy and the increase of living pressure, people have a delay of birth age, the incidence rate of mastitis is higher and higher nowadays, the clinical requirement is huge, the invention mainly takes prevention and treatment, pertinently treats infectious mastitis and guides a pregnant woman to nurse, pathogenic bacteria are quickly and rapidly found out, appropriate antibiotics are given, and biological abuse resistance is avoided.
In the mastitis treatment process, particularly, while the treatment mode is continuously improved, the existing detection mode for mastitis pathogenic bacteria is expensive and complicated, and the comprehensive strain detection is not required in the actual operation process, so that the economic burden of a patient can be greatly reduced by the improved mode of the invention. In addition, the product prepared by the invention is not only household, but also can be widely used in the places related to the nursing of pregnant women, such as outpatients, lunar centers, nursing centers and the like, and has wide market and remarkable economic benefit.
Meanwhile, the product manufactured by the invention also develops a new thought, has extremely strong transformation capability, and doctors can properly adjust the product according to different diseases and different easily-infected strains in medical work and apply the product to monitoring of pathogenic bacteria of other various diseases, such as respiratory tract infection. Meanwhile, the invention can also be popularized to other fields for reference and applied to all corners of life, for example, people lack an economical product in the existing life to simply and conveniently monitor the sanitary condition of family life, so that the invention has a strong place to use, and the feedback of whether pathogenic bacteria are contained in the refrigerator or not and food can be quickly obtained at any time by putting the test paper adjusted by the invention into the refrigerator, thereby making corresponding guarantee for the health of people and preventing the occurrence of diseases in time, and having wide market and development prospects.
Drawings
FIG. 1 is a front view of the portable MRSA detection kit for mastitis pathogen in lactation period in accordance with the present invention;
wherein: 1. a sample adding area, wherein the area contains immune nano magnetic beads; 2. a magnet adsorption area, the bottom of which is provided with a powerful magnet;
3. a lysis zone containing a staphylococcus aureus lysate; 4. a connecting zone connecting the preceding reaction part and the underlying reaction part
A color development zone; 5. a color development zone; 6. a base; 7. a drawer handle a; 8. a drawer handle b; 9. drawer handle c (note: pulling out drawer handle c will strengthen the handle
Force magnets are pulled out together); 10. a drawer handle d.
FIG. 2 is a schematic diagram of the color development zone of the portable MRSA detection kit for mastitis pathogen in lactation period;
wherein: 11. a sample pad for moving a sample along the sample pad; 12. a colloidal gold bonding pad; 13. chromatographic membrane (NC nitric acid)
Cellulose films); 14. a water-absorbing material; 15. detecting a line T; 16. a quality control line C;
note: except for the color development surface of the chromatographic membrane 13, all four sides of the whole color development area 5 structure are wrapped by PVC rubber plates
Wrap (including the other three sides of the chromatographic membrane part except the color development surface). The upper and connection regions 4, lower of the color development region structure
Is connected with the base 6.
FIG. 3 is a schematic diagram showing the detection result of the color zone of the portable MRSA detection kit for pathogenic bacteria of mastitis in lactation period;
in the figure: t, C two lines-positive; c one line — negative; no line-invalid。
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and the accompanying drawings.
(1) The magnetic nano-particles used in the embodiment of the invention are libido-modified magnetic nano-particles with the particle size of 800nm, are purchased from Wuhanjia source quantum dot technology development company PBP2a aptamer, are synthesized by Biotechnology engineering (Shanghai) GmbH, and have the address of Songjiang Kong Min-No. 698 in Shanghai city and the preservation number of: m23.
(2) The structure of the immune colloidal gold immunochromatographic test paper used in the embodiment of the present invention is shown in fig. 2, and the reaction support is 6.2cmx0.4cmPCV plate; the water absorption pad is 2cmx0.4cm oil filter paper; 1.8cm x0.4cm nitrocellulose membrane is sequentially coated with a mouse anti-PBP 2a protein (Qac protein) antibody (purchased from Dingguo biotechnology company, 2mg/ml), goat anti-mouse IgG1mg/ml (containing 1% BSA blocking agent), and a glass fiber membrane containing 0.4cm xO.4cm colloidal gold labeled mouse anti-PBP 2a protein (Qac protein) antibody (the dosage of standard gold is 5ug, PBS dilution; the optimal pH value is 8.5); the sample pad is a 2.7cmx0.4cm glass fiber membrane; thus forming the MRSA colloidal gold test paper.
(3) In this embodiment, based on the synthesized nano immunomagnetic beads and the immune colloidal gold immunochromatographic test paper, a high-sensitivity and high-selectivity MRSA detection system is established for detecting mastitis MRSA in lactation.
The specific steps of the detection method of the present invention are as follows (the detection tool used is shown in fig. 1 and 2):
(1) in thatSample application region 1Dripping 2mL of a sample to be detected, slightly shaking for 1min to ensure that the emulsion and 1mL of nano-immunomagnetic beads which are pre-placed in the first grid are fully and uniformly mixed (no precipitate can be seen by naked eyes), and then reacting for 30min at room temperature;
(2) is drawn offDrawer handle a7Let the liquid and magnetic beads fall intoMagnet adsorption zone 2In the middle, letDrawer handle c9The connected strong magnet adsorbs the magnetic beads for 3 min;
(3) pouring out the residual liquid in the second step, and then pulling outDrawer handle c9Is then pulled outDrawer handle b8The nano immunomagnetic beads (adsorbed MRSA) are all dropped intoCleavage zone 3Placing 1ml of staphylococcus aureus lysate in advance, shaking gently, mixing well, and standing at room temperature for 30 min;
(4) is drawn offDrawer handle d10Allowing the magnetic beads and the lysis solution to enter togetherJoining region 4Standing at room temperature for about 15min, and waitingColor development zone 5And (4) developing color.
In the step (1), the dosage of the IgG antibody coated magnetic nanoparticles is 1mL (10mg/mL), and the concentration of a sample to be detected is 2mL and is 108CFU/mL of Staphylococcus aureus N315 strain.
In the step (2), the magnet is a strong magnet produced by Shenzhen Yuexing magnet Limited, and the product number is as follows: 192050470.
in step (3), the Staphylococcus aureus lysis reagent was staphyloccocilysine produced by Wuhansai Rui Biotech, Inc. at 50 ug/mL.
In step iv, the immuno-colloidal gold immunochromatographic strip used was prepared by binding 5ug of monoclonal antibody per ml of colloidal gold. Wherein, PBP2a protein (Qac protein) antibody is fixed on the T line, and the concentration of the antibody is 2 mg/mL; goat anti-mouse IgG was immobilized on line C at an antibody concentration of 1 mg/mL. Preparing a T line and a C line: 500ul of each immune colloidal gold is respectively taken, and the immune colloidal gold is respectively and uniformly dripped on glass fiber with the width of 1cm, the distance between the two immune colloidal gold and the glass fiber is 2cm, and the immune colloidal gold is dried and then adsorbed on a colloidal gold binding pad (glass fiber membrane).
Analyzing the result, standing for 15min at room temperature, and prompting the infection of the drug-resistant staphylococcus aureus if the two deep red strips are strong positive; a dark and a light indicate weak positivity, possibly with infection with drug-resistant staphylococcus aureus; only one red band did not support drug-resistant staphylococcus aureus infection; no red band indicates damage. See fig. 3.
The result shows that the design of the invention can effectively realize the high-sensitivity and high-selectivity detection of MRSA.
The working principle of the invention is as follows:
1. enrichment principle:
the function of the immunomagnetic beads is mainly to combine with corresponding antigens in liquid under the action of fluid mechanics through specific antibodies on the surface, and to completely separate target antigens from other impurities through multiple magnetic separation actions, thereby obtaining highly concentrated antigens. The immunomagnetic beads can enrich various types of antigens such as nucleic acid with extremely small molecular weight, small molecular toxin, specific protein, cells or pathogenic microorganisms in a short time according to different antibodies coupled to the surfaces of the magnetic beads.
2. The principle of the colloidal gold immunochromatographic test paper is as follows:
(1) chloroauric acid (HAuCl4) can be polymerized into gold particles with certain sizes under the action of a reducing agent to form a negatively charged hydrophobic gel solution. The gold is called colloidal gold because it is in a stable colloidal state due to electrostatic interaction. The negative charges on the surface of the colloidal gold particles and the positive charge groups of the protein form firm combination due to electrostatic adsorption. The colloidal gold has strong adsorption function to protein, and high molecules such as protein and the like are adsorbed to the surface of the colloidal gold particles without covalent bonds, so that the activity of the labeled macromolecular substances is not changed.
(2) Double antibody sandwich method: first, a known specific antibody (monoclonal antibody or polyclonal antibody) is coated on a membrane in a certain amount to serve as a detection zone, and a secondary antibody capable of being combined with a gold standard serves as a quality control zone. The other single gold-labeled conjugate matched with the coated antibody is adsorbed on the gold-labeled pad and dried. One end of the dried gold pad was attached to the membrane and the other end was attached to the sample pad. The other side of the membrane is stuck with a water absorption pad. During detection, a certain amount of liquid sample is added into the sample pad, and the sample moves along the direction from the sample pad to the absorbent pad by virtue of capillary action. Firstly, drying a gold label pad to redissolve a gold label conjugate, and if an antigen to be detected is in a specimen, performing antigen-antibody reaction to form a complex A (gold particles-antibody-antigen); when the sample continues to move to reach the position of the detection zone, the sample and the coating antibody react again to form a complex B (gold particle-antibody-antigen-coating antibody), the complex B is gathered at the position of the detection zone and finally reaches the macroscopic degree, and if no antigen to be detected exists in the sample, a macroscopic red strip cannot be formed. The free gold-labeled conjugate or complex a crosses the detection zone to reach the quality control zone, and reacts with the secondary antibody to form complex C (gold particle-antibody-secondary antibody), which aggregates and produces a red band visible to the naked eye. The quality control band shows a red band regardless of whether the sample contains the substance to be detected.
Claims (5)
1. A portable mastitis pathogen MRSA detection method in lactation period is characterized in that: MRSA is rapidly detected by the combined use of an immune nano magnetic bead technology and a colloidal gold immunochromatography technology, and a monoclonal antibody obtained by a hybridoma technology is coupled to the nano magnetic bead to form nano immune magnetic bead; the other paired monoclonal antibody marked by the colloidal gold is used as a marked antibody, and the polyclonal serum and the goat anti-mouse IgG are respectively used as a detection line T and a quality control line C, and the method specifically comprises the following steps:
the method is characterized in that a portable mastitis pathogenic bacterium MRSA detection kit in the lactation period is adopted for detection, and the portable mastitis pathogenic bacterium MRSA detection kit in the lactation period comprises nanometer immunomagnetic beads, strong magnets, staphylococcus aureus lysate and colloidal gold immunochromatography test paper; the colloidal gold immunochromatographic test paper comprises a binding pad of a colloidal gold labeled antibody, a chromatographic membrane containing a detection line T and a quality control line C and a water absorption pad, when a sample is detected, specific capture and enrichment are carried out on nano immunomagnetic beads and thalli in the sample, then an enrichment liquid is added into a lysis solution for lysis, then the lysis solution is dropped on the sample pad of the colloidal gold immunochromatographic test paper, and interpretation of a macroscopic chromogenic strip is formed at the detection line T and the quality control line C according to the color of colloidal gold, so that rapid semi-quantitative detection of MRSA is realized.
2. The portable method of detecting MRSA as claimed in claim 1, wherein the method comprises the steps of: the volume ratio of the sample to be detected to the nano magnetic beads is 20:1, 10:1 and 5:1 or 2: 1; the method for capturing and enriching the thallus by the nano immunomagnetic beads comprises the following steps: after fully shaking and uniformly mixing the sample and the self-made immunomagnetic beads, standing for reaction for 30-60min, then carrying out magnetic field separation, and washing for 3 times by PBS (phosphate buffer solution) to obtain a thallus enrichment solution;
the volume ratio of the bacterial lysate to the sample to be detected is 1:20, 1:10 or 1: 1; and dripping the obtained lysate into a detection area, and reading T, C line color development after 5-15min to obtain a result.
3. The portable method of detecting a pathogenic bacteria MRSA for mastitis in lactation according to claim 1 or 2, wherein: the method adopts a portable mastitis pathogen MRSA detection kit in lactation period to detect, and comprises the following steps:
(2.1) adsorbing a sample to be detected with nano immunomagnetic beads: adding the nanometer immunomagnetic beads and the sample to be detected into a container, uniformly mixing, and reacting at 25-40 ℃ for 30-60 min;
(2.2) attracting and gathering the nano magnetic beads by a magnet: placing the container in the previous step on a strong magnet, standing for 3min, and pouring out the liquid;
(2.3) lysis of bacteria: adding 50ul/ml of staphylococcus aureus lysate into the container, and continuously reacting for 30-60min at 25-40 ℃;
(2.4) color development: pouring the lysate onto a sample pad of colloidal gold immunochromatographic test paper, carrying out chromatography for 10-15min to observe color change, and if two dark red strips are present, determining that the test paper is strong positive, thus prompting the infection of drug-resistant staphylococcus aureus; if the color is dark and light, the prompt is weak positive, and the possibility of drug-resistant staphylococcus aureus infection exists; if only one red strip is presented, the drug-resistant staphylococcus aureus infection is not supported; if no red band is present, damage is indicated.
4. The portable method of detecting a pathogenic bacteria MRSA for mastitis in lactation according to claim 1 or 2, wherein: the preparation steps of the nano immunomagnetic beads are as follows:
(3.1) preparation and purification of antibody: using MRSA as antigen to immunize mice by autoclaving, and selecting Balb/c, female mice; wherein the injection amount of the antigen is 100-200ug per mouse, monoclonal antibody cell strains are prepared and screened according to the conventional hybridoma technology and the limiting dilution method, then the MRSA-resistant specific antibody cell strains are proliferated and cultured and injected into the mouse to prepare the ascites, and the obtained ascites is purified by an octanoic acid-sulfuric acid string precipitation method; using MRSA thallus as antigen to immunize a New Zealand white rabbit, purifying the obtained anti-MRSA serum by a sulfuric acid string precipitation method, and storing the obtained polyclonal antibody at-80 ℃ for later use;
(3.2) coupling of nano magnetic beads and antibodies: selecting nano magnetic particles with the particle diameter of 10-800nm, and shaking and uniformly mixing the nano immunomagnetic beads and staphylococcus aureus protein AIgG for 6 hours according to the mass ratio of 15: 1; then adding sodium borohydride according to the mass ratio of the specific antibody to sodium borohydride of 1:1 to obtain the specific antibody coupled nano immunomagnetic beads, purifying by a column method, and storing at 4 ℃.
5. The portable method of detecting MRSA as claimed in claim 4, wherein the method comprises the steps of: the diameter of the nano magnetic particle is 60-300 nm.
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