阅读说明：本技术 一种金黄色葡萄球菌免疫磁珠洗液 (Staphylococcus aureus immunomagnetic bead washing liquor ) 是由 曲晓莹 卢勉飞 蔡芷荷 滕昆仑 吴清平 陈博 陈鲁 于 2019-10-24 设计创作，主要内容包括：本发明公开了一种金黄色葡萄球菌免疫磁珠洗液,通过组分的优化,与常规洗液相比,在洗涤过程中提高了洗液的清洗效果,并且有效降低了食品基质的干扰,而且具有特异持续性增菌的功效,尤其是目标菌含量极低的样品增菌效果显著,从而提高了金黄色葡萄球菌的检出率,为免疫磁珠在试剂检测中的应用提供了保障。(The invention discloses a staphylococcus aureus immunomagnetic bead washing liquid, which is characterized in that through component optimization, compared with a conventional washing liquid, the washing liquid improves the washing effect in the washing process, effectively reduces the interference of a food matrix, has the effect of specific and continuous bacteria increasing, and particularly has an obvious bacteria increasing effect on a sample with extremely low target bacteria content, so that the detection rate of staphylococcus aureus is improved, and the application of immunomagnetic beads in reagent detection is guaranteed.)

1. The staphylococcus aureus immunomagnetic bead washing liquor is a buffer solution, and is characterized in that a surfactant, protein, water-soluble sugar, inorganic salt and a specific bacteriostatic agent are added into the buffer solution.

2. The staphylococcus aureus immunomagnetic bead washing liquid as claimed in claim 1, wherein the buffer solution comprises 0.2-1.4 g/L of surfactant, 4.0-28.5 g/L of protein, 5.5-19.5 g/L of water-soluble sugar, 3.5-5.5 g/L of inorganic salt and 4.5-6.5 mg/L of specific bacteriostatic agent.

3. Staphylococcus aureus immunomagnetic bead wash according to claim 1 or 2,

the surfactant is at least one selected from Tween-20, TritonX-100 and OhiomasURF ON-870;

the protein is selected from at least one of tryptone, bovine serum albumin and lactalbumin;

the water-soluble sugar is at least one of lactose and glucose;

the inorganic salt is at least one selected from NaCl and KCl;

the specific bacteriostatic agent is selected from at least one of amphotericin B and polymyxin B.

4. The staphylococcus aureus immunomagnetic bead washing liquid as claimed in claim 1, wherein the pH of the washing liquid is 7.0-7.5, the solution is a buffer solution, and the buffer solution is added with 5.5-9.5 g/L lactose, 6.0-10.0 g/L glucose, 4.0-6.0 g/L tryptone, 7.5-9.5 g/L bovine serum albumin, 8.0-13.0 g/L lactalbumin, 2.0-3.0 mg/L amphotericin B, 2.5-3.5 mg/L polymyxin B, 3.5-5.5 g/L NaCl, 0.60-0.70 g/L Tween-20, 0.20-0.30 g/L triton-100, and 0.30-0.40 g/L odasurf-870.

5. The staphylococcus aureus immunomagnetic bead lotion as claimed in claim 1, wherein the lotion has a pH of 7.0-7.5, and the solution is a buffer solution, wherein 5.5g/L lactose, 6.0g/L glucose, 4.0g/L tryptone, 7.5g/L bovine serum albumin, 8.0g/L lactalbumin, 2.0mg/L amphotericin B, 2.5mg/L polymyxin B, 3.5g/L NaCl, 0.6g/L Tween-20, 0.20g/L triton x-100, and 0.30g/L ohodascurf ON-870 are added to the buffer solution.

6. The staphylococcus aureus immunomagnetic bead lotion as claimed in claim 1, wherein the lotion has a pH of 7.0-7.5, and the solution is a buffer solution, wherein the buffer solution contains 7.5g/L lactose, 8.0g/L glucose, 5.0g/L tryptone, 8.5g/L bovine serum albumin, 10.0g/L lactalbumin, 2.5mg/L amphotericin B, 3.0mg/L polymyxin B, 4.5g/L NaCl, 0.65g/L Tween-20, 0.25g/L triton x-100, and 0.35g/L ohodascurf ON-870.

7. The staphylococcus aureus immunomagnetic bead lotion as claimed in claim 1, wherein the lotion has a pH of 7.0-7.5, and the solution is a buffer solution, wherein 9.5g/L lactose, 10.0g/L glucose, 6.0g/L tryptone, 9.5g/L bovine serum albumin, 13.0g/L lactalbumin, 3.0mg/L amphotericin B, 3.5mg/L polymyxin B, 5.5g/L NaCl, 0.70g/L Tween-20, 0.30g/L triton x-100, and 0.40g/L ohodascurf ON-870 are added to the buffer solution.

8. The staphylococcus aureus immunomagnetic bead wash of claim 1, 2, 4, 5, 6 or 7, wherein the buffer is a PBS buffer or a Tris-HCl buffer.

9. A method of using an immunomagnetic bead wash according to any of claims 1 to 8, comprising the steps of:

uniformly mixing immunomagnetic beads and a sample, incubating, standing, performing magnetic separation, and removing a supernatant;

adding an immunomagnetic bead solution according to any one of claims 1 to 8, incubating, standing for magnetic separation, and removing the supernatant.

10. Use of an immunomagnetic bead lotion according to any one of claims 1 to 8 as a staphylococcus aureus detection reagent.

Technical Field

The invention belongs to the technical field of immunization, and particularly relates to staphylococcus aureus immunomagnetic bead washing liquor.

Background

Staphylococcus aureus (Staphylococcus aureus) is one of the common food-borne pathogenic microorganisms, and produces Staphylococcus aureus Enterotoxins (SEs) under proper conditions, which are the most important pathogenic factors of food-borne pathogenic bacteria. Staphylococcus aureus is widely distributed, and external environments such as air, water, human and livestock and excrement thereof are traced; the internal environment such as the body surface, mucosa, respiratory tract, nasopharynx and intestinal tract of human and animal are also distributed. 30% -50% of healthy people are staphylococcus aureus carriers, so staphylococcus aureus is ubiquitous.

In recent years, new detection technologies, especially molecular detection products, are emerging continuously, the sensitivity is improved, but the detection result is more easily influenced by a sample matrix, and the importance of sample pretreatment before detection becomes more prominent. In comparison, immunomagnetic beads are the most ideal means for sample pretreatment at present. As a separation and enrichment technology with specificity, the immunomagnetic beads have important application values in a plurality of fields, such as DNA extraction, protein purification, cell screening, food-borne pathogenic microorganism enrichment and the like. Although high-quality antibody resources are crucial to the specific enrichment of immunomagnetic beads, in practical sample application of immunomagnetic beads, complex food substrates such as minced meat and the like also interfere with the enrichment process, so that the recovery rate of the immunomagnetic beads is reduced, and the enrichment effect is reduced. Therefore, in the immune enrichment operation process, the immune magnetic bead washing liquid is required to be used for removing food impurities. However, the conventional immunomagnetic bead washing liquid does not have a good effect in practical application, and a large amount of immunomagnetic beads are lost when complex food samples such as minced meat and the like are enriched, so that the detection rate of target bacteria is reduced. Therefore, the components of the immunomagnetic bead washing solution need to be optimized to improve the washing effect of the washing solution. In addition, in actual detection, the target bacteria content of a plurality of samples is extremely low, the target bacteria content is still low after bacteria are enriched according to a national standard method, the detection rate is not high, specific bacteria-enriching components are added in washing liquor, and the effect of continuously enriching bacteria can be achieved in the washing process.

According to the existing problems, the development of staphylococcus aureus immunomagnetic bead washing liquid is a problem which needs to be solved urgently.

Disclosure of Invention

The invention aims to provide a staphylococcus aureus immunomagnetic bead washing liquid, which is used for reducing the interference of a food substrate and improving the detection rate of target bacteria staphylococcus aureus.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the invention, a staphylococcus aureus immunomagnetic bead lotion is provided. According to the embodiment of the invention, the solution of the staphylococcus aureus immunomagnetic bead washing solution is a buffer solution, and a surfactant, a protein, a water-soluble sugar, an inorganic salt and a specific bacteriostatic agent are added into the buffer solution, wherein the specific bacteriostatic agent can inhibit the growth of non-target bacteria in a sample. According to the embodiment of the invention, the washing effect of the washing liquid is improved, the interference of the food matrix is reduced, the specific continuous bacteria increasing effect is achieved, especially the bacteria increasing effect of the sample with extremely low target bacteria content is obvious, and the detection rate of staphylococcus aureus is improved.

According to the embodiment of the invention, the solution is a buffer solution, and 0.2-1.4 g/L of surfactant, 4.0-28.5 g/L of protein, 5.5-19.5 g/L of water-soluble sugar, 3.5-5.5 g/L of inorganic salt and 4.5-6.5 mg/L of specific bacteriostatic agent are added into the buffer solution.

According to an embodiment of the present invention, the surfactant is selected from at least one of Tween-20, TritonX-100 and OodASURFON-870;

the protein is selected from at least one of tryptone, bovine serum albumin and lactalbumin;

the water-soluble sugar is selected from lactose and glucose; the inorganic salt is at least one selected from NaCl and KCl;

the specific bacteriostatic agent is selected from at least one of amphotericin B and polymyxin B.

According to an embodiment of the invention, the pH of the washing solution is 7.0-7.5, the solution is a buffer solution, and 5.5-9.5 g/L lactose, 6.0-10.0 g/L glucose, 4.0-6.0 g/L tryptone, 7.5-9.5 g/L bovine serum albumin, 8.0-13.0 g/L lactalbumin, 2.0-3.0 mg/L amphotericin B, 2.5-3.5 mg/L polymyxin B, 3.5-5.5 g/L NaCl, 0.60-0.70 g/L Tween-20, 0.20-0.30 g/L TritonX-100 and 0.30-0.40 g/L LOHODASURF ON-870 are added into the buffer solution. The inventors found that the bacteria growth and washing effects are more remarkable when the mass ratio is within the above range. For further effects, the inventor further optimizes the cleaning agent, and surprisingly discovers that the proportioning effect is greatly enhanced, and the bacterium increasing and cleaning effects are more remarkable within the range.

According to the embodiment of the invention, the pH value of the washing solution is 7.0-7.5, the solution is a buffer solution, and 5.5g/L lactose, 6.0g/L glucose, 4.0g/L tryptone, 7.5g/L bovine serum albumin, 8.0g/L lactalbumin, 2.0mg/L amphotericin B, 2.5mg/L polymyxin B, 3.5g/L NaCl, 0.6g/L Tween-20, 0.20g/L LTritonX-100 and 0.30g/L OHODASURF ON-870 are added into the buffer solution. In the proportion, the bacterium increasing and cleaning effects are more obvious.

According to the embodiment of the invention, the pH value of the washing solution is 7.0-7.5, the solution is a buffer solution, and 7.5g/L lactose, 8.0g/L glucose, 5.0g/L tryptone, 8.5g/L bovine serum albumin, 10.0g/L lactalbumin, 2.5mg/L amphotericin B, 3.0mg/L polymyxin B, 4.5g/L NaCl, 0.65g/L Tween-20, 0.25g/L LTritonX-100 and 0.35g/L OHODASURF ON-870 are added into the buffer solution. In the proportion, the bacterium increasing and cleaning effects are more obvious.

According to the embodiment of the invention, the pH value of the washing solution is 7.0-7.5, the solution is a buffer solution, and 9.5g/L lactose, 10.0g/L glucose, 6.0g/L tryptone, 9.5g/L bovine serum albumin, 13.0g/L lactalbumin, 3.0mg/L amphotericin B, 3.5mg/L polymyxin B, 5.5g/L NaCl, 0.70g/L Tween-20, 0.30g/L LTritonX-100 and 0.40g/L OHODASURF ON-870 are added into the buffer solution. In the proportion, the bacterium increasing and cleaning effects are more obvious.

According to an embodiment of the invention, the buffer is a PBS buffer or a Tris-HCl buffer. In the proportion, the bacterium increasing and cleaning effects are more obvious.

In a second aspect of the present invention, the present invention provides a preparation method of the immunomagnetic bead washing liquid, wherein the immunomagnetic bead washing liquid is obtained by weighing the raw materials according to any one of the formulas and mixing.

In a third aspect, the present invention provides a method for using the aforementioned immunomagnetic bead lotion, comprising the steps of:

uniformly mixing immunomagnetic beads and a sample, incubating, standing, performing magnetic separation, and removing a supernatant;

adding the immunomagnetic bead washing solution to incubate, standing for magnetic separation, removing supernatant, and detecting staphylococcus aureus in the sample.

According to an embodiment of the present invention, the volume ratio of the immunomagnetic beads to the sample is: 20 μ L of: 1 mL.

According to the embodiment of the invention, the incubation condition is that the incubation is carried out at 35-37 ℃ for 10-15 min.

According to the embodiment of the invention, the standing magnetic separation is carried out for 1-2 min.

According to the embodiment of the invention, the volume of the immunomagnetic bead washing liquid is 1-2 mL.

In a fourth aspect, the present invention provides the use of any one of the above immunomagnetic bead lotions as a staphylococcus aureus detection reagent.

The invention has the beneficial effects that:

the staphylococcus aureus immunomagnetic bead washing liquid disclosed by the invention has the advantages that the interference of a food matrix is greatly reduced through the optimization of the components, the washing function of the washing liquid is improved, and the washing liquid is used for enriching target bacteria in the matrix. The magnetic bead washing solution can be used for continuously increasing bacteria in a sample with low target bacteria content, so that the detection rate of staphylococcus aureus is improved, and a guarantee is provided for the application of immunomagnetic beads in reagent detection.

Detailed Description

The technical solution of the present invention is clearly and completely illustrated below with reference to the following examples, but is not limited thereto.

OHODASURF ON-870 is a surfactant available from Schupport Biotech, Inc., Yangzhou under the trade designation SPBHS 17. Tryptone (cat # T819615) available from Michael corporation. Amphotericin B (cat # A8250), polymyxin B (cat # P8350), available from Solebao scientific Co., Ltd, Beijing. Other materials and reagents, unless otherwise specified, were commercially available.

The preparation method of the staphylococcus aureus immunomagnetic beads comprises the following steps:

1) putting 1mg of carboxyl magnetic beads into an EP tube, adding 1mL of ultrapure water, performing ultrasonic treatment for 30s, centrifuging to remove supernatant, taking precipitate, cleaning, and finally suspending in 100 mu L of ultrapure water;

2) slowly adding 100 μ L MIX & GO activator (cat # A-SMPN100, purchased from Sigma), and activating at room temperature for 1 hr;

3) after activation, 1mL MEST (25mM, pH6.0) buffer was added to wash 2 times and resuspended in 100. mu.L MES buffer;

4) slowly adding 50-80 mu g of anti-staphylococcus aureus antibody (purchased from Abcam, cat number ab37644), and placing on a blending instrument for room temperature coupling for 2 h;

5) washing the magnetic beads with TBST buffer solution, adding 1mL of blocking solution (PBS solution containing 1% BSA), and placing on a mixing machine for blocking for 2 h;

6) and after the sealing is finished, washing the magnetic beads by using TBST buffer solution to obtain the staphylococcus aureus immunomagnetic beads for the subsequent experiment.