阅读说明：本技术 一种大肠杆菌o157免疫磁珠洗液 (Escherichia coli O157 immunomagnetic bead washing liquor ) 是由 曲晓莹 蔡芷荷 卢勉飞 徐环 吴清平 巩路 陈鲁 于 2019-10-24 设计创作，主要内容包括：本发明公开了一种大肠杆菌O157免疫磁珠洗液,将表面活性剂、蛋白质、水溶性糖、无机盐、抑菌剂以特定比例混合,溶剂为缓冲液。与常规磁珠洗液相比较,除了提高了清洗效果,还特异持续增菌,提高了大肠杆菌O157的检出率。而且有效降低了食品基质的干扰,提高了免疫磁珠洗液的灵敏度,克服了常规磁珠洗液的缺陷。(The invention discloses Escherichia coli O157 immunomagnetic bead washing liquor, which is prepared by mixing a surfactant, protein, water-soluble sugar, inorganic salt and a bacteriostatic agent in a specific ratio, wherein a solvent is a buffer solution. Compared with the conventional magnetic bead washing liquid, the washing liquid improves the washing effect, also specifically and continuously increases bacteria, and improves the detection rate of Escherichia coli O157. And the interference of food substrates is effectively reduced, the sensitivity of the immunomagnetic bead washing liquid is improved, and the defects of the conventional magnetic bead washing liquid are overcome.)

1. The solution of the Escherichia coli O157 immunomagnetic bead washing liquid is 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 Escherichia coli O157 immunomagnetic bead washing solution as claimed in claim 1, wherein the solution is a buffer solution, and the buffer solution contains 0.2-1.45 g/L of surfactant, 0.5-0.7 g/L of inorganic salt, 4.8-33.9 g/L of protein, 5.0-6.5 g/L of water-soluble sugar, and 0.003-1.017 g/L of specific bacteriostatic agent.

3. The E.coli O157 immunomagnetic bead lotion according to claim 1 or 2,

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

the protein is at least one of tryptone, casein sodium and lactalbumin;

the water soluble sugar is selected from lactose;

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

the specific bacteriostatic agent is selected from at least one of neomycin sodium and bile salt.

4. The Escherichia coli O157 immunomagnetic bead washing solution as set forth in claim 1, wherein the pH of the washing solution is 7.0-7.5, and the solution is a buffer solution, wherein 5.0-6.5 g/L lactose, 4.8-6.8 g/L tryptone, 7.6-10.6 g/L casein sodium, 7.0-10.0 g/L lactalbumin, 0.5-0.7 g/L NaCl, 13-17 mg/L neomycin sodium, 0.6-1.0 g/L cholate, 0.60-0.75 g/L Tween-20, 0.20-0.35 g/L TritonX-100 and 0.20-0.35 g/L ODOHASURF ON-870 are added to the buffer solution.

5. The Escherichia coli O157 immunomagnetic bead washing solution according to claim 1, wherein the pH of the washing solution is 7.0-7.5, and the solution is a buffer solution, wherein 5.0g/L lactose, 4.8g/L tryptone, 7.6g/L sodium caseinate, 7.0g/L lactalbumin, 0.5g/L NaCl, 13mg/L neomycin sodium, 0.6g/L bile salt, 0.60g/L Tween-20, 0.20g/L TritonX-100, and 0.20g/L OHODASURF ON-870 are added to the buffer solution.

6. The Escherichia coli O157 immunomagnetic bead washing solution according to claim 1, wherein the pH of the washing solution is 7.0-7.5, and the solution is a buffer solution, wherein 6.5g/L lactose, 5.8g/L tryptone, 8.6g/L sodium caseinate, 8.5g/L lactalbumin, 0.65g/L NaCl, 15mg/L neomycin sodium, 0.8g/L bile salt, 0.70g/L Tween-20, 0.30g/L TritonX-100, and 0.30g/L OHODASURF ON-870 are added to the buffer solution.

7. The Escherichia coli O157 immunomagnetic bead wash according to claim 1, wherein the pH of the wash is 7.0-7.5, and the solution is a buffer solution, wherein 7.0g/L lactose, 6.8g/L tryptone, 10.6g/L sodium caseinate, 10.0g/L lactalbumin, 0.70g/L NaCl, 17mg/L neomycin sodium, 1.0g/L bile salt, 0.75g/L Tween-20, 0.35g/L TritonX-100, and 0.35g/L OHODASURF ON-870 are added to the buffer solution.

8. The E.coli O157 immunomagnetic bead wash of claim 1, 2, 4, 5, 6 or 7, wherein the buffer is PBS buffer or 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 the immunomagnetic bead lotion of any one of claims 1 to 8 as a detection reagent for Escherichia coli O157.

Technical Field

The invention belongs to the technical field of immunization, and particularly relates to Escherichia coli O157 immunomagnetic bead washing liquor.

Background

Escherichia coli O157: H7 is a more prominent serotype strain of Enterohemorrhagic Escherichia coli (EHEC), and the infection dose of Escherichia coli O157: H7 is extremely small (only 10 bacteria). The infected patients may have symptoms such as diarrhea and the like, and further develop hemorrhagic colitis, the disease condition is developed very quickly, the fatality rate is high, and serious harm is caused to the social and public health.

In food-borne EHEC infection, meat and products thereof, milk, fruits and products thereof and the like can be polluted, the pathogenic bacteria are difficult to detect in the conventional Escherichia coli detection, and in order to improve the detection rate, molecular detection products are continuously developed, so that 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.

Among a plurality of detection technologies, the immunomagnetic bead method has obvious effect. The immunomagnetic bead method is a specific separation and enrichment technology, and has important application value in various fields such as DNA extraction, protein purification, cell screening, food-borne pathogenic microorganism enrichment and the like. High-quality antibody resources are important for the specific enrichment of the immunomagnetic beads, but in practical application, different food substrates such as minced meat interfere 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 solution does not have good effect. Particularly, the minced meat sample is enriched, so that a large amount of immunomagnetic beads are lost, and the detection rate of target bacteria is reduced. But also reduces the cleaning effect and increases other pathogenic bacteria, so that the cleaning process can not meet the experimental requirements.

According to the defects of the prior art, the development of an Escherichia coli O157 immunomagnetic bead washing liquid is a problem which needs to be solved urgently.

Disclosure of Invention

The invention aims to provide Escherichia coli O157 immunomagnetic bead washing liquor so as to reduce interference of food substrates and improve the detection rate of Escherichia coli O157.

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

in a first aspect of the invention, an Escherichia coli O157 immunomagnetic bead washing solution is provided. According to the embodiment of the invention, the solution of the escherichia coli O157 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 cleaning effect of the washing liquid can be improved, the interference of a food substrate is effectively reduced, the specific and persistent bacteria increasing effect is achieved, especially the bacteria increasing effect of a sample with extremely low target bacteria content is obvious, and therefore, the detection rate of Escherichia coli O157 is improved.

According to the embodiment of the invention, the solution is a buffer solution, and 0.2-1.45 g/L of surfactant, 0.5-0.7 g/L of inorganic salt, 4.8-33.9 g/L of protein, 5.0-6.5 g/L of water-soluble sugar and 0.003-1.017 g/L of specific bacteriostatic agent are added into the buffer solution.

In accordance with an embodiment of the present invention,

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

the protein is at least one of tryptone, casein sodium and lactalbumin;

the water soluble sugar is selected from lactose;

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

the specific bacteriostatic agent is selected from neomycin sodium and bile salt.

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 5.0-6.5 g/L lactose, 4.8-6.8 g/L tryptone, 7.6-10.6 g/L sodium caseinate, 7.0-10.0 g/L lactalbumin, 0.5-0.7 g/L NaCl, 13-17 mg/L neomycin sodium, 0.6-1.0 g/L cholate, 0.60-0.75 g/L LTen-20, 0.20-0.35 g/L TritonX-100 and 0.20-0.35 g/L OHODASURF 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 lotion is 7.0-7.5, the solution is a buffer solution, and 5.0g/L lactose, 4.8g/L tryptone, 7.6g/L sodium caseinate, 7.0g/L lactalbumin, 0.5g/L NaCl, 13mg/L neomycin sodium, 0.6g/L bile salt, 0.60g/L Tween-20, 0.20g/L TritonX-100 and 0.20g/LOHODASURF 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 lotion is 7.0-7.5, the solution is a buffer solution, and 6.5g/L lactose, 5.8g/L tryptone, 8.6g/L sodium caseinate, 8.5g/L lactalbumin, 0.65g/L NaCl, 15mg/L neomycin sodium, 0.8g/L bile salt, 0.70g/L Tween-20, 0.30g/L TritonX-100 and 0.30g/LOHODASURF 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 lotion is 7.0-7.5, the solution is a buffer solution, and 7.0g/L lactose, 6.8g/L tryptone, 10.6g/L sodium caseinate, 10.0g/L lactalbumin, 0.70g/L NaCl, 17mg/L neomycin sodium, 1.0g/L bile salt, 0.75g/L Tween-20, 0.35g/L TritonX-100 and 0.35g/L LOHODASURF 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, and removing the supernatant.

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 an application of the immunomagnetic bead washing solution as a detection reagent for Escherichia coli O157.

The invention has the beneficial effects that:

the Escherichia coli O157 immunomagnetic bead washing liquor disclosed by the invention is prepared by mixing the components in a specific ratio, so that the immune cleaning effect can be improved, the specific bacteria increasing effect is realized, and the detection rate of Escherichia coli O157 is improved. Effectively reduces the interference of food substrates, improves the accuracy and the sensitivity of the immunomagnetic beads, and meets the requirements of modern industrial production.

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. Novobiocin sodium salt (cat # MB5425) purchased from Dalian Meiren Biotechnology Ltd. Bile salts (B8550) purchased from Beijing Soilebao Tech Co., Ltd; other materials and reagents, unless otherwise specified, were commercially available.

The preparation method of the Escherichia coli O157 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 an anti-Escherichia coli O157 antibody (purchased from Abcam, cat number ab30521), and placing on a mixing 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 Escherichia coli O157 immunomagnetic beads for the subsequent experiment.