CN110819687A - Escherichia coli O157 immunomagnetic bead washing liquor - Google Patents
Escherichia coli O157 immunomagnetic bead washing liquor Download PDFInfo
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- CN110819687A CN110819687A CN201911019698.0A CN201911019698A CN110819687A CN 110819687 A CN110819687 A CN 110819687A CN 201911019698 A CN201911019698 A CN 201911019698A CN 110819687 A CN110819687 A CN 110819687A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/10—Enterobacteria
Abstract
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.
Description
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.
Examples 1 to 8
Escherichia coli O157 immunomagnetic bead washing solutions of examples 1 to 8 were weighed according to formulas 1 to 8 of Table 1, and prepared into corresponding solutions using PBS buffer (0.1M, pH 7.4); and mixing, stirring and uniformly mixing to ensure that the pH of the washing solution is 7.0-7.5, thus obtaining the Escherichia coli O157 immunomagnetic bead washing solution.
The conventional wash formulation was 0.5g/L Tween-20 in PBS (0.1M, pH 7.4).
TABLE 1 Immunomagnetic bead washes for different formulations
The immunomagnetic bead washing liquid provided by the embodiment of the invention is further subjected to effect detection.
One-factor analysis of immunomagnetic bead washes
Escherichia coli O157 immunomagnetic bead washing liquid component: the single-factor levels of lactalbumin and OHODASURF ON-870 were set as shown in Table 2, and the other components were mixed with PBS buffer (0.1M, pH7.4) to prepare 6.5g/L lactose, 4.8g/L tryptone, 7.6g/L casein sodium, 0.5g/L NaCl, 13mg/L novobiocin sodium, 0.6g/L bile salt, 0.60g/L Tween-20, 0.20g/L TritonX-100, and 0.35g/L OHODASURF ON-870, respectively.
In addition, Escherichia coli O157 was set in the following groups: 0.5CFU/g, 1.0CFU/g, 2.5CFU/g, 5.0CFU/g, 10.0CFU/g, 15.0CFU/g, 20.0CFU/g, respectively, for the artificial infection of pork.
According to the experimental steps of national standard GB4789.4-2016, the infected pork sample is enriched, 1mL of enrichment liquid and 20 microliter of immunomagnetic beads are respectively added into 1mL of pork sample and mixed uniformly, incubation is carried out for 10min at 37 ℃, the mixture is placed into a magnetic frame and kept stand for 1-2 min, and the supernatant is removed; adding 1mL of immunomagnetic bead washing liquor prepared by the invention, uniformly mixing, incubating for 15min at 37 ℃, placing in a magnetic frame, standing for 1-2 min, removing supernatant, taking precipitate, and washing the precipitate twice.
The content of Escherichia coli O157 was determined according to the LAMP kit (cat # KJD05L, Kyoto Loop Kai Microbiol technologies Co., Ltd.) or other quantitative methods.
TABLE 2 Single-factor experiment of E.coli O157 immunomagnetic bead wash components
As shown in Table 2, the appropriate concentration of lactalbumin in the immunomagnetic bead washing solution is 2.5-20 g/L, the detected amount of Escherichia coli O157 is 2.5-10 CFU/g, and the sensitivity is extremely high; the appropriate concentration of CFU/g OHODASURF ON-870 is 0.1-0.4 g/L, the detected amount of Escherichia coli O157 is 2.5-10 CFU/g, and the sensitivity is extremely high.
Sensitivity test of immunomagnetic bead washing solution
Carrying out artificial pollution on pork samples according to seven levels of 0.1CFU/g, 0.5CFU/g, 2.5CFU/g, 5CFU/g, 10CFU/g, 20CFU/g and 62.5CFU/g, enriching the samples according to the national standard GB4789.4-2016, respectively taking 1mL of enriched liquid and 20 mu L of immunomagnetic beads, adding the enriched liquid and the 20 mu L of immunomagnetic beads into 1mL of samples, uniformly mixing, incubating for 10min at 37 ℃, placing the samples in a magnetic frame, standing for 1-2 min, and removing supernatant; adding 1mL of immunomagnetic bead washing liquid, uniformly mixing, incubating for 15min at 37 ℃, placing in a magnetic frame, standing for 1-2 min, removing supernatant, and repeating the washing operation twice.
And finally, performing result identification by using an LAMP kit or other quantitative methods.
The results are shown in Table 3. The magnetic bead washing solution prepared by the invention can obviously improve the sensitivity of detecting the Escherichia coli O157, wherein the positive detection rates of the formulas 4, 5 and 6 (examples 4, 5 and 6) are the most, and the effect is the best.
TABLE 3 influence of E.coli O157 immunomagnetic bead wash on the detection Rate
Actual sample validation
Collecting 50 pork samples from each of three farmer markets, enriching the samples according to national standard GB4789.4-2016, respectively taking 1mL of enriched liquid, adding 20 mu L of immunomagnetic beads into 1mL of samples, uniformly mixing, incubating at 37 ℃ for 10min, placing in a magnetic frame, standing for 1-2 min, and removing supernatant; adding 1mL of immunomagnetic bead washing solution of the embodiment of the invention, uniformly mixing, incubating at 37 ℃ for 15min, placing in a magnetic frame, standing for 1-2 min, removing supernatant, and repeating the washing operation twice.
And finally, performing result identification by using an LAMP kit or other quantitative methods.
TABLE 4 influence of immunomagnetic bead washes on Positive detection Rate
As can be seen from the results shown in Table 4, the positive samples detected by the lotions of formulas 4, 5 and 6 are significantly higher than the conventional lotions, and the immunomagnetic bead lotions prepared in the examples 4, 5 and 6 of the present invention are more beneficial to increase the detection rate of the positive samples.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
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.
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