CN110819686A - Salmonella immunomagnetic bead washing liquor - Google Patents
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Abstract
The invention discloses salmonella immunomagnetic bead washing liquor, which is characterized in that through component optimization, compared with the conventional washing liquor, the washing liquor has the advantages that the washing effect is improved in the washing process, the interference of a food matrix is effectively reduced, the specific and persistent bacteria increasing effect is realized, particularly, the bacteria increasing effect of a sample with extremely low target bacteria content is remarkable, the detection rate of salmonella is improved, and the application of immunomagnetic beads in reagent detection is guaranteed.
Description
Technical Field
The invention belongs to the technical field of immunization, and particularly relates to salmonella immunomagnetic bead washing liquor.
Background
Salmonella is the most common food-borne pathogenic bacterium causing bacterial food poisoning and is widely distributed around the world. Salmonella belongs to host-wide pathogenic bacteria, and can be directly or indirectly transmitted from animals to animals, animals to humans and humans to humans. The infection pathway of salmonella is mainly food or direct contact. Salmonella infection can cause various symptoms such as food-poisoning gastroenteritis, typhoid fever, septicemia and the like, and infants, old people, people with low immunity and people who take antibacterial drugs recently are more easily infected. Therefore, salmonella can cause great harm to human health and life safety.
Salmonellosis caused by salmonella is the most common food-borne bacterial disease worldwide. In recent years, new detection technologies, especially molecular detection products, are emerging continuously, the sensitivity of detecting salmonella is improved, but the detection result is easily influenced by a sample matrix, and the importance of sample pretreatment 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 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 very low after bacteria are enriched according to the national standard method, the detection rate is not high, specific bacteria-enriching components are added in the immunomagnetic bead washing liquid, and the effect of continuous bacteria enrichment can be achieved in the washing process.
According to the existing problems, the development of a salmonella immunomagnetic bead washing solution is a problem which needs to be solved urgently.
Disclosure of Invention
The invention aims to provide salmonella immunomagnetic bead washing liquor to reduce the interference of food substrates and improve the detection rate of target bacteria salmonella.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, a salmonella immunomagnetic bead washing liquid is provided. According to the embodiment of the invention, the solution of the salmonella 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 matrix is effectively reduced, the specific and persistent bacteria increasing effect is achieved, and especially the bacteria increasing effect of a sample with extremely low target bacteria content is obvious, so that the detection rate of salmonella is improved.
According to the embodiment of the invention, the solution is a buffer solution, and 0.2-1.4 g/L of surfactant, 2.0-35 g/L of protein, 5.5-9.5 g/L of water-soluble sugar, 0.5-0.8 g/L of inorganic salt and 6.0-33.5 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, OodASURFON-870;
the protein is at least one of tryptone, soytone, sodium caseinate and lactalbumin;
the water-soluble sugar is selected from glucose;
the inorganic salt is at least one selected from NaCl and KCl;
the specific bacteriostatic agent is at least one of neomycin sodium, vancomycin and cefsulodin sodium.
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 glucose, 3.5-6.5 g/L tryptone, 9.5-15.5 g/L soytone, 2.0-3.0 g/L sodium caseinate, 6.5-10.0 g/L lactalbumin, 0.5-0.70 g/L NaCl, 9.5-13.5 mg/L vancomycin, 8.0-12.0 mg/L neomycin sodium salt, 6.0-8.0 mg/L cefsulodin sodium, 0.6-0.7 g/L Tween-20, 0.2-0.3 g/L LTritX-100 and 0.30-0.40 g/L OHODAURF 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 glucose, 3.5g/L tryptone, 9.5g/L soytone, 2.0g/L sodium caseinate, 6.5g/L lactalbumin, 0.5g/L NaCl, 9.5mg/L vancomycin, 8.0mg/L neomycin sodium salt, 6.0mg/L cefsulodin sodium, 0.6g/L LTween-20, 0.2g/L TritonX-100 and 0.3g/L OHODUFF 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 glucose, 5.0g/L tryptone, 12.5g/L soytone, 2.5g/L sodium caseinate, 8.0g/L lactalbumin, 0.65g/L NaCl, 11.5mg/L vancomycin, 10.0mg/L neomycin sodium salt, 7.0mg/L cefsulodin sodium, 0.65g/L Tween-20, 0.25g/L TritonX-100 and 0.35g/L OHODURF 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, 9.5g/L glucose, 6.5g/L tryptone, 15.5g/L soytone, 3.0g/L sodium caseinate, 10.0g/L lactalbumin, 0.70g/L NaCl, 13.5mg/L vancomycin, 12.0mg/L neomycin sodium salt, 8.0mg/L cefsulodin sodium, 0.7g/L Tween-20, 0.30g/L TritonX-100 and 0.40g/L OHODURF 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 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 an immunomagnetic bead lotion as a salmonella detection reagent.
The invention has the beneficial effects that:
through component optimization, compared with the conventional washing liquid, the salmonella immunomagnetic bead washing liquid disclosed by the invention has the advantages that the washing effect of the washing liquid is improved in the washing process, and the interference of a food matrix is effectively reduced. More particularly, the kit has the effect of specific and continuous enrichment, and particularly has an extremely obvious enrichment effect on samples with extremely low target bacteria content, so that the detection rate of salmonella is improved, and the application of immunomagnetic beads in reagent detection is guaranteed.
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 mclin; soy peptone (cat. No. FA0030) available from Beijing Soilebao Tech Co., Ltd; sodium caseinate (cat # C8654) purchased from Sigma; lactalbumin (cat # TX00903), available from shanghai cary laboratory equipment ltd; novobiocin sodium salt (cat # MB5425) available from Dalian Meiren Biotechnology Ltd; vancomycin (cat # C29699) and cefsulodin sodium (cat # S80772) are purchased from Yuanye biosciences, and other materials and reagents are purchased from commercial sources unless otherwise specified.
The preparation method of salmonella 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-salmonella antibody (purchased from Abcam under the cat number ab35156), 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 salmonella immunomagnetic beads for the subsequent experiment.
Examples 1 to 8
The salmonella immunomagnetic bead washing solutions of examples 1 to 8 were prepared by weighing raw materials according to formulas 1 to 8 of table 1, and preparing corresponding solutions using PBS buffer (0.1M, PH 7.4); and mixing, stirring and uniformly mixing to ensure that the pH value of the washing liquid is 7.0-7.5, thus obtaining the salmonella immunomagnetic bead washing liquid.
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
The salmonella immunomagnetic bead washing liquid comprises the following components: the single-factor levels of lactalbumin and OHODASURF ON-870 are set as shown in Table 2, and other components are mixed by using PBS buffer (0.1M, pH7.4) to prepare 7.5g/L glucose, 5.0g/L tryptone, 12.5g/L soytone, 2.5g/L sodium caseinate, 0.65g/L NaCl, 11.5mg/L vancomycin, 10.0mg/L neomycin sodium salt, 7.0mg/L cefsulodin sodium, 0.65g/L Tween-20, 0.25g/L TritonX-100.
The salmonella is set according to the following groups: 0.1CFU/g, 0.5CFU/g, 2.5CFU/g, 5CFU/g, 10CFU/g, 15CFU/g, 20CFU/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 Salmonella was identified using LAMP kit (cat # KJD05L, Kyork Kai Microbiol. Co., Ltd.) instructions or other quantitative methods.
TABLE 2 Single-factor experiment of Salmonella 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 detection amount of salmonella is 5-10 CFU/g, and the sensitivity is extremely high; the appropriate concentration of CFU/g OHOODASURF ON-870 is 0.2-0.4 g/L, the detected amount of salmonella is 0.5-10 CFU/g, and the sensitivity is extremely high.
The immunomagnetic bead washing liquid provided by the embodiment of the invention is further subjected to effect detection.
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. Result identification was performed using LAMP kit or other quantitative methods.
The results are shown in Table 3. The magnetic bead washing liquid prepared by the invention can obviously improve the sensitivity of detecting salmonella, wherein the positive detection rate of the formula 5 (example 5) is the most, and the effect is the best.
TABLE 3 influence of Salmonella immunomagnetic bead wash on 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 liquor of the embodiment of the invention, uniformly mixing, incubating for 15min at 37 ℃, placing in a magnetic frame, standing for 1-2 min, removing supernatant, and repeating 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 using the washing solutions of formulas 4, 5 and 6 are significantly higher than the conventional washing solution, wherein the detection rate of formula 5 (example 5) is the highest, which is more favorable for improving the detection rate of the positive samples compared with the conventional biochemical identification method.
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 salmonella 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 salmonella immunomagnetic bead lotion according to claim 1, wherein the solution is a buffer solution, and the buffer solution is added with 0.2-1.4 g/L of surfactant, 2.0-35 g/L of protein, 5.5-9.5 g/L of water-soluble sugar, 0.5-0.8 g/L of inorganic salt and 6.0-33.5 mg/L of specific bacteriostatic agent.
3. The Salmonella immunomagnetic bead wash solution 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, soytone, sodium caseinate and lactalbumin;
the water-soluble sugar is selected from glucose;
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, vancomycin and cefsulodin sodium.
4. The salmonella 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 5.5-9.5 g/L glucose, 3.5-6.5 g/L tryptone, 9.5-15.5 g/L soytone, 2.0-3.0 g/L casein sodium, 6.5-10.0 g/L lactalbumin, 0.5-0.70 g/L NaCl, 9.5-13.5 mg/L vancomycin, 8.0-12.0 mg/L neomycin sodium salt, 6.0-8.0 mg/L cefradixdine sodium, 0.6-0.7 g/L Tween-20, 0.2-0.3 g/L trinox x-100, and 0.30-0.40 g/L ohasuron are added to the buffer solution.
5. The salmonella immunomagnetic bead lotion according to claim 1, wherein the pH of the lotion is 7.0-7.5, and the solution is a buffer solution, wherein 5.5g/L glucose, 3.5g/L tryptone, 9.5g/L soytone, 2.0g/L sodium caseinate, 6.5g/L lactalbumin, 0.5g/L NaCl, 9.5mg/L vancomycin, 8.0mg/L neomycin sodium salt, 6.0mg/L cefsulodin sodium, 0.6g/L Tween-20, 0.2g/L TritonX-100 and 0.3g/L OHODASURF ON-870 are added to the buffer solution.
6. The salmonella immunomagnetic bead lotion according to claim 1, wherein the pH of the lotion is 7.0-7.5, and the solution is a buffer solution, wherein 7.5g/L glucose, 5.0g/L tryptone, 12.5g/L soytone, 2.5g/L sodium caseinate, 8.0g/L lactalbumin, 0.65g/L NaCl, 11.5mg/L vancomycin, 10.0mg/L neomycin sodium salt, 7.0mg/L cefsulodin sodium, 0.65g/L Tween-20, 0.25g/L TritonX-100 and 0.35g/L LOHODASURF ON-870 are added to the buffer solution.
7. The salmonella immunomagnetic bead lotion according to claim 1, wherein the pH of the lotion is 7.0-7.5, and the solution is a buffer solution, wherein 9.5g/L glucose, 6.5g/L tryptone, 15.5g/L soytone, 3.0g/L sodium caseinate, 10.0g/L lactalbumin, 0.70g/L NaCl, 13.5mg/L vancomycin, 12.0mg/L neomycin sodium salt, 8.0mg/L cefsulodin sodium, 0.7g/L Tween-20, 0.30g/L TritonX-100 and 0.40g/LOHODASURF ON-870 are added to the buffer solution.
8. The salmonella 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 salmonella detection reagent.
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