A bacterial enrichment solution and its use in detecting gladiolus Burkholderia

Abstract

A bacterial enrichment solution and its use in detecting Burkholderia gladioli (Pseudomonas cocovenenans subsp. venom), the bacterial enrichment solution containing the following components: a potato dextrose aqueous solution, gentian violet, chloramphenicol, polymyxin B, and sodium dehydroacetate. The bacterial enrichment solution has a good bacterial enrichment effect on target strains and can effectively inhibit the growth of non-target bacteria that appear frequently during routine detection, reducing interference from non-target bacteria and improving the detection rate and work efficiency of target bacteria. The present invention has a simple preparation process, low cost, and strong operability, and is suitable for enriching Burkholderia gladioli (Pseudomonas cocovenenans subsp. venom) in various foods.

Description

Technical Field

[0001] The present invention relates to the field of food microbial safety monitoring, and in particular to a bacterial enrichment solution and application thereof in detecting Burkholderia gladiolus (Pseudomonas cocovenenans subsp. Background Art

[0002] Pseudomonas cocovenenans subsp. fumonisin is a Gram-negative, short, non-spore-forming bacillus with an optimal growth temperature of 37°C and an optimal pH of 5-6. It was classified as a pathogenic form of Burkholderia gladioli in 1999 and can be found in fermented cereals, spoiled Tremella fuciformis, and wood ear mushrooms. The strain produces fumonisin, a toxin with a lethality rate of 40% to 100%, for which no effective antidote has been reported. Fumonisin is primarily absorbed through the digestive tract mucosa and enters the body through the bloodstream, with the liver, brain, and kidneys being the primary target organs. Onset is acute, with an incubation period of 2 to 24 hours.

[0003] The commonly used detection method for gladiolus Burkholderia (Pseudomonas cocovenenans subsp. cerebroventriculariae) is the national standard "GB 4789.29-2020 National Food Safety Standard Food Microbiology Inspection of Gladiolus Burkholderia (Pseudomonas cocovenenans subsp. cerebroventriculariae)", which uses GVC enrichment solution for bacterial enrichment and modified potato dextrose agar medium (mPDA) and PCFA medium for selective separation. During routine testing, it was found that after the sample was enriched with GVC enrichment solution, a layer of white biofilm would appear on the surface of the enrichment solution, which was composed of bacteria and molds. At the same time, when the bacterial suspension after enrichment was streaked into the separation plate mPDA culture medium, many non-target bacteria appeared, and the characteristics of the target bacteria colonies were not obvious. The main manifestations were: non-target bacteria spread and grew, the colonies were sticky, covering the plate on the mPDA culture medium, and masking the growth of gladiolus Burkholderia (Pseudomonas cocovenenans subspecies fermentum); molds spread and grew, covering the target strains, which was not conducive to picking target bacteria; the target bacteria colonies were small, the morphology was atypical, such as dry, and the amount of target bacteria was small, which increased the difficulty of isolating and identifying the target bacteria. During the testing of nearly a thousand samples, the main non-target bacteria isolated from GVC enrichment broth and mPDA culture medium were found to be: Pseudomonas species, such as Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas oleovorans; Enterobacter species, such as Enterobacter hallii; Pantoea dispersa and Ralstonia mannitolase; and fungi, such as molds. The presence of these non-target bacteria, on the one hand, robs the target bacteria of nutrients, which to some extent affects their growth and reproduction; on the other hand, it makes it more difficult to identify suspicious colonies, and even causes some target bacteria to be covered by other bacteria and cannot be observed, seriously affecting detection rates and work efficiency. Summary of the Invention

[0004] According to the first aspect, in one embodiment, a bacterial enrichment solution is provided, wherein the bacterial enrichment solution comprises the following components: potato dextrose aqueous solution, gentian violet, chloramphenicol, polymyxin B, and sodium dehydroacetate.

[0005] According to the second aspect, in one embodiment, a method for preparing the enrichment solution of the first aspect is provided, comprising: mixing potato dextrose aqueous solution, gentian violet, chloramphenicol, polymyxin B, and sodium dehydroacetate according to the formula to prepare the enrichment solution.

[0006] According to the third aspect, in one embodiment, there is provided use of the enrichment solution of the first aspect in detecting Burkholderia gladiolus (Pseudomonas cocovenenans subsp. fermentans).

[0007] According to the fourth aspect, in one embodiment, there is provided use of the enrichment solution of the first aspect in screening for Burkholderia gladiolus (Pseudomonas cocovenenans subsp. cerevisiae).

[0008] According to the fifth aspect, in one embodiment, there is provided use of the enrichment solution of the first aspect in culturing Burkholderia gladiolus (Pseudomonas cocovenenans subsp. flavescentis).

[0009] According to the enrichment solution of the above embodiment and its use in detecting gladiolus Burkholderia (Pseudomonas cocovenenans subspecies), the enrichment solution has a good enrichment effect on the target strain, and can effectively inhibit the growth of non-target bacteria with a higher frequency during daily detection, reduce the interference of non-target bacteria, and improve the detection rate and work efficiency of target bacteria. The preparation process of the present invention is simple, low cost, and highly operable, and is suitable for the enrichment of gladiolus Burkholderia (Pseudomonas cocovenenans subspecies) in various foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a graph showing the growth of non-target mixed bacteria on TSA plates;

[0011] Figure 2 The figure shows the growth of different enrichment solutions on mPDA plates;

[0012] Figure 3 The graph shows the growth of different enrichment solutions on PCFA plates. DETAILED DESCRIPTION

[0013] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can readily appreciate that some of the features may be omitted in different circumstances, or may be replaced by other materials or methods. In some cases, some operations related to the present application are not shown or described in the specification. This is to avoid the core of the present application being overwhelmed by excessive descriptions, and for those skilled in the art, it is not necessary to describe these related operations in detail. They can fully understand the related operations based on the description in the specification and the general technical knowledge in the art.

[0014] In addition, the features, operations, or characteristics described in the specification may be combined in any appropriate manner to form various embodiments. Furthermore, the steps or actions in the method description may be reordered or adjusted in a manner readily apparent to those skilled in the art. Therefore, the various sequences in the specification and drawings are provided solely for the purpose of clearly describing a particular embodiment and are not intended to be mandatory, unless otherwise specified.

[0015] The serial numbers assigned to the components in this document, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning.

[0016] In order to minimize missed detection and improve work efficiency when picking suspicious colonies, it is necessary to improve the enrichment solution used in the current standard.

[0017] In order to overcome the situation in which the existing detection standards use GVC enrichment solution to enrich the bacteria, resulting in a large number of non-target bacteria, which increases the difficulty of subsequent separation and detection and affects work efficiency, in one embodiment, the present invention provides an enrichment solution for selectively culturing gladiolus Burkholderia (Pseudomonas cocovenenans subspecies fermentum) and a preparation method thereof. The enrichment solution is conducive to the proliferation of target strains, and at the same time effectively inhibits non-target bacteria that appear more frequently in daily detection, which is conducive to improving the detection rate and work efficiency, and has strong practicality.

[0018] According to a first aspect, in one embodiment, a bacterial enrichment solution is provided, comprising the following components: potato dextrose aqueous solution, gentian violet, chloramphenicol, polymyxin B, and sodium dehydroacetate. This bacterial enrichment solution can inhibit the reproduction of non-target bacteria, thereby selectively proliferating Burkholderia gladioli (Pseudomonas cocovenenans subsp. cerebroventis), and can be used to screen, culture, or detect Burkholderia gladioli (Pseudomonas cocovenenans subsp. cerebroventis). This bacterial enrichment solution significantly improves the detection efficiency of Burkholderia gladioli (Pseudomonas cocovenenans subsp. cerebroventis), reducing missed detections.

[0019] Burkholderia gladioli (Pseudomonas cocovenenans subsp. farino fermentans), scientific name: Burkholderia gladioli (Pseudomonas cocovenenans subsp. farino fermentans), is a Gram-negative bacterium. The biochemical characteristics of Burkholderia gladiolus (Pseudomonas cocovenenans subsp. cocovenenans) and Burkholderia gladiolus are very similar (Reference 1: Fan Lu, Luan Jie, Investigation and analysis of a food poisoning incident caused by Burkholderia gladiolus (Pseudomonas cocovenenans subsp. cocovenenans) in Yunnan Province, Journal of Food Safety and Quality, Vol. 10, No. 23, December 2019; Reference 2: Lin Jie, Fang Chenyu, Lu Jingfang, et al., Research on real-time fluorescence PCR detection of Pseudomonas cocovenenans subsp. cocovenenans in food, Journal of Food Safety and Quality, Vol. 11, No. 11, June 2020; Reference 3: Jiao Zhenquan, Cao Wei, Yu Dongmin, et al., Comparative study of the 16S-23S rRNA intergenic region sequences of Pseudomonas cocovenenans and Burkholderia gladiolus, Chinese Journal of Food Hygiene, Vol. 20, No. 3, 2008).

[0020] Polymyxin B is also known as polymyxin B. Polymyxin B mainly has an inhibitory effect on some Gram-negative bacteria. In the present invention, polymyxin B has the effect of inhibiting the growth and reproduction of non-target bacteria (i.e., microorganisms other than Burkholderia gladioli (Pseudomonas cocovenenans subsp. fermentans)).

[0021] Sodium dehydroacetate, also known as sodium dehydroacetate, can inhibit the growth and reproduction of fungi such as yeast and mold.

[0022] Gentian violet is also known as gentian violet and crystal violet. It has a killing effect on some Gram-positive bacteria such as Staphylococcus, Corynebacterium diphtheriae, Pseudomonas aeruginosa, Candida albicans, Epidermophyton, etc., but has almost no effect on other Gram-negative bacteria and acid-fast bacteria.

[0023] Chloramphenicol is an antibiotic that has inhibitory effects on some Gram-positive and Gram-negative bacteria.

[0024] Gentian violet, chloramphenicol, polymyxin B, sodium dehydroacetate, and raw materials such as potatoes and glucose used to prepare the potato glucose aqueous solution can all be purchased from the market.

[0025] In one embodiment, the Burkholderia gladioli selectively proliferated by the enrichment solution of the present invention typically includes at least one of a strain that produces fumonisin and a strain that does not produce fumonisin. Typically, after selectively proliferating Burkholderia gladioli in a food sample using the enrichment solution of the present invention, subsequent strain identification and toxicity testing are performed.

[0026] In one embodiment, the concentration of polymyxin B in the enrichment broth is ≥ 5 μg / mL. While effectively inhibiting non-target bacteria, a polymyxin B concentration as low as possible is generally selected to control costs. In the present invention, the lowest polymyxin B concentration in the enrichment broth can be 5 μg / mL.

[0027] In one embodiment, the concentration of sodium dehydroacetate in the enrichment solution is ≥ 0.01 wt %.

[0028] In one embodiment, the enrichment solution contains the following components at concentrations: 5-20 μg / mL polymyxin B and 0.01-0.03 wt% sodium dehydroacetate. The concentration of polymyxin B in the enrichment solution may include but is not limited to 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, etc., and the concentration of sodium dehydroacetate includes but is not limited to 0.01 wt%, 0.02 wt%, 0.03 wt%, etc. The aforementioned concentrations refer to the final concentrations of polymyxin B and sodium dehydroacetate in the enrichment solution.

[0029] In one embodiment, the enrichment solution contains the following components at the following concentrations: polymyxin B 10 μg / mL, sodium dehydroacetate 0.03 wt %.

[0030] In one embodiment, the enrichment solution contains the following components at the following concentrations: 10 μg / mL gentian violet and 20 μg / mL chloramphenicol.

[0031] In one embodiment, the potato dextrose aqueous solution (also called PD water) is prepared by mixing potatoes, dextrose and water and then heating and boiling.

[0032] In one embodiment, the potato dextrose aqueous solution is prepared as follows: potatoes are mixed with water, heated to boil, and then glucose is added to obtain the potato dextrose aqueous solution.

[0033] In one embodiment, after boiling, the mixture is kept for 10 to 20 minutes.

[0034] In one embodiment, 300 g of potatoes (peeled) and 20 g of glucose are added to every 1000 mL of water.

[0035] In one embodiment, peeled potatoes are cut into pieces, added to water (e.g., 1000 mL), boiled for 10-20 minutes, filtered through gauze, and water is added to the initial volume (e.g., 1000 mL). Glucose is then added and heated to dissolve to obtain the potato dextrose aqueous solution. The potato dextrose aqueous solution is typically sterilized by autoclaving (121° C. for 20 minutes) and then cooled for use in preparing the enrichment solution.

[0036] In one embodiment, the potatoes are typically peeled potatoes.

[0037] In one embodiment, the water includes but is not limited to distilled water.

[0038] In one embodiment, the pH of the potato dextrose aqueous solution is 7.0±0.2.

[0039] In one embodiment, the enrichment solution is an enrichment solution that can selectively proliferate Burkholderia gladiolus (Pseudomonas cocovenenans subsp. flavescentis).

[0040] According to the second aspect, in one embodiment, a method for preparing the enrichment solution of the first aspect is provided, comprising: mixing potato dextrose aqueous solution, gentian violet, chloramphenicol, polymyxin B, and sodium dehydroacetate according to the formula to prepare the enrichment solution.

[0041] In one embodiment, the potato dextrose aqueous solution is prepared as follows: potatoes, glucose, and water are mixed according to the formula, and the mixture is heated and boiled to obtain the potato dextrose aqueous solution.

[0042] In one embodiment, the potato dextrose aqueous solution is prepared as follows: potatoes are mixed with water, heated to boil, and then glucose is added to obtain the potato dextrose aqueous solution.

[0043] In one embodiment, after boiling, the mixture is kept for 10 to 20 minutes.

[0044] In one embodiment, the potato dextrose aqueous solution is prepared as follows: potatoes, glucose, and water are mixed according to the formula, heated to a boil, and then filtered through gauze; water is added to the filtered filtrate to make up to the initial volume; glucose is then added, heated to dissolve, mixed, and sterilized to obtain the potato dextrose aqueous solution;

[0045] In one embodiment, the sterilization is performed by high pressure steam sterilization at 121° C. for 15 to 30 minutes.

[0046] In one embodiment, 300 g of potatoes and 20 g of glucose are added to every 1000 mL of water.

[0047] In one embodiment, the method for preparing the enrichment solution comprises the following steps:

[0048] 1) Weigh 300g of peeled potatoes, chop them into pieces, add 1000mL of distilled water, boil for 10-20min, filter through gauze, add more distilled water to 1000mL, add 20g of glucose, and heat to dissolve;

[0049] 2) adding NaOH aqueous solution and / or HCl solution to the mixed solution in step 1) to adjust the pH value to 6.8-7.2, sterilizing by high pressure at 121° C. for 20 min, and cooling to 50° C. after sterilization;

[0050] 3) Supplementing additives: under sterile conditions, adding gentian violet, chloramphenicol, polymyxin B, and sodium dehydroacetate solutions that have been sterilized by filtration (the pore size of the sterile filter membrane used for filtration sterilization is not greater than 0.45 μm) to the cooled mixed solution in step 2). To each 1 L of the mixed solution prepared in step 2), add 10 mg of gentian violet, 20 mg of chloramphenicol, 10 mg of polymyxin B, and 0.3 g of sodium dehydroacetate. Mix thoroughly and aliquot to prepare an enrichment solution.

[0051] According to a third aspect, in one embodiment, a use of the enrichment solution described in the first aspect in detecting (Pseudomonas cocovenenans subsp. cocovenenans) is provided. The enrichment solution of the present invention can be used to detect Burkholderia gladioli (Pseudomonas cocovenenans subsp. cocovenenans) in food, effectively inhibiting the reproduction of non-target bacteria, improving the detection efficiency of target bacteria, and effectively reducing missed detections.

[0052] According to a fourth aspect, in one embodiment, a use of the enrichment solution described in the first aspect for screening for Burkholderia gladioli (Pseudomonas cocovenenans subsp. cerebroventris) is provided. The enrichment solution of the present invention can be used to screen for Burkholderia gladioli (Pseudomonas cocovenenans subsp. cerebroventris) from a sample and to conduct further research on the microorganism.

[0053] According to the fifth aspect, in one embodiment, there is provided a use of the enrichment solution of the first aspect in culturing Burkholderia gladioli (Pseudomonas cocovenenans subsp. cerevisiae). The enrichment solution of the present invention can be used to expand the culture of Burkholderia gladioli,

[0054] In one embodiment, the peeled potatoes and glucose in the enrichment solution provided by the present invention provide essential nutrients, such as carbon sources, nitrogen sources, inorganic salts, and growth factors, for microbial growth and reproduction. Gentian violet, chloramphenicol, and polymyxin B act as inhibitors to inhibit the growth and reproduction of non-target bacteria, while sodium dehydroacetate inhibits the growth and reproduction of fungi, such as yeasts and molds.

[0055] In one embodiment, the advantages of the present invention compared with the GVC enrichment solution in the existing detection standard include: the enrichment solution can effectively inhibit the growth of miscellaneous bacteria in the sample, and can effectively inhibit the growth of non-target microorganisms such as mold, dispersed Pantoea, Pseudomonas fluorescens, and Enterobacter hallii that appear in daily detection, which is conducive to improving the detection rate and work efficiency, and has strong practicality.

[0056] Example 1

[0057] This example provides an experiment to verify the specificity of the enrichment solution.

[0058] 1. Preparation of culture medium

[0059] Enrichment solution: Weigh 300g of peeled potatoes, chop them into pieces, add 1000mL of distilled water, boil for 10-20min, filter through gauze, add more distilled water to 1000mL, add 20g of glucose, and heat to dissolve; adjust the pH to 6.8-7.2, autoclave at 121°C for 20min, and cool to 50°C after sterilization; under aseptic conditions, filter and sterilize (the pore size of the sterile filter membrane used is no more than 0.45μm, 0.22μm in this embodiment) an aqueous solution containing 10mg of gentian violet, 20mg of chloramphenicol, 10mg of polymyxin B, and 0.3g of sodium dehydroacetate, add the mixed solution, mix thoroughly, and then aliquot into 10mL for standby use. In the resulting enrichment solution, the final concentrations of gentian violet, chloramphenicol, polymyxin B, and sodium dehydroacetate are 10μg / mL, 20μg / mL, 10μg / mL, and 0.03wt%, respectively.

[0060] Tryptone soy broth (TSB) culture medium: prepared according to the manufacturer's instructions (Beijing Luqiao, batch number: 200602);

[0061] Tryptic soy agar (TSA) medium: prepared according to the manufacturer's instructions (Beijing Luqiao, batch number: 190815).

[0062] 2. Strain activation

[0063] Eight strains of non-target bacteria isolated during routine testing (Enterobacter hallii, Pseudomonas fluorescens, Pseudomonas putida, Pantoea dispersa, Pseudomonas oleovorans, Aspergillus niger, Lucknerella nondecarboxylase, and Ralstonia mannitolase) were inoculated with Burkholderia gladioli (CICC10574), Burkholderia gladioli (83756), Burkholderia gladioli (95947), and Burkholderia gladioli (79123)) in TSB broth for activation and incubation at 36°C for 24 hours. Burkholderia gladioli (83756), Burkholderia gladioli (95947), and Burkholderia gladioli (79123) were isolated in the inventor's laboratory. CICC refers to the China Center of Industrial Culture Collection.

[0064] 3. Vaccination

[0065] Dilute the activated 12 strains 10-fold with sterile saline. Transfer 0.1 mL of the resulting bacterial suspension (concentration ranges from 1000 to 5000 CFU) to the enrichment broth and incubate at 36°C for 24 hours. Use a 3 mm diameter inoculating loop to streak onto a TSA plate and incubate at 36°C for 48 hours. Observe the growth of the strains.

[0066] 4. Results and Analysis

[0067] Burkholderia gladiolus grows well on TSA plates, as do Enterobacter hormaechei (Gram-negative bacteria, genus: Enterobacter, species epithet: hormaechei), Pseudomonas fluorescens (Gram-negative bacteria, genus: Pseudomonas, species epithet: fluorescens), Pseudomonas putida (Gram-negative bacteria, genus: Pseudomonas, species epithet: putida), Pantoea dispersa (genus: Pantoea, species epithet: dispersa), Pseudomonas oleovorans (Gram-negative bacteria, genus: : Pseudomonas, species name epithet: oleovorans), Aspergillus niger (fungus, genus: Aspergillus, species name epithet: niger), non-decarboxylating Leclercia (Gram-negative bacteria, genus: Leclercia, species name epithet: adcarboxglata), and mannitol-degrading Ralstonia (Gram-negative bacteria, genus: Pseudomonas, species name epithet: oleovorans) did not grow on the TSA plate. The results are shown in Table 1, indicating that the enrichment solution has a high specificity for Burkholderia gladiolus.

[0068] Table 1 Growth of strains in culture medium

[0069]

[0070]

[0071] Note: +: indicates growth; -: indicates no growth.

[0072] Example 2

[0073] This example provides an experiment to evaluate the selectivity of the enrichment solution.

[0074] 1. Preparation of culture medium

[0075] According to the composition and preparation method of the enrichment solution and culture medium in Example 1, the enrichment solution and tryptic soy agar (TSA) medium were prepared.

[0076] 2. Vaccination

[0077] The types of non-target bacteria and the preparation of bacterial suspensions are shown in Example 1. Finally, 1 mL of the relevant bacterial suspension was taken to prepare 1-5×10 3 Prepare a mixed bacterial solution with a CFU / mL concentration for later use. Inoculate each test tube in step 1 with 1 mL of the mixed bacterial solution, with an inoculum size of 1000 to 5000 CFU. Incubate at 36°C for 24 hours. Pipette 10 μL of the culture medium containing non-target bacteria and spread it evenly onto a non-selective TSA plate. Incubate at 36°C for 48 hours.

[0078] 3. Results and Analysis

[0079] After 48 hours of culture, the number of colonies of non-target bacteria on the TSA plate was 0 CFU (see the results). Figure 1 No growth of mixed bacteria was observed, indicating that the enrichment broth culture medium of Example 1 of the present invention had a good selectivity effect and met the detection requirements.

[0080] Example 3

[0081] This example provides a comparative experiment between the enrichment solution of Example 1 of the present invention and the GVC enrichment solution in the national standard "GB4789.29-2020 National Food Safety Standard Food Microbiology Examination of Burkholderia gladiolus (Pseudomonas cocovenenans subspecies)" (hereinafter referred to as the national standard GB 4789.29-2020) in a food contamination simulation experiment.

[0082] Rice noodles were purchased from the market (testing confirmed that they did not contain Burkholderia gladioli), and the samples were enriched using the enrichment solution of Example 1 of the present invention and the national standard GVC enrichment solution. Subsequently, the selective culture medium mPDA (modified potato dextrose agar, see Appendix A.2 of the national standard GB 4789.29-2020) and PCFA (see Appendix A.3 of the national standard) in the national standard were used for separation, and the selective enrichment effects of the two enrichment solutions during the detection process were compared.

[0083] 1. Preparation of bacterial solution

[0084] Take the fresh culture of the standard strain Burkholderia gladioli (CICC10574), prepare 1OD bacterial suspension, and dilute it to 10 -7 Dilution (bacterial concentration is 1-10 CFU / mL).

[0085] 2. Preparation of culture medium

[0086] The enrichment solution was prepared according to the preparation steps of the culture medium in Example 1. The GVC enrichment solution and the selective culture media mPDA and PCFA were prepared according to the method in Appendix A of the national standard GB 4789.29-2020.

[0087] 3. Inoculation after artificial contamination and bacterial enrichment

[0088] Take two portions of rice noodles, each 25g, add 225ml of GVC enrichment solution to one portion of rice noodles, and add the enrichment solution of Example 1 of the present invention to the other portion of rice noodles, and take 10 -7 Prepare a simulated sample by diluting 1 ml of the standard bacterial suspension, mixing well, and incubating at 36°C for 24 hours. Use an inoculating loop to directly streak the enriched solution onto mPDA and PCFA selective separation media, incubate at 36°C for 48 hours, and observe the growth of colonies on various media.

[0089] 4. Microbial identification

[0090] Five typical or suspicious colonies were picked from the selective culture media mPDA and PCFA, and the strains were identified using the laboratory's VITEKMS fully automatic rapid microbial mass spectrometry detection system (Mérieux, France).

[0091] 5. Results and Analysis

[0092] After the sample was enriched with the enrichment solution of Example 1 of the present invention, the colonies on the selective separation medium mPDA and PCFA were single and typical, as shown in FIG. Figure 2 (1) and Figure 3(1) All 5 suspected bacteria were identified as Burkholderia gladioli (toxicity test was not performed), with an identification rate of 100%. After the samples were enriched with GVC, a viscous bacterial liquid appeared in the selective separation medium mPDA, covering the entire plate. Figure 2 (2) It is not conducive to the observation and picking of suspicious strains, and the target strains are not identified; suspicious colonies and non-target colonies appear on the PCFA, see Figure 3 (2) Among the 5 suspected bacteria selected, 3 were identified as target bacteria and 2 were non-target bacteria, with an identification rate of 60%. This shows that the enrichment solution of Example 1 of the present invention has better selectivity than the GVC enrichment solution in the national standard GB 4789.29-2020, is beneficial to the subsequent separation and identification steps, improves the detection rate and work efficiency, and has strong practicality.

[0093] The above examples are used to illustrate the present invention, which are only used to help understand the present invention and are not intended to limit the present invention. Those skilled in the art can make several simple deductions, modifications or substitutions based on the concept of the present invention.

Claims

1. A bacterial enrichment solution, characterized in that: The enrichment solution is a enrichment solution that can selectively proliferate Burkholderia gladiolus (Pseudomonas cocovenenans subspecies fermentum), and the enrichment solution is composed of the following: potato glucose aqueous solution, 10 μg / mL gentian violet, 20 μg / mL chloramphenicol, 5-20 μg / mL polymyxin B, and 0.01wt%-0.03wt% sodium dehydroacetate; the pH of the potato glucose aqueous solution is 7.0±0.

2.

2. The enrichment solution according to claim 1, wherein The enrichment solution contains the following components at the following concentrations: 10 μg / mL polymyxin B and 0.03 wt % sodium dehydroacetate.

3. The enrichment solution according to claim 1, wherein The potato glucose aqueous solution is prepared by mixing potatoes, glucose and water and then heating and boiling; And / or, the potato glucose aqueous solution is prepared by mixing potatoes with water, heating and boiling, and then adding glucose to obtain the potato glucose aqueous solution; 300 g of potatoes and 20 g of glucose are added to every 1000 mL of water.

4. The method for preparing the enrichment solution according to any one of claims 1 to 3, wherein: include: The potato dextrose aqueous solution, gentian violet, chloramphenicol, polymyxin B and sodium dehydroacetate are mixed according to the formula to prepare the enrichment solution.

5. The preparation method according to claim 4, wherein The preparation method of the potato glucose aqueous solution is as follows: potatoes, glucose and water are mixed according to the formula, and heated and boiled to obtain the potato glucose aqueous solution; And / or, the potato glucose aqueous solution is prepared by the following method: mixing potatoes with water, heating and boiling, and then adding glucose to prepare the potato glucose aqueous solution; after boiling, keeping the mixture for 10 min to 20 min; And / or, the potato glucose aqueous solution is prepared by: mixing potatoes, glucose, and water according to the formula, heating and boiling, then filtering through gauze, adding water to the filtered filtrate to make up to the initial volume, then adding glucose, heating to dissolve, mixing, and sterilizing to obtain the potato glucose aqueous solution; the sterilization is performed by high-pressure steam sterilization at 121° C. for 15 to 30 minutes.

6. Use of the enrichment solution according to any one of claims 1 to 3 in the preparation of a reagent for detecting Burkholderia gladiolus (Pseudomonas cocovenenans subsp. fermentans).

7. Use of the enrichment solution according to any one of claims 1 to 3 in preparing a reagent for screening Burkholderia gladiolus (Pseudomonas cocovenenans subsp. fermentans).

8. Use of the enrichment solution according to any one of claims 1 to 3 in preparing a reagent for culturing Burkholderia gladiolus (Pseudomonas cocovenenans subsp. fermentans).

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