Liquid separation culture medium for helicobacter pylori as well as preparation method and application of liquid separation culture medium

Abstract

The invention relates to the technical field of culture of helicobacter pylori, and discloses a liquid separation culture medium for helicobacter pylori and a preparation method and application thereof, the liquid separation culture medium comprises the following raw materials by mass: 82-86.5 parts of Brinell broth, 10 parts of an antibiotic solution, 0.5-3 parts of an enrichment broth, and 3-5 parts of fetal calf serum. By adopting the helicobacter pylori liquid separation culture medium provided by the scheme, a large amount of helicobacter pylori can be rapidly cultured, and the helicobacter pylori can reach a growth logarithmic phase within 8 hours. Helicobacter pylori can also be obtained by optimizing a culture medium to culture a gastric mucosa sample, the helicobacter pylori cannot be separated from the gastric mucosa sample by a culture medium without adding fetal calf serum or enrichment fluid, and in addition, the culture effect is poor due to the fact that too much or too little fetal calf serum and enrichment fluid are added. Therefore, the liquid culture medium is optimized, separation of the helicobacter pylori does not depend on a solid agar culture medium through the synergistic effect of the fetal calf serum and the enrichment broth, and the growth speed and the culture positive rate of the helicobacter pylori are increased.

Description

Technical Field

[0001] The present invention relates to the technical field of Helicobacter pylori culture, and in particular to a liquid separation culture medium for Helicobacter pylori, a preparation method thereof and an application thereof. Background Art

[0002] Helicobacter pylori ( Helicobacterpylori H. pylori (H. pylori), a motile, helical, flagellated, Gram-negative bacterium, was discovered in the mid-to-late 19th century. However, due to its inability to be cultured in vitro, it remained largely unappreciated until its successful isolation and culture in 1982. It has been shown to be a major pathogen in various gastrointestinal diseases, including chronic gastritis and peptic ulcers, and is a high-risk factor for gastric cancer. Given its serious potential, in-depth research into the biological characteristics of H. pylori to guide clinical diagnosis and treatment is of paramount importance.

[0003] Currently, the cultivation of Helicobacter pylori primarily relies on traditional isolation media, such as Columbia agar and Brucella broth agar. However, as a microaerophilic bacterium, Helicobacter pylori requires demanding in vitro culture conditions, slow growth, and strict requirements for the gas environment and culture medium. In traditional isolation media, Helicobacter pylori metabolizes slowly, requiring 3-5 days to reach the logarithmic phase. This long growth cycle makes it highly susceptible to contamination by other bacteria, resulting in a low positive detection rate and making it difficult to meet the needs of rapid and efficient culture. For Helicobacter pylori strain identification and bacterial resistance research, the first step is to isolate and culture it from the gastric mucosa. However, under existing technologies, H. pylori cannot be isolated from gastric mucosal tissue in liquid culture medium, which greatly hinders the development of related experiments and research. Therefore, the development of a new culture medium that can improve the positive rate of Helicobacter pylori culture, achieve rapid and large-scale culture, and isolate H. pylori in liquid culture medium has become a key technical challenge that needs to be overcome. Summary of the Invention

[0004] The present invention aims to provide a liquid separation culture medium for Helicobacter pylori, its preparation method and application, in order to solve the technical problem that the growth rate of Helicobacter pylori cultured in existing culture media is too slow, for example, it takes 3 to 5 days to grow to the logarithmic phase, resulting in a long growth cycle, a decreased positive detection rate, and difficulty in meeting the needs of rapid and efficient culture.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a liquid isolation culture medium for Helicobacter pylori, comprising the following raw materials in parts by mass: 82-86.5 parts of Brucella broth, 10 parts of antibiotic solution, 0.5-3 parts of enrichment solution, and 3-5 parts of fetal bovine serum.

[0006] Preferably, as an improvement, the antibiotic solution contains the following components in concentrations: vancomycin hydrochloride 0.30-0.32 g / L, water-soluble amphotericin B 0.30-0.32 g / L, trimethoprim 0.14-0.16 g / L, polymyxin B sulfate 0.015-0.025 g / L, and the balance is sterile water.

[0007] Preferably, as an improvement, the enrichment solution contains the following components at the following concentrations: adenine 1-1.5 g / L, guanine hydrochloride 0.03-0.06 g / L, L-cysteine ​​hydrochloride 23-25 ​​mg / L, L-cystine 0.8-1.2 g / L, ferric nitrate 10-20 mg / L, L-glutathione 8-12 g / L, VB1 1.5-4 mg / L, and the balance is sterile water.

[0008] Preferably, as an improvement, the pH value of the culture medium is 7.0-7.4.

[0009] Preferably, as an improvement, the present invention further provides a method for preparing a liquid separation medium for Helicobacter pylori, comprising the following steps: S1. Dissolve Brucella broth in pure water according to the formula and sterilize to obtain a solution; S2. Cool the solution prepared in S1, add fetal bovine serum, antibiotic solution, and enrichment solution, adjust the pH to 7.0-7.4, and pour into a glass bottle to prepare a Helicobacter pylori liquid isolation culture medium.

[0010] Preferably, as an improvement, in S1, the temperature of the sterilization treatment is 111-131° C.; and the time of the sterilization treatment is 10-20 minutes.

[0011] Preferably, as an improvement, in S2, the temperature of the solution after cooling is 50-55°C.

[0012] Preferably, as an improvement, in S2, the antibiotic solution is prepared by dissolving vancomycin hydrochloride, water-soluble amphotericin B, trimethoprim and polymyxin B sulfate in sterile water.

[0013] Preferably, as an improvement, in S2, the enrichment solution is prepared by dissolving adenine, guanine hydrochloride, L-cysteine ​​hydrochloride, L-cystine, ferric nitrate, L-glutathione and VB1 in sterile water and filtering and sterilizing.

[0014] Preferably, as an improvement, the present invention further provides an application of a Helicobacter pylori liquid separation culture medium, comprising the following steps: Step (1), culturing, isolating and purifying on a plate to obtain an Hp standard strain, eluting the strain with a 0.85% NaCl solution and suspending the Hp cells, and diluting them for later use; Step (2), placing the mucosa obtained under gastroscopy into a transport medium to obtain a gastric mucosa sample for later use; Step (3), after the liquid separation culture medium is divided into aliquots, the aliquots are used to inoculate the Hp standard strain and the gastric mucosal sample respectively; Step (4), culturing under microaerobic conditions and continuously sampling to determine OD values ​​to obtain a culture OD curve.

[0015] The principles and advantages of this solution are: 1. The culture provided by the present invention contains Brucella broth, fetal bovine serum and a variety of growth-promoting factors to meet the high nutritional needs of Helicobacter pylori growth. Brucella broth, as the basic nutrient in the culture medium, provides the carbon source required for bacterial growth. Based on its rich nutritional components, the enrichment solution can provide Helicobacter pylori with the nutrients required for cell growth and proliferation, thereby promoting the rapid growth of Helicobacter pylori. Optimizing the ratio of fetal bovine serum and enrichment solution can enable Helicobacter pylori to multiply rapidly in large quantities. The separation culture medium of the present invention uses a microaerobic working platform to separate and culture Helicobacter pylori, which can improve the culture positive rate.

[0016] 2. Compared with the traditional isolation culture medium, in which Helicobacter pylori metabolism is slow and it takes 3 to 5 days to grow to the logarithmic phase, the liquid isolation culture medium prepared by this scheme can effectively promote the rapid growth of Helicobacter pylori, so that Helicobacter pylori can reach the logarithmic phase in about 8 hours of culture, significantly shortening the culture cycle, improving the culture efficiency and culture positivity rate, and fully meeting the needs of rapid and efficient culture of Helicobacter pylori.

[0017] 3. The method for preparing Helicobacter pylori liquid culture medium in this scheme contains antibiotics, which can inhibit the miscellaneous bacteria in the gastric mucosal sample, while Brucella broth, fetal bovine serum and enrichment liquid can provide rich nutrients, so that Helicobacter pylori in the gastric mucosa can be directly isolated in the liquid culture medium, further shortening the efficiency of Helicobacter pylori isolation and culture to meet the requirements of subsequent research. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a culture OD curve of the liquid separation culture medium prepared in Example 1 of the present invention.

[0019] Figure 2 This is a culture OD curve of the liquid separation culture medium prepared in Comparative Example 1 of the present invention.

[0020] Figure 3 This is a culture OD curve of the liquid separation culture medium prepared in Comparative Example 2 of the present invention.

[0021] Figure 4 This is a culture OD curve of the liquid separation culture medium prepared in Comparative Example 3 of the present invention.

[0022] Figure 5 This is a culture OD curve of the liquid separation culture medium prepared in Comparative Example 4 of the present invention.

[0023] Figure 6 This is a culture OD curve of the liquid separation culture medium prepared in Comparative Example 5 of the present invention.

[0024] Figure 7 This is a culture OD curve of the liquid separation culture medium prepared in Comparative Example 6 of the present invention.

[0025] Figure 8 This is a culture OD curve of the liquid separation culture medium prepared in Comparative Example 7 of the present invention. DETAILED DESCRIPTION

[0026] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available.

[0027] Program Overview The present invention provides a liquid isolation culture medium for Helicobacter pylori, comprising the following raw materials in parts by weight: 82-86.5 parts of Brucella broth, 10 parts of antibiotic solution, 0.5-3 parts of enrichment solution, and 3-5 parts of fetal bovine serum.

[0028] The antibiotic solution contains the following components at the following concentrations: vancomycin hydrochloride 0.30-0.32 g / L, water-soluble amphotericin B 0.30-0.32 g / L, trimethoprim 0.14-0.16 g / L, polymyxin B sulfate 0.015-0.025 g / L, with the balance being sterile water. The enrichment broth contains the following components at the following concentrations: adenine 1-1.5 g / L, guanine hydrochloride 0.03-0.06 g / L, L-cysteine ​​hydrochloride 23-25 ​​mg / L, L-cystine 0.8-1.2 g / L, ferric nitrate 10-20 mg / L, L-glutathione 8-12 g / L, and VB1 1.5-4 mg / L, with the balance being sterile water. The pH of the culture medium is 7.0-7.4.

[0029] This solution also provides a method for preparing a liquid isolation culture medium for Helicobacter pylori, comprising the following steps: S1. Dissolve Brucella broth in pure water according to the formula, and sterilize at 111-131° C. for 10-20 minutes to obtain a solution; S2. Cool the solution prepared in S1 to 50-55° C., add fetal bovine serum, antibiotic solution and enrichment solution, adjust the pH and pour into a glass bottle to prepare a Helicobacter pylori liquid isolation culture medium.

[0030] The antibiotic solution is prepared by dissolving vancomycin hydrochloride, water-soluble amphotericin B, trimethoprim, and polymyxin B sulfate in sterile water. The enrichment solution is prepared by dissolving adenine, guanine hydrochloride, L-cysteine ​​hydrochloride, L-cystine, ferric nitrate, L-glutathione, and VB1 in sterile water and filtering and sterilizing.

[0031] This solution also provides an application of a Helicobacter pylori liquid isolation culture medium, comprising the following steps: Step (1) culture, isolate and purify the Hp standard strain on Columbia blood agar plate (95% Columbia agar + 5% sterile defibrinated sheep blood), elute the strain with 0.85% NaCl solution and suspend the Hp cells, dilute to OD 600 It is reserved after 0.5; In step (2), the mucosa obtained under gastroscopy is placed in a transport medium (the formula is Brucella broth supplemented with 5% calf serum) to obtain a gastric mucosal sample for later use; Step (3) The liquid separation medium was divided into four 15 mL centrifuge tubes, each with a volume of 7.5 mL. Two of the tubes were inoculated with the H. pylori standard strain, each with an inoculation volume of 75 μL, and the other two tubes were inoculated with gastric mucosal samples; Step (4), culturing at 37° C. under microaerobic conditions for 5 days, measuring OD values ​​at 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h to obtain a culture OD curve.

[0032] Example 1 A method for preparing a liquid separation medium for Helicobacter pylori comprises the following steps: (1) Dissolve 2.81 g of Brucella broth in 65 mL of pure water and sterilize at 121°C for 15 minutes. (2) Dissolve 320 mg of vancomycin hydrochloride, 320 mg of water-soluble amphotericin B, 160 mg of trimethoprim, and 20 mg of polymyxin B sulfate in 100 mL of sterile water to prepare an antibiotic solution for later use; (3) Dissolve 0.012 g of adenine, 0.0005 g of guanine hydrochloride, 0.24 mg of L-cysteine ​​hydrochloride, 0.01 g of L-cystine, 0.1 mg of ferric nitrate, 0.1 g of L-glutathione, and 0.025 mg of VB1 in 10 mL of sterile water, filter through a 0.2 μm filter membrane to sterilize, and prepare a bacterial enrichment solution for later use; (4) The solution obtained in step (1) was cooled to 50° C., and 15 mL of fetal bovine serum, 10 mL of antibiotics, and 10 mL of enrichment solution were added to prepare 100 mL of Helicobacter pylori liquid isolation culture medium. Comparative Example 1 Compared with Example 1, the difference is that no fetal bovine serum and enrichment solution are added, and the steps are as follows: (1) Dissolve 3.85 g of Brucella broth in 90 mL of pure water and sterilize at 121°C for 15 minutes. (2) Dissolve 320 mg of vancomycin hydrochloride, 320 mg of water-soluble amphotericin B, 160 mg of trimethoprim, and 20 mg of polymyxin B sulfate in 100 mL of sterile water to prepare an antibiotic solution for later use; (3) The solution obtained in step (1) was cooled to 50° C. and 10 mL of antibiotics was added to prepare 100 mL of Helicobacter pylori liquid isolation culture medium. Comparative Example 2 Compared with Example 1, the difference is that no enrichment solution is added, and the steps are as follows: (1) Dissolve 2.81 g of Brucella broth in 75 mL of pure water and sterilize at 121°C for 15 minutes. (2) Dissolve 320 mg of vancomycin hydrochloride, 320 mg of water-soluble amphotericin B, 160 mg of trimethoprim, and 20 mg of polymyxin B sulfate in 100 mL of sterile water to prepare an antibiotic solution for later use; (3) The solution obtained in step (1) was cooled to 50° C., and 10 mL of antibiotics and 15 mL of fetal bovine serum were added to prepare 100 mL of Helicobacter pylori liquid isolation culture medium. Comparative Example 3 Compared with Example 1, the difference is that no fetal bovine serum is added, and the steps are as follows: (1) Dissolve 2.81 g of Brucella broth in 80 mL of pure water and sterilize at 121°C for 15 minutes. (2) Dissolve 320 mg of vancomycin hydrochloride, 320 mg of water-soluble amphotericin B, 160 mg of trimethoprim, and 20 mg of polymyxin B sulfate in 100 mL of sterile water to prepare an antibiotic solution for later use; (3) Dissolve 0.012 g of adenine, 0.0005 g of guanine hydrochloride, 0.24 mg of L-cysteine ​​hydrochloride, 0.01 g of L-cystine, 0.1 mg of ferric nitrate, 0.1 g of L-glutathione, and 0.025 mg of VB1 in 10 mL of sterile water, filter through a 0.2 μm filter membrane to sterilize, and prepare a bacterial enrichment solution for later use; (4) After cooling the solution obtained in step (1) to 50° C., 10 mL of antibiotics and 10 mL of bacterial enrichment solution were added to prepare 100 mL of Helicobacter pylori liquid isolation culture medium. Comparative Example 4 Compared with Example 1, the difference is that the amount of fetal bovine serum used is too small, and the steps are as follows: (1) Dissolve 2.81 g of Brucella broth in 78 mL of pure water and sterilize at 121°C for 15 minutes. (2) Dissolve 320 mg of vancomycin hydrochloride, 320 mg of water-soluble amphotericin B, 160 mg of trimethoprim, and 20 mg of polymyxin B sulfate in 100 mL of sterile water to prepare an antibiotic solution for later use; (3) Dissolve 0.012 g of adenine, 0.0005 g of guanine hydrochloride, 0.24 mg of L-cysteine ​​hydrochloride, 0.01 g of L-cystine, 0.1 mg of ferric nitrate, 0.1 g of L-glutathione, and 0.025 mg of VB1 in 10 mL of sterile water, filter through a 0.2 μm filter membrane to sterilize, and prepare a bacterial enrichment solution for later use; (4) The solution obtained in step (1) was cooled to 50° C., and 10 mL of antibiotics, 2 mL of fetal bovine serum, and 10 mL of enrichment solution were added to prepare 100 mL of Helicobacter pylori liquid isolation culture medium. Comparative Example 5 Compared with Example 1, the difference is that the amount of fetal bovine serum used is too much, and the steps are as follows: (1) Dissolve 2.81 g of Brucella broth in 52 mL of pure water and sterilize at 121°C for 15 minutes. (2) Dissolve 320 mg of vancomycin hydrochloride, 320 mg of water-soluble amphotericin B, 160 mg of trimethoprim, and 20 mg of polymyxin B sulfate in 100 mL of sterile water to prepare an antibiotic solution for later use; (3) Dissolve 0.012 g of adenine, 0.0005 g of guanine hydrochloride, 0.24 mg of L-cysteine ​​hydrochloride, 0.01 g of L-cystine, 0.1 mg of ferric nitrate, 0.1 g of L-glutathione, and 0.025 mg of VB1 in 10 mL of sterile water, filter through a 0.2 μm filter membrane to sterilize, and prepare a bacterial enrichment solution for later use; (4) The solution obtained in step (1) was cooled to 50° C., and 10 mL of antibiotics, 28 mL of fetal bovine serum, and 10 mL of enrichment solution were added to prepare 100 mL of Helicobacter pylori liquid isolation culture medium. Comparative Example 6 Compared with Example 1, the difference is that the amount of enrichment solution used is too small. The steps are as follows: (1) Dissolve 2.81 g of Brucella broth in 74 mL of pure water and sterilize at 121°C for 15 minutes. (2) Dissolve 320 mg of vancomycin hydrochloride, 320 mg of water-soluble amphotericin B, 160 mg of trimethoprim, and 20 mg of polymyxin B sulfate in 100 mL of sterile water to prepare an antibiotic solution for later use; (3) Dissolve 0.012 g of adenine, 0.0005 g of guanine hydrochloride, 0.24 mg of L-cysteine ​​hydrochloride, 0.01 g of L-cystine, 0.1 mg of ferric nitrate, 0.1 g of L-glutathione, and 0.025 mg of VB1 in 10 mL of sterile water, filter through a 0.2 μm filter membrane to sterilize, and prepare a bacterial enrichment solution for later use; (4) The solution obtained in step (1) was cooled to 50° C., and 10 mL of antibiotics, 15 mL of fetal bovine serum, and 1 mL of enrichment solution were added to prepare 100 mL of Helicobacter pylori liquid isolation culture medium. Comparative Example 7 Compared with Example 1, the difference is that the amount of enrichment solution is too much, and the steps are as follows: (1) Dissolve 2.81 g of Brucella broth in 55 mL of pure water and sterilize at 121°C for 15 minutes. (2) Dissolve 320 mg of vancomycin hydrochloride, 320 mg of water-soluble amphotericin B, 160 mg of trimethoprim, and 20 mg of polymyxin B sulfate in 100 mL of sterile water to prepare an antibiotic solution for later use; (3) Dissolve 0.024 g of adenine, 0.001 g of guanine hydrochloride, 0.48 mg of L-cysteine ​​hydrochloride, 0.02 g of L-cystine, 0.2 mg of ferric nitrate, 0.2 g of L-glutathione, and 0.05 mg of VB1 in 20 mL of sterile water, filter through a 0.2 μm filter membrane to sterilize, and prepare a bacterial enrichment solution for later use; (4) The solution obtained in step (1) was cooled to 50° C., and 10 mL of antibiotics, 15 mL of fetal bovine serum, and 20 mL of enrichment solution were added to prepare 100 mL of Helicobacter pylori liquid isolation culture medium.

[0033] Experimental Example 1: Detection of the effect of Helicobacter pylori liquid isolation culture medium The optimized culture medium of Example 1 has the following characteristics: richer nutrients, including components such as fetal bovine serum and enrichment solution.

[0034] An application of a Helicobacter pylori liquid separation culture medium for isolating, culturing, and amplifying the culture to obtain a Helicobacter pylori logarithmic phase bacterial liquid comprises the following steps: (1) Cultivate, isolate, and purify the H. pylori standard strain on a plate, elute the strain with 0.85% NaCl solution, suspend the H. pylori cells, and dilute to an OD600 of approximately 0.5 for later use.

[0035] (2) The mucosa obtained under gastroscopy is placed in transport medium (Bruch's broth supplemented with 5% calf serum) to obtain a gastric mucosal sample for later use.

[0036] (3) The culture medium prepared according to the formula and method of Example 1 was dispensed into four 15 mL centrifuge tubes, each with a volume of 7.5 mL. Two tubes were inoculated with the H. pylori standard strain, each with an inoculation volume of 75 μL, and the other two tubes were inoculated with gastric mucosal samples.

[0037] (4) Culture at 37°C under microaerobic conditions for 5 days, and measure the OD value at 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h.

[0038] The optimized medium of Example 1 was used to culture the standard strain of Hp. The strain reached the logarithmic growth phase at 8 hours and the growth peak at 72 hours (3 days), with an OD value of 0.4111. When the gastric mucosa sample was inoculated, Hp growth was observed at 8 hours and stabilized at 48 hours, with an OD value fluctuating around 0.2. The specific culture OD curve is shown in FIG. Figure 1 shown.

[0039] Experimental Example 2 Comparative Example 1 was used as the optimized culture medium, which was characterized by lack of nutrients and no addition of fetal bovine serum, enrichment solution, etc. The steps were the same as those in Experimental Example 1.

[0040] The optimized culture medium of Comparative Example 1 was used to culture Helicobacter pylori. The results showed that Helicobacter pylori hardly grew and could not be isolated from the gastric mucosa sample. The OD curve was as follows: Figure 2 shown.

[0041] Experimental Example 3 Comparative Example 2 was used as the optimized culture medium, which had the following characteristics: no enrichment solution was added. The steps were the same as those in Experimental Example 1.

[0042] When the optimized culture medium of comparative example 2 was inoculated with the standard strain of Hp, the bacteria reached the logarithmic growth phase in 24 hours and stabilized in 72 hours, with the maximum OD value of 0.3364. When the gastric mucosa sample was inoculated, the OD value did not change significantly, and no signs of Hp growth were observed. The OD curve was as follows: Figure 3 shown.

[0043] Experimental Example 4 Comparative Example 3 was used as the optimized culture medium, which had the following characteristics: no fetal bovine serum was added. The steps were the same as those in Experimental Example 1.

[0044] When the optimized culture medium of comparative example 3 was inoculated with the standard strain of Hp, the bacteria reached the logarithmic growth phase in 24 hours, stabilized in 72 hours, and the maximum OD value was 0.1156; when the gastric mucosa sample was inoculated, the OD value showed a slow growth trend, the maximum OD value was 0.073, and the OD curve was as follows: Figure 4 shown.

[0045] Experimental Example 5 Comparative Example 4 was used as the optimized culture medium, which had the following characteristics: too little fetal bovine serum was used. The steps were the same as those in Experimental Example 1.

[0046] The optimized culture medium of comparative example 4 was inoculated with the Hp standard strain. The bacteria reached the logarithmic growth phase in 24 hours and stabilized in 48 hours. The maximum OD value was 0.123. When the gastric mucosa sample was inoculated, the OD value showed a slow growth trend with a small change value. The maximum OD value was 0.077. The OD curve was as follows: Figure 5 shown.

[0047] Experimental Example 6 Comparative Example 5 was used as the optimized culture medium, which had the following characteristics: excessive amount of fetal bovine serum was used. The steps were the same as those in Experimental Example 1.

[0048] The optimized culture medium of comparative example 5 was inoculated with Hp standard strains and gastric mucosa samples, and there was no significant change in OD. The OD curve was as follows: Figure 6 shown.

[0049] Experimental Example 7 Comparative Example 6 was used as the optimized culture medium, which had the following characteristics: too little amount of enrichment solution was used. The steps were the same as those in Experimental Example 1.

[0050] When the optimized culture medium of comparative example 6 was inoculated with Hp standard strains and gastric mucosa samples, no obvious growth signs were observed. The OD curve was as follows: Figure 7 shown.

[0051] Experimental Example 8 Comparative Example 7 was used as the optimized culture medium, which had the following characteristics: too little amount of enrichment solution was used. The steps were the same as those in Experimental Example 1.

[0052] When the optimized culture medium of comparative example 7 was inoculated with the Hp standard strain, the bacteria reached the logarithmic growth phase in 4 hours and stabilized in 8 hours, with a maximum OD value of 0.156; when the gastric mucosa sample was inoculated, the growth trend of the sample was the same as that of the standard strain, with a maximum OD value of 0.096. The OD curve is as follows: Figure 7 shown.

[0053] In summary, the helicobacter pylori liquid separation culture medium that utilizes present embodiment 1 to prepare can cultivate helicobacter pylori in a large number quickly, makes its 8h arrive growth logarithmic phase.Optimizing culture medium to cultivate gastric mucosa sample also can obtain helicobacter pylori bacterium, and the culture medium that does not add foetal calf serum or enrichment liquid can not separate and obtain helicobacter pylori from gastric mucosa sample, in addition, adding foetal calf serum and enrichment liquid too much or too little all causes poor culture effect.Therefore optimizing liquid culture medium makes the separation of helicobacter pylori independent of solid agar medium by the synergistic effect of foetal calf serum and enrichment liquid, and improves growth rate and the culture positive rate of helicobacter pylori.

[0054] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A liquid separation medium for Helicobacter pylori, characterized in that: The preparation comprises the following raw materials in parts by weight: 82-86.5 parts of Brucella broth, 10 parts of antibiotic solution, 0.5-3 parts of bacterial enrichment solution, and 3-5 parts of fetal bovine serum.

2. The liquid separation medium for Helicobacter pylori according to claim 1, wherein: The antibiotic solution contains the following components in the following concentrations: 0.30-0.32 g / L of vancomycin hydrochloride, 0.30-0.32 g / L of water-soluble amphotericin B, 0.14-0.16 g / L of trimethoprim, 0.015-0.025 g / L of polymyxin B sulfate, and the balance is sterile water.

3. The liquid separation medium for Helicobacter pylori according to claim 2, wherein: The enrichment solution contains the following components at the following concentrations: 1-1.5 g / L adenine, 0.03-0.06 g / L guanine hydrochloride, 23-25 ​​mg / L L-cysteine ​​hydrochloride, 0.8-1.2 g / L L-cystine, 10-20 mg / L ferric nitrate, 8-12 g / L L-glutathione, 1.5-4 mg / L VB1, and the balance is sterile water.

4. The liquid separation medium for Helicobacter pylori according to claim 3, characterized in that: The pH value of the culture medium is 7.0-7.

4.

5. The method for preparing a liquid separation medium for Helicobacter pylori according to claim 4, characterized in that: The steps include: S1. Dissolve Brucella broth in pure water according to the formula and sterilize to obtain a solution; S2. Cool the solution prepared in S1, add fetal bovine serum, antibiotic solution and enrichment solution, adjust the pH and pour into a glass bottle to prepare a Helicobacter pylori liquid isolation culture medium.

6. The method for preparing a liquid separation medium for Helicobacter pylori according to claim 5, characterized in that: In S1, the temperature of the sterilization treatment is 111-131° C.; the time of the sterilization treatment is 10-20 minutes.

7. The method for preparing a liquid separation medium for Helicobacter pylori according to claim 6, characterized in that: In S2, the temperature of the solution after cooling is 50-55°C.

8. The method for preparing a liquid separation medium for Helicobacter pylori according to claim 7, characterized in that: In S2, the antibiotic solution is prepared by dissolving vancomycin hydrochloride, water-soluble amphotericin B, trimethoprim and polymyxin B sulfate in sterile water.

9. The method for preparing a liquid separation medium for Helicobacter pylori according to claim 8, characterized in that: In S2, the enrichment solution is prepared by dissolving adenine, guanine hydrochloride, L-cysteine ​​hydrochloride, L-cystine, ferric nitrate, L-glutathione and VB1 in sterile water and filtering and sterilizing.

10. An application of a Helicobacter pylori liquid isolation culture medium, characterized in that: The steps include: Step (1), culturing, isolating and purifying on a plate to obtain an Hp standard strain, eluting the strain with a 0.85% NaCl solution and suspending the Hp cells, and diluting them for later use; Step (2), placing the mucosa obtained under gastroscopy into a transport medium to obtain a gastric mucosa sample for later use; Step (3), packaging the liquid separation culture medium obtained by the preparation method according to any one of claims 5 to 9, and using the packaging to inoculate the Hp standard strain and the gastric mucosal sample, respectively; Step (4), culturing under microaerobic conditions and continuously sampling to determine OD values ​​to obtain a culture OD curve.

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