Helicobacter pylori allochroic isolation medium as well as preparation method and application thereof

Abstract

The invention provides a helicobacter pylori allochroic isolation medium as well as a preparation method and application thereof, and belongs to the technical field of helicobacter pylori culture. The helicobacter pylori color-changing separation culture medium is prepared from the following components in concentration: 4 to 6 g / L of bovine brain extract powder, 4 to 6 g / L of bovine heart extract powder, 10 to 15 g / L of agar, 21 to 25 g / L of casein peptone, 2.5 to 3.5 g / L of glucose, 4 to 6 g / L of sodium chloride, 2.5 to 3.5 g / L of disodium hydrogen phosphate, 0.5 to 1.5 g / L of beta-cyclodextrin, 1.5 to 2.5 g / L of urea, 0.1 to 0.3 g / L of bromothymol blue, 0.01 to 0.05 mL / L of bacteriostatic agent, 80 to 100 mL / L of calf serum and a solvent of water. The culture medium can obviously improve the separation culture success rate of the helicobacter pylori clinical strain, and has the characteristics of high efficiency, specificity, high bacterial colony activity and color change.

Description

Technical Field

[0001] The present invention relates to the technical field of Helicobacter pylori culture, and particularly relates to a Helicobacter pylori discoloration isolation medium, a preparation method thereof, and an application thereof. Background Art

[0002] Helicobacter pylori (HP) is a Gram-negative bacterium with a slender and curved spiral shape. It is mainly colonized in the human gastric mucosa, and the infection rate worldwide is 50-70%. HP can induce inflammatory activities and specific immune responses in the gastric mucosa, resulting in chronic gastritis of the gastric mucosa. If the infection persists, atypical hyperplasia and metaplasia will further occur, and ultimately some hosts will develop gastric cancer. HP infection can also cause a series of diseases, such as gastritis, peptic ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, etc. The World Health Organization listed HP as a Class I carcinogen for gastric cancer as early as 1994. The Kyoto expert consensus in 2016 defined HP gastritis as an infectious disease and pointed out that patients infected with HP should be treated.

[0003] At present, the combined use of proton pump inhibitors (PPI), antibacterial drugs (such as amoxicillin, clarithromycin, levofloxacin, metronidazole, etc.), and bismuth-containing compounds is the main treatment method for eradicating HP. However, due to the improper use of antibacterial drugs, the drug resistance rate of HP to commonly used antibacterial drugs has been increasing year by year (some have exceeded 15%), resulting in a significant decrease in the eradication rate. Therefore, the drug sensitivity test based on the isolation and culture of HP is the key to achieving precise treatment for infected patients. However, since HP is a recognized fastidious bacterium, it has strict requirements for the nutrient matrix and gas environment, and the gastric mucosa collected from the stomach is contaminated with various miscellaneous bacteria. Direct culture is easily inhibited by other miscellaneous bacteria, resulting in separation failure, low success rate of clinical specimen isolation and culture, and greatly limiting the development of HP isolation and culture and in vitro drug sensitivity tests in clinical practice.

[0004] At present, there are many diagnostic methods for HP infection. Commonly used clinically are serum antibody detection, urea breath test, rapid urease test, gastric tissue culture, polymerase chain reaction, etc. Among them, the method of culturing gastric mucosa samples in a microaerophilic environment (85% N2, 10% CO2, and 5% O2) is the most reliable, with a specificity of up to 100%, and is considered the gold standard for HP diagnosis.

[0005] The commonly used HP medium in clinical laboratories is mostly composed of commercial basic nutrients such as Columbia agar and brain heart infusion agar, plus sterile defibrinated sheep blood and HP bacteriostatic agent. Although HP can grow on these media, affected by various factors such as specimen collection and growth of miscellaneous bacteria, the detection rate of clinical specimens is low, and the culture time required is long, resulting in a long cycle for drug sensitivity reports and delaying the timely treatment of patients. The storage condition of sterile defibrinated sheep blood is 2-8°C, and its shelf life is only one month. It is very easy to expire after being ordered by the laboratory, resulting in waste. Improper selection of bacteriostatic agents will also inhibit the growth of HP, leading to a low success rate of culturing clinical specimens. It can be seen that many factors restricting the development of HP culture in laboratories need to be solved urgently.

[0006] Chromogenic medium is a new type of medium that uses the principle of the reaction between enzymes produced by microorganisms themselves and corresponding chromogenic substrates to change color to identify microorganisms. Using chromogenic medium for the screening and isolation of microorganisms, its sensitivity and specificity are much better than those of traditional media. HP can produce urease, which can decompose urea to produce ammonia and carbon dioxide, and ammonia is an alkaline substance that can increase the pH. Bromothymol blue is an acid-base indicator, and its color change range is pH 6.0-7.6. It is yellow in the acidic state and blue in the alkaline state, and its color change range just includes the optimal growth pH of HP. At the same time, bromothymol blue is acidic in the dissolved state, and this characteristic can also serve as an acid-base regulator.

[0007] Therefore, in view of the above-mentioned many restrictive problems existing in the clinical development of HP medium, the inventor has developed a solid chromogenic selective medium through a large number of experiments, which can significantly improve the success rate of culturing HP clinical specimens and at the same time shorten the HP culture cycle. Summary of the Invention

[0008] The purpose of the present invention is to provide a HP chromogenic separation medium, its preparation method and application, which can significantly improve the success rate of culturing clinical strains, and make this medium have the characteristics of high efficiency, specificity, high colony activity and color change by adding substances such as indicators, selective bacteriostatic agents and calf serum.

[0009] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0010] The present invention provides a Helicobacter pylori chromogenic separation medium, which comprises the following components in the following concentrations: bovine brain extract powder 4-6 g / L, bovine heart extract powder 4-6 g / L, agar 10-15 g / L, casein peptone 21-25 g / L, glucose 2.5-3.5 g / L, sodium chloride 4-6 g / L, disodium hydrogen phosphate 2.5-3.5 g / L, β-cyclodextrin 0.5-1.5 g / L, urea 1.5-2.5 g / L, bromothymol blue 0.1-0.3 g / L, bacteriostatic agent 0.01-0.05 mL / L, calf serum 80-100 mL / L, and the solvent is water.

[0011] Preferably, the components and concentrations of the bacteriostatic agent are: amphotericin B 2-4 mg / L, vancomycin 3-6 mg / L, polymyxin 1-2 mg / L, trimethoprim 3-5 mg / L.

[0012] Preferably, the pH value of the culture medium is 7.1-7.3.

[0013] The present invention provides a method for preparing the Helicobacter pylori discoloration isolation medium. Dissolve bovine brain extract powder, bovine heart extract powder, agar, casein peptone, glucose, sodium chloride, disodium hydrogen phosphate, β-cyclodextrin, and bromothymol blue in water, autoclave, and after cooling, add a bacteriostatic agent, urea, and calf serum. After adjusting the pH value, pour it into a disposable sterile petri dish. After cooling and solidifying, the Helicobacter pylori discoloration isolation medium can be obtained.

[0014] The present invention provides an application of the Helicobacter pylori discoloration isolation medium or the Helicobacter pylori discoloration isolation medium prepared by the method in the high-efficiency screening of clinical strains of Helicobacter pylori.

[0015] The present invention provides a method for high-efficiency screening of clinical strains of Helicobacter pylori, including the following steps:

[0016] (1) Collect gastric mucosa specimens from Helicobacter pylori-infected patients and make them into homogenates;

[0017] (2) Inoculate the homogenate obtained in step (1) on the Helicobacter pylori discoloration isolation medium or the Helicobacter pylori discoloration isolation medium prepared by the method for culture.

[0018] Preferably, in step (1), the gastric mucosa specimen is a gastric mucosa specimen from the gastric antrum and / or gastric body.

[0019] Preferably, in step (2), the inoculation amount of the homogenate is 0.1-0.3 mL.

[0020] Preferably, in step (2), the culture is carried out in a microaerophilic environment, and the microaerophilic environment is 85% N2, 10% CO2, and 5% O2; the temperature during the culture is 35-37 °C, and the time is 20-120 h.

[0021] Preferably, in step (2), if there are pinpoint-like small blue colonies growing on the culture medium and the plate turns blue, it is initially judged that the Helicobacter pylori culture is positive; if the culture medium turns yellow and no pinpoint-like small colonies are seen, it is judged that the Helicobacter pylori culture is negative.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a new type of high - efficiency HP color - changing isolation medium. Among them, calf serum is used to replace traditional defibrinated sheep blood. Calf serum is more nutritious, contains a higher content of factors promoting the growth of HP, and can be quantitatively dispensed and stored frozen. It has a longer storage period than defibrinated sheep blood and is more convenient to use. A pH indicator is added. After HP grows on the culture plate, both the colonies and the plate can change color, enabling more intuitive observation of the growth of HP. The added pH indicator is bromothymol blue, which is an acidic solution after dissolution. It not only serves as an indicator but also has the function of adjusting pH. Since the pH of calf serum is significantly higher than that of sheep blood, adding it increases the overall pH of the medium, which is not conducive to the growth of HP. After quantitatively adding bromothymol blue, the pH of the medium can be maintained at 7.1 - 7.3. The added β - cyclodextrin has characteristics such as antioxidant, increasing water solubility, and improving bioavailability, thereby improving the utilization efficiency of various nutrients in the medium.

[0024] The present invention also tested the addition amount of traditional bacteriostatic agents and improved the addition amount of each antibiotic to ensure effective inhibition of non - target bacteria without affecting the growth of HP, thereby making the success rate of primary culture of HP in clinical specimens higher.

[0025] The HP color - changing isolation medium prepared by the present invention has the color - changing property and can be used for the primary isolation culture and sub - culture of clinical HP strains. It has a high detection rate and good specificity for culturing HP in clinical specimens, can effectively shorten the HP culture cycle, and a pure HP strain can be obtained in 48 hours, saving time for conducting in vitro drug sensitivity tests and thus effectively shortening the time for patients to obtain drug sensitivity reports. The present invention provides a new method for the isolation and culture of clinical HP and has broad clinical application prospects. Brief Description of the Drawings

[0026] Figure 1 It is the appearance of the HP color - changing isolation medium prepared in Example 1;

[0027] Figure 2 It is the state of the plate and colonies when the color - changing isolation medium in Example 1 is used to culture HP standard strain positively;

[0028] Figure 3 It is the state of the plate when the color - changing isolation medium in Example 1 is used to culture the negative control;

[0029] Figure 4 It is the state of the plate and colonies when the isolation medium without indicator in Example 2 is used to culture HP standard strain positively;

[0030] Figure 5 It is a comparison diagram of the HP colonies picked from the culture obtained by the color - changing isolation medium in Example 1 and the isolation medium in Example 2. The blue ones are the bacteria picked from the color - changing plate, and the brown ones are the isolation medium without indicator;

[0031] Figure 6 The plate and colony status of culturing clinical specimens on the commercial Columbia medium for 96 h in Experimental Example 1;

[0032] Figure 7 The plate and colony status of culturing clinical specimens on the HP isolation medium prepared in Example 5 for 96 h in Experimental Example 1,

[0033] Figure 8 The plate and colony status of culturing clinical specimens on the commercial HP special medium for 96 h in Experimental Example 1;

[0034] Figure 9 The plate and colony status of culturing clinical specimens on the HP isolation medium prepared in Example 4 for 96 h in Experimental Example 1;

[0035] Figure 10 The plate and colony status of culturing clinical specimens on the HP color-changing isolation medium prepared in Example 1 for 96 h in Experimental Example 1;

[0036] Figure 11 The plate and colony status of culturing clinical specimens on the HP isolation medium prepared in Example 2 for 96 h in Experimental Example 1;

[0037] Figure 12 The plate and colony status of culturing clinical specimens on the HP isolation medium prepared in Example 3 for 96 h in Experimental Example 1;

[0038] Figure 13 The plate and colony status of subculturing cryopreserved strains on the commercial Columbia medium for 96 h in Experimental Example 2;

[0039] Figure 14 The plate and colony status of subculturing cryopreserved strains on the commercial HP special medium for 96 h in Experimental Example 2;

[0040] Figure 15 The plate and colony status of subculturing cryopreserved strains on the HP isolation medium prepared in Example 1 for 96 h in Experimental Example 2;

[0041] Figure 16 The plate and colony status of culturing clinical specimens on the HP isolation medium prepared in Example 2 for 96 h in Experimental Example 2;

[0042] Figure 17 The plate and colony status of culturing clinical specimens on the HP isolation medium prepared in Example 3 for 96 h in Experimental Example 2;

[0043] Figure 18 The plate and colony status of culturing clinical specimens on the HP isolation medium prepared in Example 4 for 96 h in Experimental Example 2;

[0044] Figure 19 For the plate and colony states after culturing clinical specimens for 96 h using the HP isolation medium prepared in Example 5 in Experimental Example 2. Specific implementation manners

[0045] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1

[0047] A preparation method of a Helicobacter pylori (HP) color-changing isolation medium comprises the following specific steps:

[0048] (1) Dissolve 5 g of bovine brain extract powder, 5 g of bovine heart extract powder, 12 g of agar, 24 g of casein peptone, 3 g of glucose, 5 g of sodium chloride, 3 g of disodium hydrogen phosphate, 0.3 g of β-cyclodextrin, and 0.2 g of bromothymol blue into 900 mL of distilled water, and sterilize it under high pressure at 121 °C for 15 min using a pressure cooker for later use.

[0049] (2) Prepare antibiotic solutions and urea stock solutions with sterile distilled water to obtain solutions with the following concentrations: amphotericin B 10 mg / mL, vancomycin 50 mg / mL, polymyxin 25 mg / mL, trimethoprim 10 mg / mL, and urea 0.2 g / mL. Filter the above-prepared solutions through a 0.2-μm filter membrane to remove bacteria, then aliquot them into sterile containers and store them at -20 °C. Restore them to room temperature before use.

[0050] (3) When the sterilized mixture in step (1) cools to 45 °C, add 100 mL of calf serum restored to room temperature, 300 μL of amphotericin B, 80 μL of vancomycin, 60 μL of polymyxin, 400 μL of trimethoprim, and 10 mL of urea. After fully mixing, quickly pour it into a sterile petri dish to obtain the HP color-changing isolation medium ( Figure 1 ).

[0051] Homogenize the gastric mucosa specimens (one piece of gastric mucosa specimen from the antrum and one from the corpus of the stomach of HP-infected patients) collected clinically and placed in a preservation solution in a fully automatic homogenizer for 40 seconds to prepare a homogenate. Use a sterile pipette to aspirate 0.2 mL of the homogenate and drop it onto the above-prepared HP color-changing isolation medium, and inoculate it in a three-zone streaking manner with a sterile loop. After inoculation, invert the plate and place it in a microaerophilic incubator (85% N2, 10% CO2, and 5% O2), and culture it at a temperature of 37 °C and a humidity of 80% RH for 5 days. At the same time, inoculate the HP standard strain 26695 and negative control (sterile normal saline) previously preserved in this laboratory into the HP color-changing isolation medium for culture according to the above method as controls.

[0052] The results are asFigure 2 , Figure 3 and Figure 5 as shown in the left figure in the middle. Among them, Figure 2 is the plate and colony state of the HP standard strain cultured on the HP color-changing isolation medium being positive, Figure 3 is the plate state of the HP color-changing isolation medium inoculated with a negative control, Figure 5 The left figure in the middle is the plate state of the HP color-changing isolation medium for culturing clinically isolated HP strains and the state after scraping with a disposable swab. It can be seen that an indicator is added to the HP color-changing isolation medium prepared in Example 1, making the medium have the characteristic of color change, and it can be used for the culture of HP standard strains and the primary isolation culture of clinical HP strains.

[0053] Example 2

[0054] The difference between this example and Example 1 is only that the pH indicator bromothymol blue and urea are not added, and the remaining steps are the same as those in Example 1, and an HP isolation medium is prepared.

[0055] According to the method described in Example 1, clinically isolated HP strains are cultured with the HP isolation medium prepared in this example. Figure 4 is the plate and colony state of the HP standard strain cultured on the medium without adding a pH indicator being positive. Figure 5 is a comparison diagram of the colors after scraping with a disposable swab of clinically isolated HP strains cultured with the media prepared in this example (right figure) and Example 1 (left figure). It can be seen that the medium prepared in this example has no color-changing characteristic when culturing HP, while Example 1 does.

[0056] Example 3

[0057] The difference between this example and Example 1 is only that in this example, sterile defibrinated sheep blood is used to replace the calf serum in Example 1, and the remaining steps are the same as those in Example 1, and an HP isolation medium is prepared.

[0058] The medium prepared in this example can be used for the subculture of HP, but the success rate of primary isolation from clinical samples is not good. Since defibrinated sheep blood is added to the medium, the medium itself is dark red, so the color change of the medium cannot be observed, but the color change of the colonies can be observed after scraping the colonies with a disposable swab.

[0059] Example 4

[0060] The difference between this example and Example 1 is only that the addition amount of the bacteriostatic agent is different. The addition amounts of the bacteriostatic agents in this example are: amphotericin B 400 μL, vancomycin 100 μL, polymyxin 80 μL, trimethoprim 500 μL, and the remaining steps are the same as those in Example 1, and an HP color-changing isolation medium is prepared.

[0061] The culture medium prepared in this example can be used for the subculture of HP. However, due to the relatively high concentration of antibiotics, the success rate of primary isolation and purification of clinical samples is poor, and the colony growth on the culture medium is weak. Although HP is naturally resistant to the above antibiotics, high concentrations will also affect the growth of HP.

[0062] Example 5

[0063] The difference between this example and Example 1 is only the addition amount of the bacteriostatic agent. The addition amounts of the bacteriostatic agent in this example are: amphotericin B 200 μL, vancomycin 60 μL, polymyxin 40 μL, trimethoprim 300 μL. The remaining steps are the same as those in Example 1, and a HP discoloration isolation culture medium is prepared.

[0064] The culture medium prepared in this example can be used for the subculture of HP. However, during the primary isolation and culture of clinical specimens, the inhibitory effect on non-target contaminants is poor, and there are many contaminants, resulting in an unsatisfactory culture success rate.

[0065] Experimental Example 1

[0066] Forty clinical specimens were cultured simultaneously with the culture media of Example 1, Example 2, Example 3, Example 4, Example 5, a commercial HP special culture medium, and a commercial Columbia culture medium of the present invention. The positive rates of culturing clinical specimens with each formulated culture medium were compared, and the growth conditions of HP in each culture medium were compared at four time points of 24 h, 48 h, 72 h, and 96 h.

[0067] Among them, the commercial HP culture medium was purchased from Shenzhen BioLotech Co., Ltd., and the commercial Columbia culture medium was purchased from Zhengzhou Antu Bio-Pharmaceutical Co., Ltd. Both products are products sold on the market.

[0068] Forty 13 gastric mucosa specimens of C urea breath test positive patients stored in a -80 °C refrigerator in the early stage of this laboratory were selected. After restoring to room temperature, they were homogenized in a full-automatic homogenizer for 40 seconds to prepare a homogenate. At the same time, they were inoculated into the culture media prepared in Example 1, Example 2, Example 3, Example 4, Example 5, the commercial HP special culture medium, and the finished Columbia culture medium. 0.2 mL of the homogenate was quantitatively inoculated on each plate, and a disposable sterile swab was used to inoculate each culture medium by the three-zone streaking method. After standing for 15 min, waiting for the homogenate to be fully absorbed, it was placed in a microaerophilic incubator (85% N2, 10% CO2, 5% O2, temperature 37 °C, humidity 75% RH) for 96 h. The growth conditions of HP in each culture medium were observed and compared at four time points of 24 h, 48 h, 72 h, and 96 h. The culture results are shown in Table 1.

[0069] Table 1 Growth conditions of HP in each culture medium

[0070]

[0071] Note: "+", "++", "+++", "++++" indicate the degree of contamination by miscellaneous bacteria. The degree of contamination by miscellaneous bacteria is the lightest with "+", and the heaviest with "++++". "-" indicates no contamination by miscellaneous bacteria.

[0072] During the cultivation process, when tiny needle-like blue colonies are observed on the HP color-changing isolation medium and the culture plate turns blue, it is initially judged that the HP culture is positive. The cultivated colonies are subjected to Gram staining and the bacterial morphology is observed under the microscope. At the same time, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and PCR methods are used to identify HP.

[0073] The morphological features of the above-mentioned HP colonies under Gram staining microscopy are red bacteria that are slender, curved, S-shaped or in the shape of a seagull spreading its wings. The similarity identified by the mass spectrometer is 99.99%. The target fragment is obtained by PCR amplification, and the urease test, oxidase test, and catalase test are all positive.

[0074] During the cultivation of gastric mucosa specimens from 40 clinical patients, after 96 hours of cultivation, 12 cases and 18 cases were positive respectively on the finished Columbia blood agar plate and the medium prepared in Example 5. As Figures 6 - 7 shown in the culture plate, both media showed contamination by miscellaneous bacteria. In particular, the finished Columbia medium was the most severely contaminated because no antibacterial agent was added. HP colonies are tiny and have high nutritional requirements. The overgrowth of miscellaneous bacteria competitively inhibits the growth of HP, resulting in a lower detection rate. After 96 hours of cultivation, 18 cases and 26 cases were positive respectively on the commercialized special HP medium and the medium prepared in Example 4. As Figures 8 - 9 shown in the culture plate, the detection rates of the two media for clinical specimens were not ideal, probably because the added antibiotic concentration was slightly too high, which inhibited the growth of miscellaneous bacteria and to a certain extent affected the growth of HP. After 96 hours of cultivation, 35 cases and 33 cases were positive respectively on the media prepared in Example 1 and Example 2. As Figures 10 - 11 shown in the culture plate, the detection rates were both above 80%, and the colony morphology of the media was plump and the activity was high. After 96 hours of cultivation, 20 cases were positive on the medium prepared in Example 3. As Figure 12 shown in the culture plate, its formula is the same as that of Example 1 except that defibrinated sheep blood is added, indicating that calf serum is more conducive to promoting the growth of HP than sheep blood.

[0075] It should be noted that although all 40 patients were HP-infected, the cultivation rate did not reach 100%. However, since the cultivation rate is not only related to the medium, but also related to specimen collection, specimen transportation, preservation, etc., a detection rate of more than 80% is already quite high in clinical applications.

[0076] In this Experimental Example 1, 40 clinical specimens were cultured using the culture media prepared with the respective formulations in the Examples and two commercial culture media under the same conditions. The culture success rates of the respective culture media and the growth states of HP at each time point were compared. Finally, it was concluded that the color-changing isolation culture medium prepared in Example 1 was the optimal culture medium. The reasonable combination of components such as calf serum, indicator, and bacteriostatic agent made the culture medium have the characteristics of high culture success rate, short culture period, and long colony activity maintenance time.

[0077] Experimental Example 2

[0078] Thirty clinical strains that had been confirmed as HP and cryopreserved in the preliminary experiment were subcultured using the culture media of Example 1, Example 2, Example 3, Example 4, Example 5 of the present invention, the commercial HP-specific culture medium, and the commercial Columbia culture medium. The positive rates of the respective formulated culture media were compared, and the growth conditions of HP in each culture medium were compared at four time points of 24 h, 48 h, 72 h, and 96 h.

[0079] Among them, the commercial HP culture medium was purchased from Shenzhen Bolot Biotechnology Co., Ltd., and the commercial Columbia culture medium was purchased from Antu Biotechnology Co., Ltd. Both products are products sold on the market.

[0080] Thirty clinical strains that had been confirmed as HP and cryopreserved at -80°C in the preliminary isolation and culture of this laboratory were selected. The cryopreserved bacterial solution was restored to room temperature. At the same time, the culture media prepared in Example 1, Example 2, Example 3, Example 4, Example 5, the commercial HP-specific culture medium, and the finished Columbia culture medium were inoculated. 0.2 mL of the bacterial solution was quantitatively inoculated per plate and evenly spread onto each culture plate with a disposable sterile swab, and cultured in a 37°C microaerophilic incubator for 96 h. The growth conditions of HP in each culture medium were compared at four time points of 24 h, 48 h, 72 h, and 96 h. The culture results are shown in Table 2.

[0081] Table 2 Growth conditions of HP in each culture medium

[0082]

[0083] Note: "+", "++", "+++", "++++" indicate the degree of contaminating bacteria. "+" indicates the lightest contaminating bacteria, and "++++" indicates the heaviest contaminating bacteria. "-" indicates no contaminating bacteria. Color change means the culture medium changes from green to blue.

[0084] During the culture process, when tiny, blue colonies like the tip of a needle were observed on the HP color-changing isolation culture medium and the culture plate turned blue, it was initially judged that the HP culture was positive. The cultured colonies were subjected to Gram staining and the bacterial morphology was observed under the microscope. At the same time, they were identified as HP by MALDI-TOF MS and PCR methods.

[0085] The above-mentioned HP colonies showed Gram-negative staining and appeared as slender, curved, S-shaped or seagull-wing-shaped red bacteria under the microscope. The urease test, oxidase test, and catalase test were all positive. The similarity identified by MALDI-TOF MS was 99.99%. The target gene was obtained by PCR amplification of the ureA fragment, and all were confirmed to be HP.

[0086] According to Figures 13 - 19 As shown, in the subculture experiments of 30 clinically preserved bacterial strains, after 96 hours of culture, the media prepared in Example 1, Example 2, Example 3, Example 4, Example 5, the commercial HP-specific medium, and the ready-to-use Columbia medium had 30, 30, 26, 28, 30, 27, and 26 positive cases respectively, and the positive culture rates all reached over 85%. The success rates of subculture in Example 1, Example 2, and Example 5 were higher than those of the two currently available ready-to-use media, all reaching 100%. This indicates that the medium of the present invention performs very well during the subculture of bacterial strains.

[0087] Through the comparison of the formulated media in the present invention and the two ready-to-use media in the above Experimental Example 1 and Experimental Example 2, it shows that the color-changing isolation medium prepared in Example 1 of the present invention not only has a high isolation success rate in the primary isolation and culture of clinical specimens, but also can significantly shorten the culture period. Its color-changing property makes positive specimens easier to identify. Therefore, this color-changing isolation medium is a highly efficient and practical medium urgently needed for clinical HP culture.

[0088] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A Helicobacter pylori discoloration isolation medium, characterized in that, Comprising components with the following concentrations: bovine brain infusion powder 4 - 6 g / L, bovine heart infusion powder 4 - 6 g / L, agar 10 - 15 g / L, peptone 21 - 25 g / L, glucose 2.5 - 3.5 g / L, sodium chloride 4 - 6 g / L, disodium hydrogen phosphate 2.5 - 3.5 g / L, β-cyclodextrin 0.5 - 1.5 g / L, urea 1.5 - 2.5 g / L, bromothymol blue 0.1 - 0.3 g / L, bacteriostatic agent 0.01 - 0.05 mL / L, calf serum 80 - 100 mL / L, and the solvent is water.

2. The Helicobacter pylori color-changing isolation medium according to claim 1, wherein The components and their concentrations of the bacteriostatic agent are: amphotericin B 2 - 4 mg / L, vancomycin 3 - 6 mg / L, polymyxin 1 - 2 mg / L, trimethoprim 3 - 5 mg / L.

3. The Helicobacter pylori color-changing isolation medium according to claim 2, wherein The pH value of the medium is 7.1 - 7.

3.

4. A method for preparing the Helicobacter pylori discoloring isolation medium according to any one of claims 1 to 3, characterized in that, Dissolve bovine brain infusion powder, bovine heart infusion powder, agar, peptone, glucose, sodium chloride, disodium hydrogen phosphate, β-cyclodextrin, and bromothymol blue in water, sterilize by high pressure, add the bacteriostatic agent, urea, and calf serum after cooling, adjust the pH value, and pour it into a disposable sterile petri dish. After cooling and solidifying, the Helicobacter pylori color-changing isolation medium can be obtained.

5. Use of the Helicobacter pylori color-changing isolation medium according to any one of claims 1 - 3 or the Helicobacter pylori color-changing isolation medium prepared by the method according to claim 4 in the high-efficiency screening of clinical strains of Helicobacter pylori.

6. A method for efficiently screening clinical strains of Helicobacter pylori, characterized in that, Comprising the following steps: (1) Collect gastric mucosa specimens from Helicobacter pylori-infected patients and make them into homogenates. (2) Inoculate the homogenate obtained in step (1) onto the Helicobacter pylori color-changing isolation medium according to any one of claims 1 - 3 or the Helicobacter pylori color-changing isolation medium prepared by the method according to claim 4 and culture it.

7. The method according to claim 6, characterized in that, The gastric mucosa specimen in step (1) is a gastric mucosa specimen from the gastric antrum and / or gastric body.

8. The method according to claim 6, wherein The inoculation amount of the homogenate in step (2) is 0.1 - 0.3 mL.

9. The method according to claim 6, wherein The culture in step (2) is carried out in a microaerophilic environment, and the microaerophilic environment is 85% N2, 10% CO2, and 5% O2; the temperature during the culture is 35 - 37 °C, and the time is 20 - 120 h.

10. The method according to any one of claims 6 to 9, characterized in that, In step (2), if there are pinpoint-like small blue colonies growing on the medium and the plate turns blue, it is preliminarily judged that the Helicobacter pylori culture is positive; if the medium turns yellow and no pinpoint-like small colonies are seen, it is determined that the Helicobacter pylori culture is negative.

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