Helicobacter pylori agar plate without micro-aerobic environment culture

Abstract

The invention relates to the technical field of biology, in particular to a helicobacter pylori culture agar plate without a micro-aerobic environment. The invention provides a culture medium added with growth factors and a reducing reagent, an antibacterial scheme is improved, an indicator is also added, activated carbon and animal blood do not need to be added to a flat plate, the product is transparent, bacterial colonies are easy to observe, the bacterial colonies are colored, blue or red and easy to distinguish, and the helicobacter pylori separation effect of a product sample is good. The sensitivity is high, the culture time is short, the antibacterial activity is strong, and the influence of infectious microbes is less.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a Helicobacter pylori agar plate which does not require microaerobic environment for cultivation. Background Art

[0002] In 1983, Warren and Marshall cultured and isolated Helicobacter pylori (hereinafter referred to as Hp) from gastric mucosal specimens. This is a pathogenic bacterium that can colonize the gastric mucosa for a long time. It is Gram-negative, curved or slightly spiral, 2.5 to 5 mm long and 0.5 mm thick. The bacteria are usually distributed in uneven groups in the pits and glandular cavities of the stomach. It can secrete a variety of exotoxins, which damage the gastric mucosal epithelium. The secreted catalase, lipase, phosphatase, proteolytic enzymes and toxins can damage gastric epithelial cells and cause inflammatory changes in the gastric mucosa. Studies have confirmed that Hp infection is closely related to digestive system diseases such as peptic ulcer, chronic gastritis with gastric mucosal atrophy and erosion, gastric cancer, gastric mucosa-associated lymphoid tissue lymphoma, and is also closely related to more than 50% of extra-digestive diseases such as idiopathic thrombocytopenic purpura and unexplained iron deficiency anemia in children and adults.

[0003] Existing detection methods include culture, immunological testing, nucleic acid testing, and urea breath test. Among them, bacterial culture has high specificity and is often used as the "gold standard" for Hp detection. At present, Hp has a serious clinical drug resistance, resulting in poor drug efficacy and inability to effectively eradicate Hp. Therefore, Hp culture and drug sensitivity testing are of great significance for clinical eradication of Hp.

[0004] Helicobacter pylori lives in the complex environment of the stomach. During primary culture, the gas requirements are relatively strict, generally 2% to 5% oxygen (microaerobic), and the maximum does not exceed 3% to 7%. The pH value of the culture medium is best when it is neutral or slightly alkaline. At present, sterile test tubes or sterile broth tubes are often used as transport fluids for bacterial culture specimens to transport specimens in clinical practice. However, these two methods cannot be used to transport Helicobacter pylori culture specimens: because sterile test tubes or sterile broth are used as transport fluids at room temperature for more than 5 hours, the Helicobacter pylori in the specimens will die, resulting in false negative culture results. Routine culture of Helicobacter pylori cannot be carried out clinically, mainly because there is no suitable transport fluid for Helicobacter pylori culture of gastric biopsy tissue specimens and the corresponding suitable culture medium.

[0005] The clinical strains of Helicobacter pylori isolated and cultured can be used for basic research such as bacterial typing, pathogenic mechanism research, construction of animal models, and identification of pathogenic factors. However, Helicobacter pylori has very strict requirements for growth conditions. It needs to place the inoculated plate in a microaerobic environment for closed culture. This culture method requires special instruments: nitrogen, oxygen, carbon dioxide and microaerobic incubators. In addition, Hp has high requirements for nutritional conditions, which results in problems such as low positive rate of Helicobacter pylori culture, affecting further research on clinical strains of Helicobacter pylori. Existing detection and identification technologies include:

[0006] 1. Bacterial culture method: The method of isolating, culturing and identifying bacteria has high specificity and is the "gold standard" for Hp detection. Commonly used culture media include selective culture media and non-selective culture media. Selective culture media have antibacterial substances added to them, which are not easy for bacteria to grow and are conducive to improving the detection of Hp. After obtaining pure culture of Hp, drug sensitivity testing can be performed. Existing drug sensitivity testing methods include agar dilution method, paper strip method, and microplate method.

[0007] 2. Immunological testing: The basis of immunological testing methods is that Hp infection can induce systemic immune response, causing the host's serum to produce Hp-related antibodies such as IgG, IgA and IgM. At present, the commonly used immunological testing method for detecting Hp in clinical practice is the ELISA method for qualitative or quantitative detection of HpIgG antibodies in the blood. It is generally believed that ELISA has high sensitivity and specificity for detecting anti-HpIgG in peripheral blood, and is easy to promote and apply. Other immunological testing methods include detecting soluble antigens after human infection with Hp, spot gold label immunoassay for HpIgG, etc. However, a positive test for HpIgG can only indicate that the patient has been infected with Hp, and cannot indicate current infection. In addition to the detection of Hp antigens and antibodies in the blood, immunological testing also includes detection of fecal Hp antigens, urine anti-Hp antibodies, saliva anti-Hp antibodies and other multi-site samples.

[0008] Fecal antigen detection test (Hp-SA) is a non-invasive diagnostic method. Since Hp resides on the surface of gastric epithelial cells, and gastric epithelial cells are renewed very quickly, during the renewal process, Hp colonized on the surface of epithelial cells is shed with the cells and excreted from the body with feces through the intestines. Therefore, Hp-SA can be detected by enzyme-linked immunosorbent assay of anti-Hp antibodies. Studies have reported that the sensitivity of Hp fecal antigen detection test is 90.0% to 98.2%, and the specificity is 75.0% to 100%. Hp-SA is an ideal non-invasive diagnostic method for Hp. It is easy to operate, time-saving, and does not require expensive instruments. It has good application value in many aspects such as epidemiological surveys of Hp infection, detection of indigestion in children, or screening before endoscopic examination. It is especially suitable for the detection of Hp infection in infants and young children, and can be used as an evaluation indicator for the long-term curative effect. Advantages of the Hp-SA test: The cost of the test is significantly lower than that of the C13 breath test; the accuracy of the urea breath test is limited in patients with a history of partial gastrectomy, while the Hp-SA test is not affected; the urea breath test requires the cooperation of the test subject, which may be difficult for young children, while the Hpi-SA test only requires the collection of stool specimens. Disadvantages of the Hp-SA test: Since this method detects Hp antigen in the stool, when the patient undergoes Hp eradication treatment, even if the patient's Hp has been eradicated, about 6% of the patients may still have Hp antigen detected in their stool 4 weeks after the end of treatment, resulting in false positive results. If the patient is tested 6 to 8 weeks after the end of treatment when using this method to judge the efficacy, the false positive rate of the test can be significantly reduced.

[0009] Urine anti-Hp antibody test. In 1993, Alemohammad et al. first reported 306 cases of urine anti-H. pylori antibody test with a sensitivity and specificity of 95.9% and 90%, respectively, confirming its usefulness. Urine anti-Hp antibody test is fast, low-cost, reliable, and easy to operate for diagnosing Hp infection in children, and is valuable for large-scale epidemiological screening of children.

[0010] For a long time, most scholars believed that the stomach was the main storage place for Hp. Hp was detected in gastric mucosa, epithelium, gastric juice, gastric reflux, vomitus and feces of patients with gastritis and even infants. In 1983, Krajden et al. first isolated and cultured Hp from dental plaque of patients with gastritis, and speculated that the oral cavity might be another gathering place for Hp in the human body. Since then, the study of Hp in the oral cavity has gradually become a hot topic. In 1993, Ferguson et al. first cultured 1 viable Hp from the saliva of 9 patients with gastritis. The following year, Patel et al. also reported that the consistency of anti-Hp IgG in saliva and anti-Hp IgG in serum reached 91%, with a good positive correlation, which could correctly judge the patient's infection with Hpi.

[0011] 3. RUT test: Hp can produce a large amount of urease, which decomposes trace urea in gastric juice to cause changes in pH, produce CO2, and increase the local pH value. RUT test is designed based on this principle and can be used to diagnose Hp infection during gastroscopy. This method is one of the invasive detection methods, and studies have shown that the sample must contain 10 4 Only Hp above CFU / mL can show positive results, and factors such as the size of the specimen, the length of the reaction time, and the high or low ambient temperature can affect the test results. When using this method to detect Hp, it should be noted that when there is active bleeding in the stomach, the change in the pH value in the stomach caused by bleeding will affect the sensitivity and specificity of the urease test.

[0012] 4. Urea breath test: Urea breath test, also known as C13, C14-UBT test, was first reported by Dr. Garham and Klein in the United States in 1986. Its principle is that Hp produces urease in the body. Patients infected with Hp take isotope-labeled urea solution orally. The isotope CO2 produced after the decomposition of urea is exhaled from the lungs. Hp infection is diagnosed by collecting breath specimens and detecting the amount of isotope CO2. According to different markers, it is divided into C13 breath test and C14 breath test. This test is currently considered to be the "gold standard" for diagnosing H. pylori infection in addition to culture. It is widely used in clinical practice, does not require endoscopic specimen collection, has low technical requirements, and has the disadvantage of high cost. The advantages of C13-UBT are: (1) the high-precision gas isotope ratio mass spectrometer used has an analysis accuracy of up to one in 100,000, with high accuracy; (2) the response is the "real-time" status of the "whole stomach". In the consensus opinions on Hp in China and Europe, this method is recommended as the first choice for the diagnosis of Hp current infection and the determination of Hp eradication as a non-invasive method; (3) the operation is simple and fast, with a high degree of automation, and the results can be obtained in 30 minutes; (4) C13 is a stable isotope, suitable for subjects of all ages; (5) the breath samples are collected in a special gas collection bottle, which can be mailed to patients in other regions without such equipment for Hp testing. Therefore, after C13-UBT passed the review of the US Food and Drug Administration (FDA) in 1996, it was soon widely used in the clinical diagnosis of Hp infection. The disadvantage is that the test may be affected by drugs, upper gastrointestinal bleeding, and other bacteria in the stomach (such as Helicobacter Helimannii), which may result in false positive and false negative results. In addition, the test requires a large mass spectrometer and expensive reagents, which greatly limits its use in primary hospitals. The C14-UBT test is inexpensive, but the test is radioactive and has a long half-life. Although the radioactivity is low, the radiation dose received in a breath test is only 1 / 60 of that received in a chest X-ray. However, large-scale application may cause environmental pollution. In addition, this method should be used with caution in pregnant women, children, and those with active gastric bleeding.

[0013] 5. N15-urea ammonia elimination test: This method was first invented by Wu Jicong and others in my country and applied in clinical practice. Its principle is basically the same as the carbon-labeled CO2 breath test, that is, the tester takes N15-urea orally, which is decomposed into N15-labeled NH3 and CO2 by urease in the stomach, absorbed by the digestive system and enters the blood circulation, while N15-labeled ammonia is converted into ammonia or urea by the body's nitrogen metabolism and then eliminated by the kidneys, and detected by chromatography. This method is also a non-invasive test, without radioactive pollution, with high sensitivity and specificity, but the instrument is expensive and has not yet been popularized.

[0014] 6. Molecular biological methods: Hoshina et al. first used polymerase chain reaction (PCR) technology for the diagnosis of Hp. PCR technology has significantly improved the sensitivity (equivalent to 100 bacterial cells) and specificity of Hp diagnosis, and has low requirements for raw materials. In addition to fresh biopsy specimens, saliva, gastric juice, paraffin-embedded specimens and specimens that have undergone rapid urease tests can also be used, and Hp DNA in feces and the environment can be detected. At present, PCR instruments and primers have been commercialized and can be promoted on a large scale for use in inspections and epidemiological surveys. However, there is also a problem, that is, Hp DNA that has been killed by antibacterial drugs but still remains in the stomach will also be amplified to produce positive results, so PCR is not suitable for judging the short-term effect of treatment. Molecular biological methods include single PCR method, reverse transcription PCR method, nested PCR method, and oligonucleotide probe hybridization method.

[0015] There are also other morphological examination methods. The mucosal surface of the biopsy gastric mucosal specimen is smeared and stained, and typical curved or spiral bacteria are observed under an oil microscope. This can quickly determine the presence of Hp infection and the presence of gastric flora and inflammatory cell infiltration.

[0016] In addition to the bacterial culture method, other methods such as immunological testing, RUT testing, urea breath test, N15-urine ammonia exclusion test, molecular biology methods, morphological methods, etc., are unable to perform drug sensitivity tests. The bacterial culture method is the prerequisite for drug sensitivity testing. The strains isolated on the culture medium are identified, and then the agar dilution method, paper strip method, and microplate method are used for drug sensitivity. The drug sensitivity results can more accurately guide clinical medication, improve the clinical cure rate of Helicobacter pylori, effectively control the abuse of antibiotics, reduce drug resistance, greatly reduce the physical pain of patients, and also reduce the mental stress of patients caused by repeated treatment.

[0017] Therefore, Helicobacter pylori isolation and culture is necessary for drug sensitivity testing. Drug sensitivity testing of Helicobacter pylori strains in different patients is an effective method to develop individualized and precise treatment plans.

[0018] However, although the prior art has a method for culture identification and drug sensitivity detection, the culture medium is based on brain heart infusion agar, Columbia agar, tryptone soy agar or Wilkins-Chalgren, all of which are dry powder culture medium reagents, and the dry powder basis is affected by the raw materials and production processes of dry powder production from different manufacturers. Helicobacter pylori is a picky microaerophilic bacterium. In addition to strict requirements on the gas environment during in vitro culture, the requirements for the culture medium are also quite strict. The culture medium must not only meet the nutritional needs of Helicobacter pylori, but also provide a suitable pH tolerance range. Traditional separation culture media cannot meet the requirements for the isolation and culture of Helicobacter pylori. Commonly used culture media such as Columbia agar medium, brain heart infusion agar medium and modified Skirrow agar medium have no advantages in culturing Helicobacter pylori, and the positive detection rate is extremely low. The culture medium is mostly liquid culture medium, and it is not easy to distinguish whether the color development is caused by miscellaneous bacteria or Helicobacter pylori, and the drug sensitivity results are easily affected by miscellaneous bacteria, resulting in inaccurate results. Summary of the invention

[0019] In view of this, the present invention provides a Helicobacter pylori agar plate that does not require microaerobic environment cultivation.

[0020] The first purpose is to eliminate the need to add activated carbon and animal blood to the culture medium and to reduce the dependence of Helicobacter pylori on the microaerobic environment. Helicobacter pylori can be separated from the sample in a 5% to 10% carbon dioxide incubator.

[0021] The second purpose is to improve the growth performance of the target bacteria, increase sensitivity, improve the antibiotic regimen, and increase the detection rate.

[0022] The third purpose is to add indicators so that the colonies appear blue or red, thereby increasing the detection rate and recognition rate of the target bacteria.

[0023] The fourth purpose is that the bacteria cultured on the plate can be directly tested for drug sensitivity without affecting the drug sensitivity results.

[0024] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0025] The present invention provides a Helicobacter pylori culture medium, comprising peptone, extract powder, carbohydrates, amino acids, metal ions, enzymes, antibiotics, reducing agents, salts, serum, agar, indicators, and buffers, but excluding:

[0026] (1) blood; and

[0027] (2), carbon; and

[0028] (3) Activated carbon.

[0029] In some specific embodiments of the present invention, the above-mentioned Helicobacter pylori culture medium:

[0030] The peptone includes at least one of animal peptone and plant peptone; and / or

[0031] The powder comprises at least one of ox brain powder, ox heart powder and beef powder; and / or

[0032] The carbohydrates include at least one of glucose, cyclodextrin, glycerol, sodium pyruvate, and α-ketoglutaric acid; and / or

[0033] The amino acids include at least one of cysteine ​​and arginine; and / or

[0034] The metal ions include at least one of ferrous ions, zinc ions and nickel ions; and / or

[0035] The enzyme comprises at least one of catalase and SOD; and / or

[0036] The antibiotics include at least one of vancomycin, teicoplanin, lincomycin, bacitracin, cefsulodin sodium, nalidixic acid, trimethoprim, polymyxin, and amphotericin; and / or

[0037] The reducing agent comprises at least one of N-acetyl L-cysteine, sodium thiosulfate and sodium sulfite; and / or

[0038] The salt comprises sodium chloride; and / or

[0039] The serum comprises at least one of horse serum and bovine serum; and / or

[0040] The indicator comprises at least one of bromothymol blue and TTC; and / or

[0041] The buffer comprises a phosphate buffer; and / or

[0042] The Helicobacter pylori culture medium also includes heme.

[0043] In some specific embodiments of the present invention, the above-mentioned Helicobacter pylori culture medium:

[0044] The weight portion of the peptone is 1 to 10 parts; and / or

[0045] The weight portion of the soaking powder is 1 to 10 parts; and / or

[0046] The weight portion of the glucose is 1 to 10 parts; and / or

[0047] The weight portion of the cyclodextrin is 5 to 15 parts; and / or

[0048] The weight portion of the glycerol is 1 to 10 parts; and / or

[0049] The weight portion of the sodium pyruvate is 1 to 5 parts; and / or

[0050] The weight portion of the α-ketoglutaric acid is 0.2 to 2 parts; and / or

[0051] The weight portion of cysteine ​​is 0.1 to 1 part; and / or

[0052] The weight portion of arginine is 0.5 to 2 parts; and / or

[0053] The weight portion of ferrous sulfate is 0.006 to 0.4 parts; and / or

[0054] The weight portion of the zinc sulfate is 0.001 to 0.01 parts; and / or

[0055] The weight portion of the nickel chloride is 0.001 to 0.01 parts; and / or

[0056] The weight portion of the vancomycin is 0.001 to 0.01 parts; and / or

[0057] The weight portion of teicoplanin is 0.001 to 0.01 parts; and / or

[0058] The weight portion of the lincomycin is 0.0001 to 0.0015 parts; and / or

[0059] The weight portion of the cefsulodin sodium is 0.001 to 0.01 parts; and / or

[0060] The weight portion of the nalidixic acid is 0.001 to 0.01 parts; and / or

[0061] The weight portion of the trimethoprim is 0.001 to 0.01 parts; and / or

[0062] The weight portion of the N-acetyl L-cysteine ​​is 0.01 to 0.1 parts; and / or

[0063] The weight portion of the sodium thiosulfate is 0.01 to 0.1 parts; and / or

[0064] The weight portion of the sodium sulfite is 0.01 to 0.1 parts; and / or

[0065] The weight portion of the sodium chloride is 5 parts; and / or

[0066] The weight portion of the horse serum is 50 to 200 parts; and / or

[0067] The weight portion of the bovine serum is 50 to 200 parts; and / or

[0068] The weight portion of the bromothymol blue is 0.01 to 0.05 parts; and / or

[0069] The weight portion of TTC is 0.01 to 0.1 parts; and / or

[0070] The weight portion of the amphotericin is 0.005 to 0.02 parts;

[0071] The weight portion of the hemoglobin is 0.001 to 0.01 parts.

[0072] In some specific embodiments of the present invention, the above-mentioned Helicobacter pylori culture medium:

[0073] The catalase content is 5000-50000 IU / L; and / or

[0074] The SOD content is 10000-100000 IU / L; and / or

[0075] The bacitracin content is 500 to 3000 IU / L; and / or

[0076] The polymyxin is 1000 to 20000 IU / L.

[0077] In some specific embodiments of the present invention, the pH value of the Helicobacter pylori culture medium is 6.0-7.5.

[0078] The present invention also provides a method for preparing the above-mentioned Helicobacter pylori culture medium, comprising: mixing raw materials to prepare a solid culture medium to obtain the Helicobacter pylori culture medium;

[0079] The raw materials include the peptone, the extract powder, the carbohydrate, the amino acid, the metal ion, the enzyme, the antibiotic, the reducing agent, the salt, the serum, the agar, the indicator and the buffer.

[0080] The present invention also provides a kit, comprising the above Helicobacter pylori culture medium.

[0081] The present invention also provides a method for isolating and culturing Helicobacter pylori, comprising: culturing based on the above-mentioned Helicobacter pylori culture medium or the above-mentioned kit.

[0082] In some specific embodiments of the present invention, the culture conditions in the above-mentioned separation and culture method do not include microaerobic conditions.

[0083] In some specific embodiments of the present invention, the culture conditions of the above separation and culture method include: the carbon dioxide content in the culture environment is 5% to 10%.

[0084] The present invention also provides a method for detecting Helicobacter pylori infection, which is based on the above Helicobacter pylori culture medium or the above kit.

[0085] The culture medium of the present invention has the following effects:

[0086] Existing culture technology has high requirements for the gas environment, solid culture requires a microaerobic environment, the cost for clinical development is relatively high, and most of the culture medium is added with activated carbon or animal blood plates, which have no color development function and a low clinical isolation rate. The present invention reduces the requirements for the gas environment, and an ordinary carbon dioxide incubator is sufficient, and culture separation can be achieved clinically without the need for additional equipment. During the first clinical isolation, some strains cannot grow on conventional culture media, resulting in clinical missed detection. The present invention supplements the growth factors and environmental conditions required for the first isolation of strains, which reduces the requirements for the gas environment and also achieves a significant improvement in the sensitivity of strain separation. Existing culture technology has no color development function and has a low sensitivity to the cultivation of Helicobacter pylori. The culture medium of the present invention can effectively distinguish between target bacteria and non-target bacteria through indicators, thereby improving the detection rate of target bacteria.

[0087] The existing culture technology has low specificity, and the target bacteria cannot be distinguished from the miscellaneous bacteria or the distinction is poor. The present invention adds an optimized antibacterial agent to inhibit the interference of most miscellaneous bacteria, and adds an indicator to make Helicobacter pylori color. At the same time, carbohydrates such as glucose or glycerol are added to make the miscellaneous bacteria color and distinguish them from the target bacteria.

[0088] The present invention does not add activated carbon or animal blood, has a simple process, avoids the problem of short product shelf life due to the instability of animal blood and the problem of activated carbon being responsible for the production process. No microaerobic environment is required, carbon dioxide culture can be achieved, and the sensitivity of clinical separation is greatly improved. It has a color development function, can distinguish target bacteria from non-target bacteria, and the cultured bacteria can be directly used to identify drug sensitivity. By verifying the drug sensitivity results, the strains grown on the plate have no effect on the drug sensitivity results.

[0089] The product of the present invention does not need to add activated carbon and animal blood. The product is transparent, and the colonies are easy to observe. The colonies are blue or red in color and easy to distinguish. The product of the present invention has good effect in separating Helicobacter pylori, high sensitivity, short culture time, strong antibacterial property, less influence of miscellaneous bacteria, and the culture effect is as follows: Figure 1 shown. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0091] Figure 1 Shows the growth of the flat plate of the present invention;

[0092] Figure 2 The growth of blood plate in comparative example 1 is shown;

[0093] Figure 3 The growth of the activated carbon plate added in Comparative Example 2 is shown. DETAILED DESCRIPTION

[0094] The present invention discloses an agar plate for culturing Helicobacter pylori without a microaerobic environment. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0095] The invention reduces the dependence of Helicobacter pylori on a microaerobic environment and improves its growth performance by adding growth factors and reducing agents required for the growth of Helicobacter pylori into a culture medium, so that the Helicobacter pylori can grow well in a carbon dioxide incubator. The growth factors include cyclodextrin, glucose, sodium pyruvate, α-ketoglutaric acid, L-cysteine, L-arginine, glycerol, catalase, superoxide dismutase, nickel chloride, zinc sulfate, heme and serum, which can improve the growth ability of target bacteria; the reducing agents include N-acetyl L-cysteine, sodium thiosulfate, sodium sulfite and ferrous sulfate, which can reduce the requirements of Helicobacter pylori on a microaerobic environment.

[0096] The present invention also improves the antibiotic scheme and improves the inhibitory ability against positive bacteria, negative bacteria and fungi. The antibiotics include nalidixic acid, trimethoprim, vancomycin, teicoplanin, lincomycin, bacitracin, amphotericin B, polymyxin B / E, which improve the inhibitory ability against negative bacteria such as Neisseria, positive bacteria such as Moniliforme, and fungi such as Candida in the sample.

[0097] The present invention further adds an indicator, which includes bromothymol blue, 2,3,5-triphenyltetrazolium chloride (TTC) or bromothymol blue+TTC, and Helicobacter pylori appears blue or red.

[0098] Specifically, the plate of the present invention is composed of peptones, extract powders, carbohydrates, amino acids, metal ions, reducing agents, enzymes, indicators, antibiotics, serum, buffer and the like.

[0099] The peptones include one or more of trypticase, peptone, and soy peptone, and the content units are all g / L:

[0100] Trypticase content 1-10, the best is 5;

[0101] Peptone content 1-10, optimal is 5;

[0102] The soy peptone content is 1 to 10, with 5 being the optimum.

[0103] The powders include one or more of ox brain powder, ox heart powder and beef powder, and the content units are all g / L:

[0104] The content of beef brain powder is 1 to 10, with the best being 5;

[0105] The content of beef heart powder is 1 to 10, with the best being 5;

[0106] The beef extract powder content is 1 to 10, with the optimum being 5;

[0107] The carbohydrates include one or more of glucose, cyclodextrin, glycerol, sodium pyruvate, and α-ketoglutaric acid, and the content units are all g / L:

[0108] Glucose content 1-10, optimal is 3;

[0109] The cyclodextrin content is 5-15, and the optimal content is 10;

[0110] Sodium pyruvate content 1-5, optimal is 3;

[0111] α-ketoglutaric acid content 0.2-2, optimal is 1;

[0112] The glycerol content is 1-10, with 5 being the optimum.

[0113] The amino acids include one or more of cysteine ​​and arginine, and the content unit is g / L:

[0114] Cysteine ​​content 0.1-1, optimal is 0.3;

[0115] The arginine content is 0.5-2, with 1 being the optimum.

[0116] The metal ions include one or more of ferrous ions, heme, zinc ions, and nickel ions, and the content units are all mg / L:

[0117] The ferrous sulfate content is 6-40, with the optimum being 12;

[0118] Hemoglobin content 1-10, optimal is 5;

[0119] Zinc sulfate content 1-10, optimal is 5;

[0120] The nickel chloride content is 1-10, and the optimal value is 5.

[0121] The enzymes include one or more of catalase and superoxide dismutase (SOD), and the content unit is IU / L:

[0122] Catalase content 5000-50000 IU / L, optimal is 10000 IU / L;

[0123] The SOD content is 10000~100000IU / L, and the optimal is 50000IU / L.

[0124] The indicator includes one or more of bromothymol blue and TTC, and the content unit is mg / L:

[0125] The content of bromothymol blue is 10-50, and the optimum is 30;

[0126] The TTC content is 10-100, with 50 being the optimum.

[0127] The reducing agent includes one or more of N-acetyl L-cysteine, sodium thiosulfate, and sodium sulfite, and the content unit is g / L:

[0128] N-acetyl L-cysteine ​​content 0.01-0.1, optimal is 0.05;

[0129] Sodium thiosulfate content 0.01~0.1, optimal is 0.05;

[0130] The sodium sulfite content is 0.01-0.1, and the optimum is 0.05.

[0131] The antibiotics include one or more of vancomycin, teicoplanin, lincomycin, bacitracin, cefsulodin sodium, nalidixic acid, trimethoprim, polymyxin, and amphotericin:

[0132] Vancomycin content 1-10 mg / L, optimal is 5 mg / L;

[0133] Teicoplanin content 1-10 mg / L, optimal is 5 mg / L;

[0134] Lincomycin content 0.1-1.5 mg / L, the optimum is 0.5 mg / L;

[0135] Bacitracin content 500-3000 IU / L, optimal is 1500 IU / L;

[0136] The content of cefsulodin sodium is 1-10 mg / L, and the optimal content is 3 mg / L;

[0137] Polymyxin 1000-20000 IU / L, with 10000 IU / L being the optimum;

[0138] Amphotericin content 5-20 mg / L, optimal is 10 mg / L;

[0139] The nalidixic acid content is 1 to 10 mg / L, with the optimum being 5 mg / L;

[0140] The trimethoprim content is 1-10 mg / L, and the optimal content is 5 mg / L.

[0141] The serum includes one or more of horse serum and bovine serum, and the content unit is g / L:

[0142] Horse serum content is 50-200, with 100 being optimal;

[0143] The bovine serum content is 50-200, with 100 being optimal.

[0144] The pH value of the plate of the present invention ranges from 6.0 to 7.5, and is optimally 6.8.

[0145] In some embodiments, the steps of preparing the plate include:

[0146] Take the amount of purified water mentioned in the formula, and then add the materials one by one. After complete dissolution, adjust the pH value to the appropriate range, sterilize at 121°C for 15 minutes, and fill the plates when the temperature drops to 50°C. Before filling the plates, aseptically add the filtered and sterilized serum solution and antibiotic solution.

[0147] In some embodiments of the present invention, agar powder mainly plays a role of coagulation, and different types of agar powder can be used in different amounts according to specific conditions. Buffer mainly plays a role of stabilizing pH, and the amount can be adjusted according to pH conditions.

[0148] The Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus, Neisseria oralis, and Haemophilus influenzae mentioned are Gram-negative bacteria; Staphylococcus aureus, Enterococcus faecalis, Leuconostoc lactis, Lactobacillus salivarius, and Streptococcus sanguinis are Gram-positive bacteria, and Candida albicans is a fungus. The above bacteria are common pathogens of the respiratory tract.

[0149] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0150] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0151] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0152] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0153] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0154] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0155] The present invention will be further described below in conjunction with the embodiments.

[0156] Example 1: Formulation 1

[0157] Purified water 900g / L + trypticase 10g / L + yeast extract powder 3g / L + beef extract powder 3g / L + starch 1g / L + phosphate buffer 0.9g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L, pH 6.8.

[0158] Example 1-1: Formulation 1-1

[0159] Purified water 900g / L + trypticase 10g / L + yeast extract powder 3g / L + beef extract powder 3g / L + phosphate buffer 0.9g / L + starch 1g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L, pH 6.8.

[0160] Example 1-2: Formulation 1-2

[0161] Purified water 900g / L + trypticase 10g / L + yeast extract powder 3g / L + beef extract powder 3g / L + phosphate buffer 0.9g / L + starch 1g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + TTC 10mg / L, pH 6.8.

[0162] Example 1-3: Formulation 1-3

[0163] Purified water 900g / L + trypticase 10g / L + yeast extract powder 3g / L + beef extract powder 3g / L + phosphate buffer 0.9g / L + starch 1g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0164] Example 2: Formulation 2

[0165] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + beef extract powder 3g / L + phosphate buffer 0.9g / L + starch 1g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0166] Example 3: Formulation 3

[0167] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + starch 1g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0168] Example 4: Formulation 4

[0169] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0170] Example 5: Formulation 5

[0171] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0172] Example 6: Formulation 6

[0173] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0174] Example 7: Formulation 7

[0175] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0176] Example 8: Formulation 8

[0177] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0178] Example 9: Formulation 9

[0179] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0180] Example 10: Formulation 10

[0181] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0182] Example 11: Formulation 11

[0183] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0184] Example 12: Formulation 12

[0185] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0186] Example 13: Formulation 13

[0187] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + hemoglobin 5mg / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0188] Example 14: Formulation 14

[0189] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + hemoglobin 5mg / L + zinc sulfate 5mg / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0190] Example 15: Formulation 15

[0191] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L + zinc sulfate 5mg / L + nickel chloride 5mg / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0192] Example 16: Formulation 16

[0193] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L + zinc sulfate 5mg / L + nickel chloride 5mg / L + catalase 10000IU / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0194] Example 17: Formulation 17

[0195] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L + zinc sulfate 5mg / L + nickel chloride 5mg / L + catalase 10000IU / L + SOD16000IU / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0196] Example 18: Formulation 18

[0197] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L + zinc sulfate 5mg / L + nickel chloride 5mg / L + catalase 10000IU / L + SOD16000IU / L + vancomycin 5mg / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0198] Example 19: Formulation 19

[0199] Purified water 900g / L + trypticase 10g / L + bovine brain extract powder 5g / L + bovine heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L + zinc sulfate 5mg / L + nickel chloride 5mg / L + catalase 10000IU / L + SOD16000IU / L + vancomycin 5mg / L + teicoplanin 5mg / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0200] Example 20: Formulation 20

[0201] Purified water 900g / L + trypticase 10g / L + ox brain extract powder 5g / L + ox heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + Hemoglobin 5mg / L + zinc sulfate 5mg / L + nickel chloride 5mg / L + catalase 10000IU / L + SOD16000IU / L + vancomycin 5mg / L + teicoplanin 5mg / L + lincomycin 0.5mg / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0202] Example 21: Formulation 21

[0203] Purified water 900g / L + trypticase 10g / L + ox brain extract powder 5g / L + ox heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L+Zinc sulfate 5mg / L+Nickel chloride 5mg / L+Catalase 10000IU / L+SOD16000IU / L+Vancomycin 5mg / L+Teicoplanin 5mg / L+Lincomycin 0.5mg / L+Bacitracin 1500IU / L+Sodium chloride 5g / L+Horse serum 10%+Agar powder 13g / L+Bromothymol blue 10mg / L+TTC 10mg / L, pH6.8.

[0204] Example 22: Formulation 22

[0205] Purified water 900g / L + trypticase 10g / L + ox brain extract powder 5g / L + ox heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L + zinc sulfate 5mg / L+Nickel chloride 5mg / L+Catalase 10000IU / L+SOD 16000IU / L+Vancomycin 5mg / L+Teicoplanin 5mg / L+Lincomycin 0.5mg / L+Bacitracin 1500IU / L+Polymyxin 1000IU / L+Sodium chloride 5g / L+Horse serum 10%+Agar powder 13g / L+Bromothymol blue 10mg / L+TTC 10mg / L, pH 6.8.

[0206] Example 23: Formulation 23

[0207] Purified water 900g / L + trypticase 10g / L + ox brain extract powder 5g / L + ox heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L + zinc sulfate 5mg / L + Nickel chloride 5mg / L + catalase 10000IU / L + SOD 16000IU / L + vancomycin 5mg / L + teicoplanin 5mg / L + lincomycin 0.5mg / L + bacitracin 1500IU / L + polymyxin 1000IU / L + amphotericin 10mg / L + sodium chloride 5g / L + horse serum 10% + agar powder 13g / L + bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0208] Example 24: Formulation 24

[0209] Purified water 900g / L + trypticase 10g / L + ox brain extract powder 5g / L + ox heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L + zinc sulfate 5mg / L + nickel chloride 5 mg / L+catalase 10000IU / L+SOD16000IU / L+vancomycin 5mg / L+teicoplanin 5mg / L+lincomycin 0.5mg / L+bacitracin 1500IU / L+polymyxin 1000IU / L+amphotericin 10mg / L+nalidixic acid 5mg / L+sodium chloride 5g / L+horse serum 10%+agar powder 13g / L+bromothymol blue 10mg / L+TTC10mg / L, pH6.8.

[0210] Example 25: Formulation 25

[0211] Purified water 900g / L + trypticase 10g / L + ox brain extract powder 5g / L + ox heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L + zinc sulfate 5mg / L + nickel chloride 5mg / L + Catalase 10000IU / L+SOD 16000IU / L+Vancomycin 5mg / L+Teicoplanin 5mg / L+Lincomycin 0.5mg / L+Bacitracin 1500IU / L+Polymyxin 1000IU / L+Amphotericin 10mg / L+Nalidixic acid 5mg / L+Trimethoprim 5mg / L+Sodium chloride 5g / L+Horse serum 10%+Agar powder 13g / L+Bromothymol blue 10mg / L+TTC 10mg / L, pH 6.8.

[0212] Example 26: Formulation 26

[0213] Purified water 900g / L + trypticase 10g / L + ox brain extract powder 5g / L + ox heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L + zinc sulfate 5mg / L + nickel chloride 5mg / L + catalase 1 0000IU / L+SOD16000IU / L+Vancomycin5mg / L+Teicoplanin5mg / L+Lincomycin0.5mg / L+Bacitracin1500IU / L+Polymyxin1000IU / L+Amphotericin10mg / L+Nalidixic acid5mg / L+Trimethoprim5mg / L+Cefsulodin Sodium3mg / L+Sodium Chloride5g / L+Horse Serum10%+Agar Powder13g / L+Bromothymol Blue10mg / L+TTC10mg / L, pH6.8.

[0214] Example 27: Formulation 27

[0215] Purified water 900g / L + trypticase 10g / L + ox brain extract powder 5g / L + ox heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L + zinc sulfate 5mg / L + nickel chloride 5mg / L + catalase 10000IU / L + SOD16000IU / L+Vancomycin 5mg / L+Teicoplanin 5mg / L+Lincomycin 0.5mg / L+Bacitracin 1500IU / L+Polymyxin 1000IU / L+Amphotericin 10mg / L+Nalidixic acid 5mg / L+Trimethoprim 5mg / L+Cefsulodin Sodium 3mg / L+N-acetyl L-cysteine ​​0.05g / L+Sodium chloride 5g / L+Horse serum 10%+Agar powder 13g / L+Bromothymol blue 10mg / L+TTC 10mg / L, pH 6.8.

[0216] Example 28: Formulation 28

[0217] Purified water 900g / L + trypticase 10g / L + ox brain extract powder 5g / L + ox heart extract powder 5g / L + phosphate buffer 0.9g / L + cyclodextrin 10g / L + soy peptone 5g / L + glucose 3g / L + sodium pyruvate 3g / L + α-ketoglutaric acid 1g / L + glycerol 5g / L + L-cysteine ​​0.3g / L + L-arginine 1g / L + ferrous sulfate 0.006g / L + heme 5mg / L + zinc sulfate 5mg / L + nickel chloride 5mg / L + catalase 10000IU / L + SOD160 00IU / L+Vancomycin 5mg / L+Teicoplanin 5mg / L+Lincomycin 0.5mg / L+Bacitracin 1500IU / L+Polymyxin 1000IU / L+Amphotericin 10mg / L+Nalidixic Acid 5mg / L+Trimethoprim 5mg / L+Cefsulodin Sodium 3mg / L+N-Acetyl L-cysteine ​​0.05g / L+Sodium thiosulfate 0.05g / L+Sodium Chloride 5g / L+Horse Serum 10%+Agar Powder 13g / L+Bromothymol Blue 10mg / L+TTC10mg / L, pH6.8.

[0218] Example 29: Formulation 29

[0219] Purified water 900g / L+trypticase 10g / L+beef brain extract powder 5g / L+beef heart extract powder 5g / L+phosphate buffer 0.9g / L+cyclodextrin 10g / L+soybean peptone 5g / L+glucose 3g / L+sodium pyruvate 3g / L+α-ketoglutaric acid 1g / L+glycerol 5g / L+L-cysteine ​​0.3g / L+L-arginine 1g / L+ferrous sulfate 0.006g / L+heme 5mg / L+zinc sulfate 5mg / L+nickel chloride 5mg / L+catalase 10000IU / L+SOD 16000IU / L + Vancomycin 5mg / L + Teicoplanin 5mg / L + Lincomycin 0.5mg / L + Bacitracin 1500IU / L + Polymyxin 1000IU / L + Amphotericin 10mg / L + Nalidixic acid 5mg / L + Trimethoprim 5mg / L + Cefsulodin sodium 3mg / L + N-acetyl L-cysteine ​​0.05g / L + Sodium thiosulfate 0.05g / L + Sodium sulfite 0.05g / L + Sodium chloride 5g / L + Horse serum 10% + Agar powder 13g / L + Bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0220] Example 30: Formulation 30

[0221] Purified water 900g / L+trypticase 10g / L+beef brain extract powder 5g / L+beef heart extract powder 5g / L+phosphate buffer 0.9g / L+cyclodextrin 10g / L+soybean peptone 5g / L+glucose 3g / L+sodium pyruvate 3g / L+α-ketoglutaric acid 1g / L+glycerol 5g / L+L-cysteine ​​0.3g / L+L-arginine 1g / L+ferrous sulfate 0.006g / L+heme 5mg / L+zinc sulfate 5mg / L+nickel chloride 5mg / L+catalase 10000IU / L+SOD 16000IU / L + Vancomycin 5mg / L + Teicoplanin 5mg / L + Lincomycin 0.5mg / L + Bacitracin 1500IU / L + Polymyxin 1000IU / L + Amphotericin 10mg / L + Nalidixic acid 5mg / L + Trimethoprim 5mg / L + Cefsulodin sodium 3mg / L + N-acetyl L-cysteine ​​0.05g / L + Sodium thiosulfate 0.05g / L + Sodium sulfite 0.05g / L + Sodium chloride 5g / L + Horse serum 10% + Agar powder 13g / L + Bromothymol blue 10mg / L + TTC 10mg / L, pH 6.0.

[0222] Example 31: Formulation 31

[0223] Purified water 900g / L+trypticase 10g / L+beef brain extract powder 5g / L+beef heart extract powder 5g / L+phosphate buffer 0.9g / L+cyclodextrin 10g / L+soybean peptone 5g / L+glucose 3g / L+sodium pyruvate 3g / L+α-ketoglutaric acid 1g / L+glycerol 5g / L+L-cysteine ​​0.3g / L+L-arginine 1g / L+ferrous sulfate 0.006g / L+heme 5mg / L+zinc sulfate 5mg / L+nickel chloride 5mg / L+catalase 10000IU / L+SOD 16000IU / L + Vancomycin 5mg / L + Teicoplanin 5mg / L + Lincomycin 0.5mg / L + Bacitracin 1500IU / L + Polymyxin 1000IU / L + Amphotericin 10mg / L + Nalidixic acid 5mg / L + Trimethoprim 5mg / L + Cefsulodin sodium 3mg / L + N-acetyl L-cysteine ​​0.05g / L + Sodium thiosulfate 0.05g / L + Sodium sulfite 0.05g / L + Sodium chloride 5g / L + Horse serum 10% + Agar powder 13g / L + Bromothymol blue 10mg / L + TTC 10mg / L, pH 7.5.

[0224] Example 32: Formulation 32

[0225] Purified water 900g / L+trypticase 1g / L+beef brain extract powder 1g / L+beef heart extract powder 1g / L+phosphate buffer 0.9g / L+cyclodextrin 5g / L+soybean peptone 1g / L+glucose 1g / L+sodium pyruvate 1g / L+α-ketoglutaric acid 0.2g / L+glycerol 1g / L+L-cysteine ​​0.1g / L+L-arginine 0.5g / L+ferrous sulfate 0.006g / L+heme 1mg / L+zinc sulfate 1mg / L+nickel chloride 1mg / L+catalase 5000IU / L+SOD 10000IU / L+Vancomycin 1mg / L+Teicoplanin 1mg / L+Lincomycin 0.1mg / L+Bacitracin 500IU / L+Polymyxin 1000IU / L+Amphotericin 5mg / L+Nalidixic acid 1mg / L+Trimethoprim 1mg / L+Cefsulodin sodium 1mg / L+N-acetyl L-cysteine ​​0.01g / L+Sodium thiosulfate 0.01g / L+Sodium sulfite 0.01g / L+Sodium chloride 5g / L+Horse serum 20%+Agar powder 13g / L+Bromothymol blue 50mg / L+TTC 100mg / L, pH 6.8.

[0226] Example 33: Formulation 33

[0227] Purified water 900g / L + trypticase 10g / L + ox brain extract powder 10g / L + ox heart extract powder 10g / L + phosphate buffer 0.9g / L + cyclodextrin 15g / L + soy peptone 10g / L + glucose 10g / L + sodium pyruvate 5g / L + α-ketoglutaric acid 2g / L + glycerol 10g / L + L-cysteine ​​1g / L + L-arginine 2g / L + ferrous sulfate 0.04g / L + heme 10mg / L + zinc sulfate 10mg / L + nickel chloride 10mg / L + catalase 50000IU / L + SOD 100000IU / L+Vancomycin 10mg / L+Teicoplanin 10mg / L+Lincomycin 1.5mg / L+Bacitracin 3000IU / L+Polymyxin 20000IU / L+Amphotericin 20mg / L+Nalidixic acid 10mg / L+Trimethoprim 10mg / L+Cefsulodin sodium 10mg / L+N-acetyl L-cysteine ​​0.1g / L+Sodium thiosulfate 0.1g / L+Sodium sulfite 0.1g / L+Sodium chloride 5g / L+Horse serum 5%+Agar powder 13g / L+Bromothymol blue 10mg / L+TTC10mg / L, pH6.8.

[0228] Example 34: Formulation 34

[0229] Purified water 900g / L+trypticase 10g / L+beef brain extract powder 5g / L+beef heart extract powder 5g / L+phosphate buffer 0.9g / L+cyclodextrin 10g / L+soybean peptone 5g / L+glucose 3g / L+sodium pyruvate 3g / L+α-ketoglutaric acid 1g / L+glycerol 5g / L+L-cysteine ​​0.3g / L+L-arginine 1g / L+ferrous sulfate 0.012g / L+heme 5mg / L+zinc sulfate 5mg / L+nickel chloride 5mg / L+catalase 10000IU / L+SOD 16000IU / L + Vancomycin 5mg / L + Teicoplanin 5mg / L + Lincomycin 0.5mg / L + Bacitracin 1500IU / L + Polymyxin 1000IU / L + Amphotericin 10mg / L + Nalidixic acid 5mg / L + Trimethoprim 5mg / L + Cefsulodin sodium 3mg / L + N-acetyl L-cysteine ​​0.05g / L + Sodium thiosulfate 0.05g / L + Sodium sulfite 0.05g / L + Sodium chloride 5g / L + Bovine serum 5% + Agar powder 13g / L + Bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0230] Example 35: Formulation 35

[0231] Purified water 900g / L+trypticase 10g / L+beef brain extract powder 5g / L+beef heart extract powder 5g / L+phosphate buffer 0.9g / L+cyclodextrin 10g / L+soybean peptone 5g / L+glucose 3g / L+sodium pyruvate 3g / L+α-ketoglutaric acid 1g / L+glycerol 5g / L+L-cysteine ​​0.3g / L+L-arginine 1g / L+ferrous sulfate 0.012g / L+heme 5mg / L+zinc sulfate 5mg / L+nickel chloride 5mg / L+catalase 10000IU / L+SOD 16000IU / L + Vancomycin 5mg / L + Teicoplanin 5mg / L + Lincomycin 0.5mg / L + Bacitracin 1500IU / L + Polymyxin 1000IU / L + Amphotericin 10mg / L + Nalidixic acid 5mg / L + Trimethoprim 5mg / L + Cefsulodin sodium 3mg / L + N-acetyl L-cysteine ​​0.05g / L + Sodium thiosulfate 0.05g / L + Sodium sulfite 0.05g / L + Sodium chloride 5g / L + Bovine serum 20% + Agar powder 13g / L + Bromothymol blue 10mg / L + TTC 10mg / L, pH 6.8.

[0232] Comparative Example 1: Culture medium currently used for Helicobacter pylori (including animal blood culture medium)

[0233] Purified water 930g / L, containing trypticase 10g / L + yeast extract powder 3g / L + beef extract powder 3g / L + phosphate buffer 0.9g / L + starch 1g / L + sodium chloride 5g / L + animal blood 7% + agar powder 13g / L + vancomycin 5mg / L + polymyxin 1000IU / L + amphotericin 10mg / L, pH 7.3±0.2.

[0234] Comparative Example 2: Culture medium currently used for Helicobacter pylori (containing activated carbon culture medium)

[0235] Purified water 1000g / L, containing trypticase 10g / L + yeast extract powder 3g / L + beef extract powder 3g / L + phosphate buffer 0.9g / L + starch 1g / L + sodium chloride 5g / L + activated carbon 2g / L + agar powder 13g / L + vancomycin 5mg / L + polymyxin 1000IU / L + amphotericin 10mg / L, pH 6.8±0.2.

[0236] Effect Example 1: Inoculate Helicobacter pylori on plates of Examples 1 to 35 and compare the growth effects

[0237] The cultured Helicobacter pylori was inoculated on the above-mentioned comparative example 1 and comparative example 2 formulation plates, and cultured at 35-37° C. under microaerobic conditions for 48-72 hours and the results were observed.

[0238] The cultured Helicobacter pylori was inoculated onto the plates of the formulations of Examples 1 to 35, and cultured at 35 to 37° C. and 5% to 10% CO 2 for 48 to 72 hours, and the results were observed.

[0239] The results are shown in Table 1.

[0240] Table 1

[0241]

[0242]

[0243] From the results, it can be seen that adding reagents can promote the growth of target bacteria, and the plate provided by the present invention can significantly improve the growth of target bacteria.

[0244] Effect Example 2: Non-target bacteria were inoculated on plates from Examples 1 to 35 to compare the growth effects

[0245] The cultured non-target bacteria were inoculated on the above-mentioned comparative example 1, comparative example 2 and examples 17 to 35 formulation plates, and cultured at 35 to 37° C. and 5% to 10% CO 2 for 48 to 72 hours and the results were observed.

[0246] The results are shown in Table 2.

[0247] Table 2

[0248]

[0249]

[0250]

[0251] From the results, it can be seen that the plate provided by the present invention has a more obvious inhibitory ability on non-target bacteria, can reduce the coverage and inhibition of target bacteria, and can improve the detection rate of target bacteria.

[0252] Effect Example 3: Inoculate Helicobacter pylori on plates using Examples 1 to 35, and compare the growth effect and drug sensitivity test effect

[0253] The cultured Helicobacter pylori was inoculated on the above-mentioned comparative example 1 and comparative example 2 formulation plates, and cultured at 35-37° C. under microaerobic conditions for 48-72 hours and the results were observed.

[0254] The cultured Helicobacter pylori was inoculated onto the plates of the formulations of Examples 1 to 35, and cultured at 35 to 37° C. and 5% to 10% CO 2 for 48 to 72 hours, and the results were observed.

[0255] The above-cultured Helicobacter pylori strains were used to prepare 2-Merckie suspensions, and agar dilution method drug sensitivity test was performed according to CLSI requirements. The suspensions were compared with the control examples to see whether they met the CLSI standard requirements. The results showed that the bacteria on the plates of the example formulations did not affect the drug sensitivity results and met the CLSI standard requirements.

[0256] The results are shown in Table 3.

[0257] Table 3

[0258]

[0259]

[0260] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Helicobacter pylori culture medium, characterized in that: Including peptone, extract powder, carbohydrates, amino acids, metal ions, enzymes, antibiotics, reducing agents, salts, serum, agar, indicators, buffers; Excludes: (1) blood; and (2), carbon; and (3) Activated carbon.

2. The Helicobacter pylori culture medium according to claim 1, characterized in that include: The peptone comprises at least one of animal peptone and plant peptone; and / or The powder comprises at least one of ox brain powder, ox heart powder and beef powder; and / or The carbohydrates include at least one of glucose, cyclodextrin, glycerol, sodium pyruvate, and α-ketoglutaric acid; and / or The amino acids include at least one of cysteine ​​and arginine; and / or The metal ions include at least one of ferrous ions, zinc ions and nickel ions; and / or The enzyme comprises at least one of catalase and SOD; and / or The antibiotics include at least one of vancomycin, teicoplanin, lincomycin, bacitracin, cefsulodin sodium, nalidixic acid, trimethoprim, polymyxin, and amphotericin; and / or The reducing agent comprises at least one of N-acetyl L-cysteine, sodium thiosulfate and sodium sulfite; and / or The salt comprises sodium chloride; and / or The serum comprises at least one of horse serum and bovine serum; and / or The indicator comprises at least one of bromothymol blue and TTC; and / or The buffer comprises a phosphate buffer; and / or The Helicobacter pylori culture medium also includes heme.

3. The Helicobacter pylori culture medium according to claim 2, characterized in that include: The weight portion of the peptone is 1 to 10 parts; and / or The weight portion of the soaking powder is 1 to 10 parts; and / or The weight portion of the glucose is 1 to 10 parts; and / or The weight portion of the cyclodextrin is 5 to 15 parts; and / or The weight portion of the glycerol is 1 to 10 parts; and / or The weight portion of the sodium pyruvate is 1 to 5 parts; and / or The weight portion of the α-ketoglutaric acid is 0.2 to 2 parts; and / or The weight portion of cysteine ​​is 0.1 to 1 part; and / or The weight portion of arginine is 0.5 to 2 parts; and / or The weight portion of ferrous sulfate is 0.006 to 0.04 parts; and / or The weight portion of the zinc sulfate is 0.001 to 0.01 parts; and / or The weight portion of the nickel chloride is 0.001 to 0.01 parts; and / or The weight portion of the vancomycin is 0.001 to 0.01 parts; and / or The weight portion of teicoplanin is 0.001 to 0.01 parts; and / or The weight portion of the lincomycin is 0.0001 to 0.0015 parts; and / or The weight portion of the cefsulodin sodium is 0.001 to 0.01 parts; and / or The weight portion of the nalidixic acid is 0.001 to 0.01 parts; and / or The weight portion of the trimethoprim is 0.001 to 0.01 parts; and / or The weight portion of the N-acetyl L-cysteine ​​is 0.01 to 0.1 parts; and / or The weight portion of the sodium thiosulfate is 0.01 to 0.1 parts; and / or The weight portion of the sodium sulfite is 0.01 to 0.1 parts; and / or The weight portion of the sodium chloride is 5 parts; and / or The weight portion of the horse serum is 50 to 200 parts; and / or The weight portion of the bovine serum is 50 to 200 parts; and / or The weight portion of the bromothymol blue is 0.01 to 0.05 parts; and / or The weight portion of TTC is 0.01 to 0.1 parts; and / or The weight portion of the amphotericin is 0.005 to 0.02 parts; The weight portion of the hemoglobin is 0.001 to 0.01 parts.

4. The Helicobacter pylori culture medium according to claim 2 or 3, characterized in that include: The catalase content is 5000-50000 IU / L; and / or The SOD content is 10000-100000 IU / L; and / or The bacitracin content is 500 to 3000 IU / L; and / or The polymyxin is 1000 to 20000 IU / L.

5. The Helicobacter pylori culture medium according to any one of claims 1 to 4, characterized in that The pH value is 6.0~7.

5.

6. The method for preparing a Helicobacter pylori culture medium according to any one of claims 1 to 5, characterized in that: include: Mixing the raw materials to prepare a solid culture medium to obtain the Helicobacter pylori culture medium; The raw materials include the peptone, the extract powder, the carbohydrate, the amino acid, the metal ion, the enzyme, the antibiotic, the reducing agent, the salt, the serum, the agar, the indicator and the buffer.

7. A kit, characterized in that The invention comprises the Helicobacter pylori culture medium according to any one of claims 1 to 5.

8. A method for isolating and culturing Helicobacter pylori for non-disease diagnosis or diagnosis purposes, characterized in that: include: Cultivated based on the Helicobacter pylori culture medium according to any one of claims 1 to 5 or the kit according to claim 7.

9. The separation and culture method according to claim 8, characterized in that: The culture conditions do not include microaerobic conditions.

10. The separation and culture method according to claim 9, characterized in that: The culture conditions include: the carbon dioxide content in the culture environment is 5% to 10%.

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