Helicobacter pylori culture medium, culture method and application thereof

Abstract

The invention provides a helicobacter pylori culture medium, a culture method and application thereof, and relates to the technical field of microbiological culture.The helicobacter pylori culture medium comprises a basic culture medium and antibiotics, and when the basic culture medium is a brain heart infusion agar culture medium, the basic culture medium is a solid culture medium and is used for activation by adding defiberized sheep blood or goat serum; when the basic culture medium is a Brinell broth culture medium, the basic culture medium is a liquid culture medium, essential amino acid is provided by adding L-cysteine, and the basic culture medium is used as a reducing agent, so that enrichment culture of anaerobic bacteria is facilitated. Meanwhile, ferric pyrophosphate, L-cysteine and antibiotics are combined for use, so that rapid growth of the helicobacter pylori is promoted, pollution caused by infectious microbes is effectively reduced, and a more stable growth environment is provided for the helicobacter pylori; data show that when the culture medium is used for culturing helicobacter pylori, compared with a traditional solid culture method, the yield of collected wet thalli is increased by nearly three times, and the high survival rate can be kept while high-speed multiplication culture of helicobacter pylori can be achieved.

Description

Technical Field

[0001] This invention relates to the field of microbial culture technology, and in particular to a culture medium, culture method and application of Helicobacter pylori. Background Technology

[0002] Helicobacter pylori (Hp) is the only known microorganism that can survive in the human stomach. It is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization and is a major cause of chronic gastritis, peptic ulcers, and other ailments. Hp is a Gram-negative, microaerophilic bacterium requiring 5-8% oxygen and cannot grow in atmospheric or strictly anaerobic environments. Diagnostic detection of Hp is currently an important research direction in this field. Culture media for Hp include both non-selective and selective media. Commonly used non-selective media include brain heart infusion agar, Columbia agar, tryptone soy agar, and Wilkins-Chalgren agar. Selective media involve adding certain antibiotics to the above media, such as vancomycin, citric acid, amphotericin B, polymyxin B, and trimethoprim (TMP).

[0003] To improve culture efficiency, existing technologies typically add essential nutrients for the growth and development of Helicobacter pylori to traditional culture media. This increases the bacterial proliferation rate by up to two times compared to traditional methods and maintains the morphology of Helicobacter pylori cells unchanged for 96 hours. However, the operation of this culture reagent is complex, and while increasing the growth rate, it cannot guarantee the survival rate.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a culture medium for Helicobacter pylori, in order to solve the technical problem that in the prior art, the survival rate of Helicobacter pylori cannot be guaranteed while it grows rapidly in the culture medium.

[0006] The second objective of this invention is to provide a method for culturing Helicobacter pylori.

[0007] A third objective of this invention is to provide the application of the above-mentioned Helicobacter pylori culture medium or the above-mentioned culture method in the preparation of Helicobacter pylori antigen.

[0008] The fourth objective of this invention is to provide a method for preparing Helicobacter pylori antigen.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a Helicobacter pylori culture medium, comprising a basal culture medium and an antibiotic; The basic culture medium includes brain heart infusion agar medium or Büchner broth medium; The antibiotics mentioned include vancomycin, trimethoprim, amphotericin B, and polymyxin B; When the basal culture medium is brain heart infusion agar medium, the Helicobacter pylori culture medium also includes defibrinated sheep blood or goat serum; When the basal medium is Brucella broth, it also includes L-cysteine, ferric pyrophosphate, and fetal bovine serum.

[0010] Furthermore, based on the total volume of the Helicobacter pylori culture medium, the content of vancomycin is 2.5~10 mg / L, the content of trimethoprim is 2.5~10 mg / L, the content of amphotericin B is 2.5~10 mg / L, and the content of polymyxin B is 0.25~1 mg / L.

[0011] Furthermore, the amount of defibrinated sheep blood and goat serum added is 80-120 ml per 1000 ml of Helicobacter pylori culture medium, preferably 100 ml.

[0012] Furthermore, based on the total volume of the Helicobacter pylori culture medium, the contents of L-cysteine ​​and ferric pyrophosphate were independently 30-50 g / L; Preferably, the amount of fetal bovine serum added is 80-120 ml per 1000 ml of Helicobacter pylori culture medium, and more preferably 100 ml.

[0013] Secondly, the present invention provides a method for culturing Helicobacter pylori, comprising activating Helicobacter pylori using a Helicobacter pylori culture medium with the aforementioned basic culture medium being brain heart infusion agar medium, and amplifying and culturing the activated Helicobacter pylori using a Helicobacter pylori culture medium with the aforementioned basic culture medium being Brucella broth medium.

[0014] Furthermore, the activation of Helicobacter pylori includes spreading frozen Helicobacter pylori on a Helicobacter pylori culture medium containing defibrinated sheep blood as the basic culture medium for preliminary activation culture, and then inoculating the preliminarily activated colonies on a Helicobacter pylori culture medium containing goat serum as the basic culture medium for secondary activation culture.

[0015] Furthermore, the amplification culture includes inoculating the Helicobacter pylori obtained from the activated culture into a Helicobacter pylori culture medium with Brucella broth as the basal medium for amplification culture; Preferably, the inoculation amount is 1 ml of a 1.5 × 10⁻⁶ solution per 100 ml of liquid culture medium. 6 bacterial suspension with cfu / ml; Preferably, the rotation speed for amplification culture is 100~140 r / min, and more preferably 120 r / min.

[0016] Furthermore, the conditions for activation culture and amplification culture independently include 35~38℃, 8~12%CO2, 3~7%O2, preferably 37℃, 10%CO2, 5%O2; Preferably, the humidity of the activation culture and the amplification culture are each 87-93%, and more preferably 90%. Preferably, the activation culture and amplification culture times are each 46-50 hours independently.

[0017] Thirdly, the present invention provides the application of the above-mentioned Helicobacter pylori culture medium or the above-mentioned culture method in the preparation of Helicobacter pylori antigen.

[0018] Fourthly, the present invention provides a method for preparing Helicobacter pylori antigen, comprising culturing Helicobacter pylori using the above-mentioned Helicobacter pylori culture medium or according to the above-mentioned culture method, collecting wet bacterial cells by centrifugation and performing ultrasonic destruction to obtain Helicobacter pylori antigen; Preferably, the centrifugation speed is 8000~12000 r / min, more preferably 10000 r / min; Preferably, the centrifugation time is 13-17 minutes, more preferably 15 minutes.

[0019] This invention provides a Helicobacter pylori culture medium. When the basal medium is brain heart infusion agar, it is a solid medium, suitable for activating Helicobacter pylori by adding defibrinated sheep blood or goat serum. When the basal medium is Brucella broth, it is a liquid medium, providing essential amino acids by adding L-cysteine, which can also act as a reducing agent, exhibiting antioxidant effects, removing free oxygen from the environment, lowering the redox potential, and promoting the proliferation of anaerobic bacteria. Simultaneously, the combined use of ferric pyrophosphate, L-cysteine, and appropriate concentrations of antibiotics promotes the rapid growth of Helicobacter pylori, effectively reducing contamination by other bacteria and providing a more stable growth environment for Helicobacter pylori. Data shows that, compared to traditional solid culture methods, the culture medium of this invention increases the yield of collected wet cells by nearly three times, maintaining a high survival rate while rapidly expanding and culturing Helicobacter pylori. Detailed Implementation

[0020] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0021] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] The present invention provides a culture medium for Helicobacter pylori, comprising a basal culture medium and antibiotics; the basal culture medium includes brain heart infusion agar medium or Brucella broth medium; the antibiotics include vancomycin, trimethoprim, amphotericin B and polymyxin B. When the basal culture medium is brain heart infusion agar medium, the Helicobacter pylori culture medium also includes defibrinated sheep blood or goat serum; when the basal culture medium is Brucella broth medium, it also includes L-cysteine, ferric pyrophosphate and fetal bovine serum.

[0024] When the basal culture medium is brain heart infusion agar, it is a solid culture medium. The addition of defibrinated sheep blood or goat serum is suitable for activating Helicobacter pylori. When the basal culture medium is Brucella broth, it is a liquid culture medium. The addition of L-cysteine ​​provides essential amino acids and can also act as a reducing agent, exhibiting antioxidant properties. It removes free oxygen from the environment, lowering the redox potential and promoting the proliferation of anaerobic bacteria. Simultaneously, the combined use of ferric pyrophosphate, L-cysteine, and appropriate concentrations of antibiotics promotes the rapid growth of Helicobacter pylori, effectively reducing contamination by other bacteria and providing a more stable growth environment for Helicobacter pylori. Data shows that the culture medium of this invention, compared to traditional solid culture methods, increases the yield of collected wet cells by nearly three times, maintaining a high survival rate while rapidly expanding the culture of Helicobacter pylori.

[0025] In some specific embodiments, based on the total volume of the Helicobacter pylori culture medium, the content of vancomycin is 2.5~10 mg / L, the content of trimethoprim is 2.5~10 mg / L, the content of amphotericin B is 2.5~10 mg / L, and the content of polymyxin B is 0.25~1 mg / L.

[0026] The content of vancomycin can be, but is not limited to, 2.5 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L or 10 mg / L, or any value between 2.5 and 10 mg / L.

[0027] The content of the trimethoprim can be, but is not limited to, 2.5 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L or 10 mg / L, or any value between 2.5 and 10 mg / L.

[0028] The content of the amphotericin B can be, but is not limited to, 2.5 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L or 10 mg / L, or any value between 2.5 and 10 mg / L.

[0029] The content of polymyxin B can be, but is not limited to, 0.25 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1 mg / L, or any value between 0.25 and 1 mg / L.

[0030] In some specific embodiments, the amount of defibrinated sheep blood and goat serum added is 80-120 ml per 1000 ml of Helicobacter pylori culture medium, preferably 100 ml.

[0031] In some specific implementations, the content of L-cysteine ​​and ferric pyrophosphate is 30-50 g / L, respectively, based on the total volume of the Helicobacter pylori culture medium.

[0032] The contents of L-cysteine ​​and ferric pyrophosphate can be independently, but are not limited to, 30 g / L, 32 g / L, 34 g / L, 36 g / L, 38 g / L, 40 g / L, 42 g / L, 44 g / L, 46 g / L, 48 g / L or 50 g / L, or any value between 30 and 50 g / L.

[0033] In some specific embodiments, the amount of fetal bovine serum added is 80-120 ml per 1000 ml of Helicobacter pylori culture medium, preferably 100 ml.

[0034] The above-mentioned method for preparing Helicobacter pylori culture medium includes adding antibacterial agents and defibrinated sheep blood or goat serum to the basic culture medium according to the formula amount; or adding antibacterial agents and L-cysteine, ferric pyrophosphate and fetal bovine serum, and mixing them evenly.

[0035] According to another aspect of the present invention, a method for culturing Helicobacter pylori is also provided, comprising activating Helicobacter pylori using a Helicobacter pylori culture medium with the above-mentioned basic culture medium being brain heart infusion agar medium, and amplifying and culturing the activated Helicobacter pylori using a Helicobacter pylori culture medium with the above-mentioned basic culture medium being Brucella broth medium.

[0036] A two-stage culture method was adopted. First, Helicobacter pylori culture medium with brain heart infusion agar as the basic medium was used to activate the strain and enhance its viability, so as to obtain a sufficient number of cultures. Then, the strain was inoculated again into Helicobacter pylori culture medium with Brucella broth as the basic medium for further expansion culture. This method can accelerate the culture growth and reproduction rate of Helicobacter pylori, maintain the survival rate, facilitate the collection of wet cells, and reduce production costs.

[0037] In some specific embodiments, the activation of Helicobacter pylori includes spreading frozen Helicobacter pylori on a Helicobacter pylori culture medium containing defibrinated sheep blood as the basic culture medium for preliminary activation culture, and then inoculating the preliminarily activated colonies on a Helicobacter pylori culture medium containing goat serum as the basic culture medium for secondary activation culture.

[0038] Two types of activation media were used, depending on the type of blood added: defibrinated sheep blood and goat serum. Cultivation in a basal medium containing defibrinated sheep blood promoted the activation and growth of Helicobacter pylori. However, due to the complex composition of whole blood, many impurities remained when collecting Helicobacter pylori from blood agar medium. Therefore, the colonies from the initial activation culture were inoculated again into a Helicobacter pylori medium containing goat serum for a second activation culture. This two-step activation process allowed Helicobacter pylori to reach its optimal activation state.

[0039] In some specific embodiments, the amplification culture includes inoculating the Helicobacter pylori obtained from activation culture onto a Helicobacter pylori culture medium with Brucella broth as the basal medium for amplification culture.

[0040] Activated Helicobacter pylori can be obtained through rinsing, which facilitates inoculation. Rinsing methods include rinsing with sterile PBS.

[0041] In some specific implementations, the inoculation volume is 1 ml of a 1.5 × 10⁻⁶ solution per 100 ml of liquid culture medium. 6 bacterial suspension with cfu / ml; In some specific implementations, the rotation speed of the amplification culture is 100~140 r / min, preferably 120 r / min.

[0042] In some specific implementations, the conditions for activation culture and amplification culture independently include 35~38℃, 8~12%CO2, 3~7%O2, preferably 37℃, 10%CO2, 5%O2; In some specific embodiments, the humidity of the activation culture and the amplification culture are independently 87-93%, preferably 90%; In some specific implementations, the activation culture and amplification culture times are each 46 to 50 hours independently.

[0043] The *Helicobacter pylori* bacteria obtained by the above-described culture method exhibit rapid growth and high activity, making them suitable for preparing *Helicobacter pylori* antigens. According to another aspect of the present invention, the application of the above-described *Helicobacter pylori* culture medium or the above-described culture method in the preparation of *Helicobacter pylori* antigens is also provided.

[0044] According to another aspect of the present invention, a method for preparing Helicobacter pylori antigen is also provided, comprising culturing Helicobacter pylori using the above-described Helicobacter pylori culture medium or according to the above-described culture method, collecting wet bacterial cells by centrifugation and performing ultrasonic destruction to obtain Helicobacter pylori antigen.

[0045] The prepared antigens can be used to establish chemiluminescent immunoassay methods and prepare reagents or kits, with positive and negative detection rates, sensitivity, and specificity all reaching 100%.

[0046] In some specific embodiments, the centrifugation speed is 8000~12000 r / min, preferably 10000 r / min; In some specific embodiments, the centrifugation time is 13 to 17 minutes, preferably 15 minutes.

[0047] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0048] The Helicobacter pylori standard strain was purchased from Beina Chuanglian Biotechnology Co., Ltd. (ATCC 43504).

[0049] Preparation of antibiotics: Weigh out 0.05 g of vancomycin, 0.05 g of trimethoprim, 0.05 g of amphotericin B, and 0.005 g of polymyxin B using an electronic balance. Add 10 mL of DMSO and mix thoroughly until no particles remain. Dissolution may be slow; incubation at 4°C overnight is recommended. Aliquot and store at -20°C for later use.

[0050] I. Helicobacter pylori culture medium The component contents of the Helicobacter pylori culture medium in Examples 1-3 are shown in Table 1.

[0051] Table 1

[0052] Comparative Example 1 Unlike Example 2, L-cysteine ​​was not added to the amplification medium.

[0053] Comparative Example 2 Unlike Example 2, ferric pyrophosphate was not added to the amplification medium.

[0054] Following the formulations and proportions of the above examples and comparative examples, dissolve all components except antibiotics and serum (or blood) in distilled water. Autoclave at 121°C for 20 minutes, then lower the temperature to 45-60°C. In a biosafety cabinet, add antibiotics and serum (or blood), mix well, and the mixture is ready. For solid culture medium, pour 10 ml per petri dish and allow to solidify for later use.

[0055] Examples 4-6 The Helicobacter pylori culture method is performed according to the following steps, using the specific methods described in Examples 1-3: 1. Activation (1) Preliminary activation of the strain: The strain was activated using a single activation medium. The activation steps were as follows: take a frozen Helicobacter pylori strain, spread it on the single activation medium, place it in an incubator, and set the incubator parameters as follows: 37℃, 90% humidity, 10% CO2, 5% O2, and activate for 48 hours.

[0056] (2) Secondary activation of the strain: The strain is activated a second time using a secondary activation medium. The secondary activation step is to use a sterile brush to brush the colonies obtained in step (1) onto a new secondary activation medium and place them in an incubator. The incubator parameters are: 37℃, 90% humidity, 10% CO2, 5% O2, and culture for 48 hours.

[0057] 2. Amplification The colonies obtained from the secondary activation were repeatedly rinsed and blown down with a pipette tip to remove the bacterial growth. After mixing, the mixture was diluted to 1.5 × 10⁻⁶. 6CFU / ml, add 1 ml of bacterial culture to 100 ml of liquid culture medium; place the shake flask in an incubator with the following parameters: 37℃, 90% humidity, 10% CO2, 5% O2, rotation speed 120 r / min, and culture for 48 hours.

[0058] Comparative Examples 3-4 The difference from Example 5 is that the activation culture medium and amplification culture medium of Comparative Examples 1 and 2 were used respectively.

[0059] Comparative Example 5 The difference from Example 5 is that in the activation culture step, only the activated blood culture medium (one-time activation culture medium) is activated, while the amplification method remains the same.

[0060] Comparative Example 6 The difference from Example 5 is that in the activation culture step, only the activated serum culture medium (secondary activation culture medium) is activated, while the amplification method remains unchanged.

[0061] Comparative Example 7 The difference from Example 5 is that no activation culture was performed; instead, the culture was directly cultured using amplification medium, and the cells were passaged every 48 hours for a total of 3 times, totaling 144 hours.

[0062] Comparative Example 8 The difference from Example 5 is that, instead of activation and amplification culture, only activated blood culture medium (the activation culture medium of Example 5) is used for culture, i.e., blood solid culture. The culture is passaged once every 48 hours, for a total of 3 times, for a total of 144 hours.

[0063] Comparative Example 9 The difference from Example 5 is that, instead of activation and amplification culture, only activated serum culture medium (the secondary activation culture medium of Example 5) is used for culture, i.e., serum solid culture. The culture is passaged once every 48 hours, for a total of 3 times, for a total of 144 hours.

[0064] Experiment 1 The cultures of Examples 4-6 and Comparative Examples 3-7 were used as samples for testing.

[0065] 1. Assay of bacterial strain activity Take 0.5 ml of the culture after 48 hours of amplification, centrifuge at 12000 r / min for 5 min; discard the supernatant and resuspend the bacterial cells in the same volume of sterile PBS. Zero the spectrophotometer using sterile PBS as a blank control, and measure the concentration at 600 nm (OD200). 600 The absorbance of the bacterial solution was measured at a certain point.

[0066] Take 300 mL of the culture after 48 hours of amplification, centrifuge at 12000 rpm / min at 4℃ for 10 min in a 500 mL centrifuge bottle, discard the supernatant, resuspend the precipitate with PBS, centrifuge at 12000 rpm / min at 4℃ for 10 min, discard the supernatant, and weigh the bacterial sludge; the absorbance and bacterial weight results are shown in Table 2.

[0067] Table 2

[0068] 2. Survival rate determination of cultured strains The broth culture of *Brucella breviculae*, amplified for 48 hours, was serially diluted 10-fold. 100 μL of each concentration gradient was evenly spread onto brain heart infusion agar medium. After incubation under the above conditions for 48 hours, the results were observed and counted. The colonies were translucent pinpoints. Colony counts were performed for each concentration gradient, and the results are shown in Table 3.

[0069] Table 3

[0070] Data shows that Examples 4-6 are superior to their respective comparative examples, with Example 5 showing the best OD. 600 The highest values ​​for bacterial count, bacterial weight, and viable cell count indicate that its biomass accumulation efficiency is optimal and that it can maintain strain activity. Compared with Example 5, Comparative Example 3 used an amplification medium without L-cysteine, and Comparative Example 4 used an amplification medium without ferric pyrophosphate, resulting in a significant decrease in strain amplification efficiency and a corresponding decrease in viable cell count. This indicates that L-cysteine ​​and ferric pyrophosphate in the amplification medium have a synergistic promoting effect on improving strain amplification efficiency and maintaining strain activity. In the activation phase, Comparative Example 5 used the activation medium only once, and the OD value was... 600 Compared to Comparative Example 6, which used only a secondary activation medium and had no impurities in the harvested cells, the viable cell count decreased and impurities were present in the harvested cells. 600 The significantly reduced viable bacterial count indicates that only sequential activation with primary and secondary activation media can activate Helicobacter pylori activity, promoting its amplification efficiency and maintaining strain viability. Comparative Example 7 used liquid amplification medium, lacking the activation step, and its OD was significantly lower than in Example 5. 600 The OD values ​​and bacterial weights were significantly lower than those of traditional solid-state cultures, namely Comparative Examples 8 and 9, compared to Example 5. 600 The bacterial count and bacterial weight were significantly lower, with the bacterial weight decreasing by about 3 times (the bacterial weight of bacteria with impurities in the bacterial sludge is not used as a reference), and the number of live bacteria was much lower than in Example 5.

[0071] Experiment 3 1. Preparation of Helicobacter pylori antigen Take 300 mL of the culture from Example 5 and centrifuge it in a 500 mL centrifuge bottle at 12000 rpm / min 4℃ for 10 min. Discard the supernatant, resuspend the precipitate with PBS, centrifuge it at 12000 rpm / min 4℃ for 10 min, and discard the supernatant. Collect the wet bacterial cells and sonicate them to prepare the Helicobacter pylori natural antigen.

[0072] 2. Chemiluminescence detection performance of Helicobacter pylori antigen This kit uses tubular chemiluminescent immunoassay technology and an indirect method to detect Helicobacter pylori antibodies in samples. Specifically, it involves conjugating natural Helicobacter pylori antigens onto magnetic beads to create a chemiluminescent immunoassay kit, the composition of which is as follows: 1) Magnetic bead coupling Take the magnetic beads to be used and wash them three times with cross-linking buffer. The concentration of magnetic beads during washing is 10 mg / mL.

[0073] Resuspend in cross-linking buffer solution, mix well, and then sonicate (sonication time is 2 min). After sonication, add Helicobacter pylori antigen to the magnetic beads, mix well, and react at room temperature overnight (18 h).

[0074] Add a sealing agent, mix well, and sonicate. Mix at 37°C and react overnight (18 h).

[0075] After sealing, wash three times with TBST, resuspend in magnetic bead preservation solution, mix well, and sonicate (sonication time 2 min). After sonication, mix well and store at 2~8℃ for later use.

[0076] 2) Acridinium ester-labeled mouse anti-human IgG antibody Add AE solution to mouse anti-human IgG antibody and mix well at room temperature in the dark for 1 hour.

[0077] Add 1% (by volume) of 1M lysine and mix well. Let stand for 30 minutes to react.

[0078] Desalting and purification (requires two steps) or ultrafiltration purification into the storage solution removes excess AE.

[0079] Take the acridinium ester diluent, add the labeled acridinium ester antibody stock solution to make a final concentration of 0.1 ug / mL, and vortex to mix.

[0080] 3. Dilute the sample with sample diluent for chemiluminescence detection. Specificity of negative and positive control sera = detection of true negatives / (detection of true negatives + detection of false positives). Sensitivity = True positives detected / (True positives detected + False negatives detected)

[0081] The results of the detection performance verification of Helicobacter pylori natural antigen are shown in Table 4.

[0082] Table 4

[0083] A chemiluminescent immunoassay method was established using Helicobacter pylori natural antigens. The results showed a positive and negative detection rate of 100%, sensitivity of 100%, and specificity of 100%.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for Helicobacter pylori, characterized in that, Includes basal culture medium and antibiotics; The basic culture medium includes brain heart infusion agar medium or Büchner broth medium; The antibiotics mentioned include vancomycin, trimethoprim, amphotericin B, and polymyxin B; When the basal culture medium is brain heart infusion agar medium, the Helicobacter pylori culture medium also includes defibrinated sheep blood or goat serum; When the basal medium is Brucella broth, it also includes L-cysteine, ferric pyrophosphate, and fetal bovine serum.

2. The Helicobacter pylori culture medium according to claim 1, characterized in that, Based on the total volume of the Helicobacter pylori culture medium, the content of vancomycin is 2.5~10 mg / L, the content of trimethoprim is 2.5~10 mg / L, the content of amphotericin B is 2.5~10 mg / L, and the content of polymyxin B is 0.25~1 mg / L.

3. The Helicobacter pylori culture medium according to claim 1 or 2, characterized in that, The amount of defibrinated sheep blood and goat serum added is 80-120 ml per 1000 ml of Helicobacter pylori culture medium, preferably 100 ml.

4. The Helicobacter pylori culture medium according to claim 1 or 2, characterized in that, Based on the total volume of the Helicobacter pylori culture medium, the contents of L-cysteine ​​and ferric pyrophosphate were independently 30-50 g / L; Preferably, the amount of fetal bovine serum added is 80-120 ml per 1000 ml of Helicobacter pylori culture medium, and more preferably 100 ml.

5. A method for culturing Helicobacter pylori, characterized in that, This includes activating Helicobacter pylori using a culture medium with brain heart infusion agar as the basal medium as described in any one of claims 1 to 4, and amplifying and culturing activated Helicobacter pylori using a culture medium with Brucella broth as the basal medium as described in any one of claims 1 to 4.

6. The cultivation method according to claim 5, characterized in that, The activation of Helicobacter pylori involves spreading frozen Helicobacter pylori onto a Helicobacter pylori culture medium containing defibrinated sheep blood as the basal medium for initial activation culture, and then inoculating the initially activated colonies onto a Helicobacter pylori culture medium containing goat serum as the basal medium for secondary activation culture.

7. The cultivation method according to claim 5, characterized in that, The amplification culture includes inoculating the Helicobacter pylori obtained from the activation culture into a Helicobacter pylori culture medium with Brucella broth as the basal medium for amplification culture. Preferably, the inoculation amount is 1 ml of a 1.5 × 10⁻⁶ solution per 100 ml of liquid culture medium. 6 bacterial suspension with cfu / ml; Preferably, the rotation speed for amplification culture is 100~140 r / min, and more preferably 120 r / min.

8. The cultivation method according to claim 6 or 7, characterized in that, The activation culture and amplification culture conditions independently include 35~38℃, 8~12%CO2, 3~7%O2, preferably 37℃, 10%CO2, 5%O2; Preferably, the humidity of the activation culture and the amplification culture are each 87-93%, and more preferably 90%. Preferably, the activation culture and amplification culture times are each 46-50 hours independently.

9. The use of the Helicobacter pylori culture medium according to any one of claims 1 to 4 or the culture method according to any one of claims 5 to 8 in the preparation of Helicobacter pylori antigen.

10. A method for preparing Helicobacter pylori antigen, characterized in that, The method includes culturing Helicobacter pylori using the culture medium for Helicobacter pylori as described in any one of claims 1 to 4 or the culture method as described in claims 5 to 8, collecting the wet bacterial cells by centrifugation and then subjecting them to ultrasonic destruction to obtain Helicobacter pylori antigen. Preferably, the centrifugation speed is 8000~12000 r / min, more preferably 10000 r / min; Preferably, the centrifugation time is 13-17 minutes, more preferably 15 minutes.

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