High-specificity group B streptococcus chromogenic medium and preparation method thereof
By introducing specific chromogenic substrates and antibacterial agents into the chromogenic medium, combined with osmotic pressure regulators, the false positive problem of traditional culture media was solved, enabling rapid and accurate detection of group B streptococci.
Patent Information
- Application Number
- CN202511481632.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-16
- Publication Date
- 2025-12-05
AI Technical Summary
When traditional chromogenic media are used to isolate and culture group B streptococci, non-target bacteria such as Escherichia coli and Klebsiella pneumoniae produce β-galactosidase, leading to frequent false positive results and affecting the specificity and accuracy of the test.
A mixture of specific chromogenic substrates, 4-chloro-3-indolyl-β-D-galactopyranoside and halogen-β-D-galactopyranoside, was designed, combined with antibacterial agents polymyxin B and nalidixic acid, and osmotic pressure regulators sodium chloride, choline chloride, and isotridecyl alcohol polyoxyethylene ether, to inhibit the growth of contaminating bacteria and improve the specificity and accuracy of detection.
Chromogenic culture media can rapidly visualize group B streptococcal colonies, shorten detection time, reduce false positives, and improve detection efficiency and accuracy. It is suitable for accurate identification of low-concentration samples.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of culture medium, and in particular to a high-specificity group B streptococcus chromogenic culture medium and a preparation method thereof. BACKGROUND
[0002] At present, group B streptococcus (GBS) is a common pathogenic bacterium and an important pathogen causing neonatal infection and adverse pregnancy outcomes in pregnant women. Traditional group B streptococcus detection methods mainly include blood plate culture method, PCR method and immunological method. These methods have advantages respectively, but have problems such as complex operation, long time consumption or high cost. In comparison, the chromogenic culture medium has advantages in operation, time consumption and cost.
[0003] When the traditional chromogenic culture medium is used to separate and culture group B streptococcus, some non-target bacteria such as Escherichia coli, Klebsiella pneumoniae and intestinal flora may also produce beta-galactosidase, resulting in frequent false positive results and affecting the specificity and accuracy of detection. SUMMARY
[0004] In order to solve the above technical problems, the present application provides a high-specificity group B streptococcus chromogenic culture medium and a preparation method thereof.
[0005] In a first aspect, the present application provides a high-specificity group B streptococcus chromogenic culture medium, which specifically comprises the following components by weight: 6-10 g / L of proteose peptone, 2-6 g / L of yeast powder, 0.02-0.08 g / L of chromogenic substrate, 13-17 g / L of agar, 0.01-0.05 g / L of bacteriostatic agent and 3-7 g / L of osmotic pressure regulator; the pH of the group B streptococcus chromogenic culture medium is 7.2±0.2. The chromogenic substrate is composed of 4-chloro-3-indoxyl-beta-D-galactopyranoside and resorcinol-beta-D-galactopyranoside at a weight ratio of 6-9:1-4. The bacteriostatic agent is composed of polymyxin B and nalydic acid at a weight ratio of 1-5:0.2-0.6. The osmotic pressure regulator is composed of sodium chloride, choline chloride and isomeric tridecyl polyoxyethylene ether at a weight ratio of 8-12:0.1-0.5:1-5.
[0006] Based on the principle of enzyme substrate specific reaction, the present application designs a selective chromogenic substrate which is only decomposed by the specific enzyme of group B streptococcus to produce a chromogenic product. The bacteriostatic agent and the osmotic pressure regulator are integrated to inhibit the metabolic interference of other microorganisms, accelerate the division cycle of the target group B streptococcus, shorten the chromogenic time and improve the sensitivity of the chromogenic reaction.
[0007] The group B streptococcus chromogenic culture medium provided in the application adds specific chromogenic substrates composed of 4-chloro-3-indolyl-beta-D-galactopyranoside and tetrazolium-beta-D-galactopyranoside in the basic culture medium. When the beta-galactosidase secreted by the group B streptococcus during growth meets the enzyme substrate, a specific enzymatic reaction occurs, thereby generating a chromogenic product, so as to identify the group B streptococcus. Different enzyme substrates correspond to different chromogenic products, and some non-target bacteria (such as Escherichia coli, Klebsiella pneumoniae and other intestinal flora) may also produce beta-galactosidase, which may lead to false positives; the selective factor inhibitor in the culture medium is needed to inhibit the growth of mixed bacteria. If the effect of the selective factor is not good, it is easy to interfere with the judgment of the results. Therefore, the application adds an inhibitor composed of a specific type and a specific weight ratio of polymyxin B and nalydic acid in the chromogenic culture medium, and optimizes the concentration, effectively inhibits the growth of mixed bacteria, and does not affect the growth and chromogenic of the group B streptococcus, thereby improving the accuracy of the detection results and reducing false positives.
[0008] In the process of identifying the group B streptococcus, if the osmotic pressure and the substance for adjusting the osmotic pressure in the chromogenic culture medium are not suitable, it will lead to non-specific protein aggregation or precipitation, increase the background noise, and reduce the sensitivity and detection efficiency. Therefore, the osmotic pressure regulator is specifically screened in the application. The osmotic pressure regulator composed of sodium chloride, choline chloride and isomeric tridecyl polyoxyethylene ether can effectively inhibit the growth of non-target bacteria, reduce the competition of mixed bacteria for the nutrients of the culture medium and the interference of the target bacteria; the provided growth environment helps the group B streptococcus resist osmotic stress, is more suitable for the growth and reproduction of the group B streptococcus, and speeds up the speed of the beta-galactosidase secreted by the group B streptococcus during growth, so that the beta-galactosidase in the sample combines with the chromogenic substrate faster, so that the group B streptococcus colony can show a typical color at a smaller time, thereby shortening the identification time and improving the sensitivity of identification.
[0009] Preferably, the high-specificity group B streptococcus chromogenic culture medium specifically comprises the following components in weight parts: 7-9 g / L of proteose peptone, 3-5 g / L of yeast powder, 0.03-0.07 g / L of chromogenic substrate, 14-16 g / L of agar, 0.02-0.04 g / L of bacteriostatic agent, and 4-6 g / L of osmotic pressure regulator.
[0010] Preferably, the high-specificity group B streptococcus chromogenic culture medium specifically comprises the following components in weight parts: 7.5-8.5 g / L of proteose peptone, 3.5-4.5 g / L of yeast powder, 0.04-0.06 g / L of chromogenic substrate, 14.5-15.5 g / L of agar, 0.025-0.035 g / L of bacteriostatic agent, and 4.5-5.5 g / L of osmotic pressure regulator.
[0011] Preferably, the chromogenic substrate is composed of 4-chloro-3-indolyl-beta-D-galactopyranoside and resorcylic acid-beta-D-galactopyranoside in a weight ratio of 7-8:2-3.
[0012] In a specific embodiment, the weight ratio of 4-chloro-3-indolyl-beta-D-galactopyranoside and resorcylic acid-beta-D-galactopyranoside in the chromogenic substrate can be 6:1, 6:2, 6:3, 6:4, 7:1, 7:2, 7:3, 7:4, 8:1, 8:2, 8:3, 9:1, 9:2, 9:4.
[0013] Preferably, the bacteriostatic agent is composed of polymyxin B and nalfon in a weight ratio of 2-4:0.3-0.5.
[0014] In a specific embodiment, the weight ratio of polymyxin B and nalfon in the bacteriostatic agent can be 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 2:0.2, 2:0.3, 2:0.4, 2:0.5, 2:0.6, 3:0.2, 3:0.4, 3:0.5, 4:0.2, 4:0.3, 4:0.5, 4:0.6, 5:0.2, 5:0.3, 5:0.4, 5:0.6.
[0015] Through experimental analysis, it is found that the bacteriostatic agent composed of polymyxin B and nalfon in the above weight ratio can further improve the effect of the chromogenic culture medium in detecting and identifying group B streptococcus.
[0016] Preferably, the osmotic pressure regulator is composed of sodium chloride, choline chloride, and isomeric tridecyl polyoxyethylene ether in a weight ratio of 9-11:0.2-0.4:2-4.
[0017] In a specific embodiment, the weight ratio of sodium chloride, choline chloride, and isomeric tridecyl polyoxyethylene ether in the osmotic pressure regulator can be 8:0.1:1, 8:0.2:4, 11:0.3:5, 11:0.4:2, 9:0.1:1, 9:0.2:4, 10:0.3:5, 10:0.4:2, 12:0.1:1, 12:0.3:5.
[0018] Through experimental analysis, it is found that the osmotic pressure regulator composed of sodium chloride, choline chloride, and isomeric tridecyl polyoxyethylene ether in the above weight ratio can further improve the effect of the chromogenic culture medium in detecting and identifying group B streptococcus.
[0019] In a second aspect, the application provides a preparation method of the group B streptococcus chromogenic culture medium, and the specific steps are as follows: weighing each raw material component according to the corresponding weight, respectively, adding proteose peptone, yeast powder, chromogenic substrate, agar, and osmotic pressure regulator into sterile water, melting, mixing uniformly, adjusting pH, boiling, cooling to 48-52℃, adding bacteriostatic agent, pouring flat plate, and standby.
[0020] In a third aspect, the application provides a group B streptococcus detection kit, comprising the candida chromogenic culture medium.
[0021] In a fourth aspect, the application provides a detection method of group B streptococcus, which utilizes the group B streptococcus chromogenic culture medium or the group B streptococcus detection kit for detection.
[0022] In a fifth aspect, the application provides the group B streptococcus chromogenic culture medium or the group B streptococcus detection kit in the application for detecting group B streptococcus.
[0023] In summary, the technical scheme of the application has the following effects: The group B streptococcus chromogenic culture medium provided by the technical scheme of the application can effectively inhibit the growth of mixed bacteria while not affecting the growth and color development of group B streptococcus when detecting and identifying group B streptococcus, thereby improving the accuracy of the detection result, reducing false positives, and reducing misjudgment.
[0024] The group B streptococcus chromogenic culture medium provided by the technical scheme of the application can quickly react with the chromogenic substrate when detecting and identifying group B streptococcus, and the target bacteria can form colonies with typical color visible to the naked eye in a short time, which shortens the time for detecting and identifying group B streptococcus and greatly improves the detection efficiency.
[0025] The group B streptococcus chromogenic culture medium provided by the technical scheme of the application can detect group B streptococcus with a detection limit of 10 3 CFU / mL, which solves the problems of color development delay and missed detection caused by background bacterial interference in low-concentration group B streptococcus detection, while maintaining the simplicity and rapid detection characteristics of the operation. DETAILED DESCRIPTION
[0026] The application will be further described in detail below in combination with examples, comparative examples, and performance detection tests, and these examples cannot be understood as limiting the scope of the application.
[0027] The CAS number of 4-chloro-3-indolyl-beta-D-galactopyranoside used in the present application is 135313-63-2, and the CAS number of resorcylic-beta-D-galactopyranoside is 95079-19-9; isomeric tridecanol polyoxyethylene ether and isomeric alcohol polyoxyethylene polyoxypropylene ether are purchased from Henan Daorun New Material Technology Co., Ltd.; the remaining raw materials are commonly used substances in the industry and can be obtained by commercial purchase. Embodiment
[0028] Embodiment 1 Embodiment 1 provides a group B streptococcus chromogenic culture medium and a preparation method thereof.
[0029] The group B streptococcus chromogenic culture medium in the embodiment is composed of the following components at the following concentrations: 8 g / L of proteose peptone, 4 g / L of yeast powder, 0.05 g / L of chromogenic substrate (the chromogenic substrate is composed of 4-chloro-3-indolyl-beta-D-galactopyranoside and resorcylic-beta-D-galactopyranoside at a weight ratio of 7:3), 15 g / L of agar, 0.03 g / L of bacteriostatic agent (the bacteriostatic agent is composed of polymyxin B and nalonixic acid at a weight ratio of 3:0.4), and 5 g / L of osmotic pressure regulator (the osmotic pressure regulator is composed of sodium chloride, choline chloride, and isomeric tridecanol polyoxyethylene ether at a weight ratio of 10:0.3:3).
[0030] The preparation method of the chromogenic culture medium for Candida in the embodiment is as follows: respective weights of each raw material component are weighed, the proteose peptone, yeast powder, chromogenic substrate, agar, and osmotic pressure regulator are added to 1000 mL of sterile water, melted, uniformly mixed, the pH is adjusted to 7.2±0.2, high-pressure sterilized for 20 min, cooled to about 50°C, and the filtered and sterilized polymyxin B and nalonixic acid are added, and then poured into a flat plate for standby use.
[0031] Embodiments 2-8 Embodiments 2-8 respectively provide a group B streptococcus chromogenic culture medium and a preparation method thereof.
[0032] In embodiment 2: the concentration of the bacteriostatic agent is 0.01 g / L, and the concentration of the osmotic pressure regulator is 7 g / L.
[0033] In embodiment 3: the concentration of the bacteriostatic agent is 0.05 g / L, and the concentration of the osmotic pressure regulator is 3 g / L.
[0034] In embodiment 4: the bacteriostatic agent is composed of polymyxin B and nalonixic acid at a weight ratio of 1:0.6.
[0035] In embodiment 5: the bacteriostatic agent is composed of polymyxin B and nalonixic acid at a weight ratio of 2:0.3.
[0036] In Example 6: the osmotic pressure regulator is composed of sodium chloride, choline chloride, isomeric tridecyl polyoxyethylene ether with a weight ratio of 8:0.5:1.
[0037] In Example 7: the osmotic pressure regulator is composed of sodium chloride, choline chloride, isomeric tridecyl polyoxyethylene ether with a weight ratio of 12:0.1:5.
[0038] In Example 8: the osmotic pressure regulator is composed of sodium chloride, choline chloride, isomeric tridecyl polyoxyethylene ether with a weight ratio of 9:0.4:2.
[0039] The other process parameters in the above examples are the same as those in Example 1.
[0040] Comparative Example Comparative Examples 1-6 Comparative Examples 1-6 respectively provide a group B streptococcus chromogenic culture medium and a preparation method thereof.
[0041] The above comparative examples differ from Example 1 in that the specific differences are as shown below.
[0042] In Comparative Example 1: the concentration of the bacteriostatic agent is 0.005 g / L, and the concentration of the osmotic pressure regulator is 10 g / L.
[0043] In Comparative Example 2: the concentration of the bacteriostatic agent is 0.1 g / L, and the concentration of the osmotic pressure regulator is 1 g / L.
[0044] In Comparative Example 3: the bacteriostatic agent is composed of gentamicin and naflicin with a weight ratio of 3:0.4.
[0045] In Comparative Example 4: the bacteriostatic agent is composed of polymyxin B and naflicin with a weight ratio of 0.4:3.
[0046] In Comparative Example 5: the osmotic pressure regulator is composed of sodium chloride, choline chloride, isomeric tridecyl polyoxyethylene ether with a weight ratio of 10:3:0.3.
[0047] In Comparative Example 6: the osmotic pressure regulator is composed of sodium chloride, choline chloride, isomeric tridecyl polyoxyethylene ether with a weight ratio of 10:0.3:3.
[0048] The other process parameters in the above comparative examples are the same as those in Example 1.
[0049] Performance detection test (1) Accuracy detection The single strain group B streptococcus to be tested is prepared into a bacterial solution with a concentration of 1x10 7The suspension of 1 x 10
[0050] The results show that each single strain in the medium is characterized by showing purple-red colonies after 14h of incubation. The above results show that the chromogenic medium prepared in the present application can accurately detect single colonies of group B streptococcus, which is consistent with the typical growth characteristics of group B streptococcus species.
[0051] (2) Specific detection Prepare 8 strains (group B streptococcus, enterococcus, lactobacillus, leuconostoc, lactococcus, escherichia coli, staphylococcus aureus, and candida albicans) to be tested into suspensions with a bacterial concentration of 1 x 10 7 The suspensions are streaked onto 8 chromogenic medium plates (examples or comparative examples) in turn. Then the inoculated chromogenic medium plates are incubated in an incubator at 36°C ± 1°C, and during the 8-48h incubation period, the colonies formed are observed by naked eye every 2 hours. The growth of these colonies is recorded.
[0052] The results show that each single strain in the medium is characterized by showing purple-red colonies after 14h of incubation. The above results show that the chromogenic medium prepared in the present application can accurately detect single colonies of group B streptococcus, which is consistent with the typical growth characteristics of group B streptococcus species.
[0053] (3) Sensitivity detection Prepare the strain to be tested (group B streptococcus) into suspensions with a bacterial concentration of 1 x 10 7 CFU / mL, 1 x 10 6 CFU / mL, 1 x 10 5 CFU / mL, 1 x 10 4 CFU / mL, 1 x 10 3 CFU / mL, 1 x 10 2 CFU / mL, respectively, and streak them onto a chromogenic medium plate (example or comparative example) in turn. Then the inoculated chromogenic medium plate is incubated in an incubator at 36°C ± 1°C, and during the 8-48h incubation period, the colonies formed are observed by naked eye every 2 hours. The growth of these colonies is recorded, and the time when each colony shows the typical growth characteristics is observed.
[0054] The detection results are shown in Table 1.
[0055] Table 1 detection results of the sensitivity performance of the chromogenic culture medium in the examples and comparative examples From the detection results in Table 1 above, it can be seen that the chromogenic culture medium prepared by using the technical scheme provided in the present application has strong sensitivity in the detection and identification of group B streptococcus, and the identification time is relatively short; thus, the chromogenic culture medium prepared in the present application has broad practical application value.
[0056] By comparing the detection results of Comparative Examples 1-3 and Comparative Examples 1-2, it can be seen that the concentrations of the bacteriostatic agent and the osmotic pressure regulator have a great influence on the application performance of the chromogenic culture medium. In Comparative Example 1-2, the concentrations of the bacteriostatic agent and the osmotic pressure regulator are not matched, and the application performance of the chromogenic culture medium is poor. In contrast, the present application controls the concentration of the bacteriostatic agent to be 0.01-0.05 g / L and the concentration of the osmotic pressure regulator to be 3-7 g / L, and the application performance of the chromogenic culture medium prepared is excellent.
[0057] By comparing the detection results of Comparative Examples 1, 4-5 and Comparative Examples 3-4, it can be seen that the type of the bacteriostatic agent has a great influence on the application performance of the chromogenic culture medium. In Comparative Example 3, the bacteriostatic agent is composed of gentamicin and nalidixic acid in a weight ratio of 3:0.4, and in Comparative Example 4, the bacteriostatic agent is composed of polymyxin B and naldixic acid in a weight ratio of 0.4:3, and the application performance of the chromogenic culture medium is poor. In contrast, the present application uses a bacteriostatic agent composed of polymyxin B and naldixic acid in a weight ratio of 1-5:0.2-0.6, and the application performance of the chromogenic culture medium prepared is excellent.
[0058] By comparing the detection results of Comparative Examples 1, 6-8 and Comparative Examples 5-6, it can be seen that the type of the osmotic pressure regulator has a great influence on the application performance of the chromogenic culture medium. In Comparative Example 5, the osmotic pressure regulator is composed of sodium chloride, choline chloride and isomeric tridecanol polyoxyethylene ether in a weight ratio of 10:3:0.3, and in Comparative Example 6, the osmotic pressure regulator is composed of sodium chloride, choline chloride and isomeric alcohol polyoxyethylene polyoxypropylene ether in a weight ratio of 10:0.3:3, and the application performance of the chromogenic culture medium is poor. In contrast, the present application uses an osmotic pressure regulator composed of sodium chloride, choline chloride and isomeric tridecanol polyoxyethylene ether in a weight ratio of 8-12:0.1-0.5:1-5, and the application performance of the chromogenic culture medium prepared is excellent.
[0059] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various modifications or changes can be made therein without departing from the spirit and scope thereof. Accordingly, it is intended that all such modifications and changes be included within the scope of the application as claimed.
Claims
1. A highly specific chromogenic culture medium for group B streptococci, characterized in that, Specifically, it includes the following components in parts by weight: peptone 6-10 g / L, yeast extract 2-6 g / L, chromogenic substrate 0.02-0.08 g / L, agar 13-17 g / L, antibacterial agent 0.01-0.05 g / L, and osmotic pressure regulator 3-7 g / L; the pH of the group B streptococcal chromogenic medium is 7.2 ± 0.2; The chromogenic substrate is composed of a mixture of 4-chloro-3-indolyl-β-D-galactopyranoside and halogen-β-D-galactopyranoside in a weight ratio of 6-9:1-4. The antibacterial agent is composed of a mixture of polymyxin B and nalidixinone acid in a weight ratio of 1-5:0.2-0.6; The osmotic pressure regulator is composed of a weight ratio of 8-12: It is composed of a mixture of sodium chloride, choline chloride, and isomeric tridecyl alcohol polyoxyethylene ether at concentrations of 0.1-0.5:1-5.
2. The highly specific group B streptococcal chromogenic culture medium according to claim 1, characterized in that, Specifically, it includes the following components by weight: peptone 7-9 g / L, yeast extract 3-5 g / L, chromogenic substrate 0.03-0.07 g / L, agar 14-16 g / L, antibacterial agent 0.02-0.04 g / L, and osmotic pressure regulator 4-6 g / L.
3. The highly specific group B streptococcal chromogenic culture medium according to claim 2, characterized in that, Specifically, it includes the following components by weight: peptone 7.5-8.5 g / L, yeast extract 3.5-4.5 g / L, chromogenic substrate 0.04-0.06 g / L, agar 14.5-15.5 g / L, antibacterial agent 0.025-0.035 g / L, and osmotic pressure regulator 4.5-5.5 g / L.
4. The highly specific group B streptococcal chromogenic culture medium according to claim 1, characterized in that, The chromogenic substrate is composed of a mixture of 4-chloro-3-indolyl-β-D-galactopyranoside and halogen-β-D-galactopyranoside in a weight ratio of 7-8:2-3.
5. The highly specific group B streptococcal chromogenic culture medium according to claim 1, characterized in that, The antibacterial agent is composed of a mixture of polymyxin B and nalidixic acid in a weight ratio of 2-4:0.3-0.
5.
6. The highly specific group B streptococcal chromogenic culture medium according to claim 1, characterized in that, The osmotic pressure regulator is composed of sodium chloride, choline chloride, and isomeric tridecyl alcohol polyoxyethylene ether in a weight ratio of 9-11:0.2-0.4:2-4.
7. A method for preparing a chromogenic culture medium for group B streptococci as described in any one of claims 1-6, characterized in that, The specific steps are as follows: Weigh the corresponding weights of each raw material component, add peptone, yeast powder, chromogenic substrate, agar, and osmotic pressure regulator to sterile water, dissolve and mix evenly, adjust the pH, boil, cool to 48-52℃, add antibacterial agent, pour into plates, and set aside.
8. A group B streptococcus detection kit, characterized in that, The Candida chromogenic culture medium comprising any one of claims 1-6.
9. A method for detecting group B streptococci, characterized in that, The detection was performed using the chromogenic culture medium for group B streptococci according to any one of claims 1-6 or the group B streptococci detection kit according to claim 8.
10. The use of the chromogenic culture medium for group B streptococci as described in any one of claims 1-6 or the group B streptococci detection kit as described in claim 8 in the detection of group B streptococci.