Method for detecting burkholderia cepacia complex in fluoxetine hydrochloride oral solution

Abstract

The application relates to the technical field of bacterial population detection, and particularly discloses a method for checking Burkholderia cepacia complex in fluoxetine hydrochloride oral liquid, which comprises the following steps: S1: preparing a diluent; S2: preparing a bacterial solution; S3: preparing a test sample group; S4: performing a positive control test; and S5: performing a negative control test. The method is verified as follows: the three test strains in the positive control test group grow well under the condition of a specified temperature and the shortest culture time, and the characteristics meet the growth characteristics of the Burkholderia cepacia complex on a Burkholderia cepacia complex selective culture medium.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, specifically a method for detecting Burkholderia cepacia in fluoxetine hydrochloride oral solution. Background Technology

[0002] Burkholderia cepacia is a group of Hordeum bacteria with similar biochemical reactions, currently including more than 20 species (including Burkholderia cepacia, Burkholderia polyphaga, Burkholderia mysterioides, etc.). This group has strong drug resistance and can even use some antibiotics as a carbon source for growth. If this bacterium is present in a drug, there is a risk that it may become ineffective. Fluoxetine hydrochloride oral solution is a high water activity preparation, and Burkholderia cepacia needs to be controlled.

[0003] Chinese invention patent CN116515940A, published on August 1, 2023, discloses a method for detecting Burkholderia cepacia, comprising the following steps: S1-Preparation of bacterial suspension; S2-Preparation of test solution; S3-Enrichment culture; S4-Selection and isolation culture; S5-Negative control test; S6-Positive control test; S7-Result determination. Using this method, the presence of Burkholderia cepacia in pharmaceuticals can be detected, preventing diseases such as sepsis, endocarditis, pneumonia, wound infection, and abscesses from occurring in patients with cystic fibrosis, chronic granulomatosis, or immunocompromised individuals due to the consumption of pharmaceuticals containing Burkholderia cepacia, thus ensuring drug safety.

[0004] However, the aforementioned publicly available solutions have the following shortcomings: the existing testing process is complex and takes more time, which cannot meet the high precision requirements of testing, resulting in pharmaceutical products that cannot meet higher precision requirements. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned technical problems and provide a method for detecting Burkholderia cepacia in fluoxetine hydrochloride oral solution.

[0006] To achieve the above objectives, the present invention employs the following technical solutions;

[0007] The technical solution adopted by this invention to solve its technical problem is: a method for detecting Burkholderia cepacia in fluoxetine hydrochloride oral solution, comprising the following steps:

[0008] S1: Preparation of diluent;

[0009] S2: Preparation of bacterial culture;

[0010] S3: Test sample group;

[0011] S4: Positive control test;

[0012] S5: Negative control test.

[0013] Furthermore, the preparation of the S1 diluent includes: pH 7.0 sterile sodium chloride-peptone buffer: weigh 14.6g of pH 7.0 sodium chloride-peptone buffer powder, add water to dissolve to 1000ml, autoclave at 121℃ for 20min, and set aside.

[0014] Further, in step S2: bacterial suspension preparation; fresh cultures of Burkholderia cepacia, Burkholderia neonicota, and Burkholderia mysterio are inoculated into tryptic soy peptone liquid medium and cultured at 30–35°C for 18–24 hours. The cultures are then serially diluted with pH 7.0 sodium chloride-peptone buffer to 10⁻⁶ and 10⁻⁷ to prepare a bacterial suspension with a bacterial count of no more than 100 CFU per ml.

[0015] The present invention provides a method for detecting Burkholderia cepacia in fluoxetine hydrochloride oral solution, which has the following beneficial effects:

[0016] 1. The present invention was verified by the above method. Under the specified temperature and the shortest culture time, the three test strains in the positive control test group grew well and their characteristics were consistent with the growth characteristics of Burkholderia cepacia on Burkholderia cepacia selective culture medium. Attached Figure Description

[0017] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings; Figure 1 This is a schematic diagram of a method for detecting Burkholderia cepacia in fluoxetine hydrochloride oral solution. Detailed Implementation

[0019] It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. 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.

[0021] It should be noted that in the embodiments of the present invention, all directional indications (such as up-down-left-right-forward-backward...) are only used to explain the relative positional relationship and movement between the components in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indication will also change accordingly. The connection can be a direct connection or an indirect connection.

[0022] Please see Figure 1 As shown, a method for detecting Burkholderia cepacia in fluoxetine hydrochloride oral solution includes the following steps:

[0023] S1: Preparation of diluent;

[0024] S2: Preparation of bacterial culture;

[0025] S3: Test sample group;

[0026] S4: Positive control test;

[0027] S5: Negative control test.

[0028] S1 dilution buffer preparation: Includes pH 7.0 sterile sodium chloride-peptone buffer: Weigh 14.6g of pH 7.0 sodium chloride-peptone buffer powder, dissolve in water to 1000ml, autoclave at 121℃ for 20min, and set aside. By maintaining osmotic pressure and pH consistent with the physiological environment, bacterial cell inactivation due to osmotic shock or acid-base imbalance is avoided. Peptone provides trace protein protection, reducing non-specific adsorption and aggregation of microorganisms during dilution. This step solves the problems of bacterial cell damage, low recovery rate, and decreased detection sensitivity caused by traditional dilution buffers (such as pure water or physiological saline), ensuring that trace amounts of target bacteria in the test sample are completely preserved, laying the foundation for subsequent enrichment and isolation.

[0029] S2: Preparation of bacterial suspension; Fresh cultures of *Burkholderia cepacia*, *Neo-Burkholderia cepacia*, and *Burkholderia mysterioides* were inoculated into tryptic soy peptone broth and incubated at 30–35°C for 18–24 hours. The cultures were then serially diluted with pH 7.0 sodium chloride-peptone buffer to 10⁻⁶ and 10⁻⁷ to prepare a bacterial suspension with a bacterial count not exceeding 100 CFU per ml. TSB is rich in nitrogen, vitamins, and amino acids, which can efficiently support the rapid proliferation of the slow-growing *Burkholderia cepacia* population with low nutritional requirements at low inoculum levels, ensuring precise and controllable bacterial suspension concentration. This step solves the problem of positive control failure and method validation failure caused by growth inhibition or death during the dilution process of low-concentration bacteria, ensuring the reproducibility and reliability of subsequent experiments.

[0030] S3 Test Sample Group; Test Solution Preparation: Add 10ml of this product to pH 7.0 sodium chloride-peptone buffer to 100ml, mix well, and prepare a 1:10 test solution; Take 10ml of the 1:10 test solution and inoculate it into 100ml of tryptic soy peptone liquid medium, and incubate at 30-35℃ for 48-72 hours; Selection and Separation Culture: Streak the above pre-culture onto Burkholderia cepacia selective medium plates, and incubate at 30-35℃ for 48-72 hours; The two-stage culture strategy achieves a synergistic effect of "enrichment-separation": the enrichment stage increases the detection probability of trace contaminants, while the selective medium achieves efficient separation of target bacteria from other bacteria. This step solves the problem of Bcc bacterial concentration in oral liquids.

[0031] S4 Positive Control Test: Following the same procedure as the test sample group, add no more than 100 CFU of *Burkholderia cepacia* (23005), *Burkholderia neocarpica* (23006), and *Burkholderia mysterioides* (23010) bacterial suspensions respectively, mix well, and incubate at 30–35°C for 48 hours. Selection and Isolation Culture: Streak the above pre-culture onto *Burkholderia cepacia* selective culture medium plates and incubate at 30–35°C for 48 hours. Based on the method suitability test results (if all three test strains are detected in the above method suitability test, then test the test sample according to the preparation and testing methods of this test solution), select one test strain as the positive control strain. The positive control test should detect the corresponding test strain.

[0032] S5 Negative Control Test: Use pH 7.0 sodium chloride-peptone buffer solution instead of the test solution for the negative control test. No bacterial growth should be observed in the negative control test.

[0033]

[0034] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0035] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for detecting Burkholderia cepacia in fluoxetine hydrochloride oral solution, characterized in that; Includes the following steps: S1: Preparation of diluent; S2: Preparation of bacterial culture; S3: Test sample group; S4: Positive control test; S5: Negative control test.

2. The method for detecting Burkholderia cepacia in fluoxetine hydrochloride oral solution according to claim 1, characterized in that: The S1 diluent is prepared as follows: pH 7.0 sterile sodium chloride-peptone buffer: Weigh 14.6g of pH 7.0 sodium chloride-peptone buffer powder, add water to dissolve to 1000ml, autoclave at 121℃ for 20min, and set aside.

3. The method for detecting Burkholderia cepacia in fluoxetine hydrochloride oral solution according to claim 1, characterized in that: S2: Preparation of bacterial suspension; Fresh cultures of Burkholderia cepacia, Burkholderia neonavi, and Burkholderia mysterio are inoculated into tryptic soy peptone liquid medium and cultured at 30-35°C for 18-24 hours. The above cultures are serially diluted with pH 7.0 sodium chloride-peptone buffer to 10⁻⁶ and 10⁻⁷ to prepare a bacterial suspension with a bacterial count of no more than 100 CFU per ml.

4. The method for detecting Burkholderia cepacia in fluoxetine hydrochloride oral solution according to claim 1, characterized in that: The S3 test sample group; test solution preparation: take 10 ml of this product and add pH 7.0 sodium chloride-peptone buffer to 100 ml, mix well, and prepare a 1:10 test solution; take 10 ml of the 1:10 test solution and inoculate it into 100 ml of tryptic soy peptone liquid medium, and incubate at 30-35℃ for 48-72 hours; selection and isolation culture: take the above pre-culture and streak it onto Burkholderia cepacia selective medium plates, and incubate at 30-35℃ for 48-72 hours.

5. The method for detecting Burkholderia cepacia in fluoxetine hydrochloride oral solution according to claim 1, characterized in that: The S4 positive control test was conducted in the same manner as the test sample group. No more than 100 CFU of *Burkholderia cepacia* (23005), *Burkholderia neonicota* (23006), and *Burkholderia mysterioides* (23010) bacterial suspensions were added and mixed thoroughly. The mixture was then incubated at 30–35°C for 48 hours. For selection and isolation culture, the pre-culture was streaked onto a *Burkholderia cepacia* selective culture medium plate and incubated at 30–35°C for 48 hours. Based on the results of the method suitability test (if all three test strains were detected in the above method suitability test, the test sample was tested according to the preparation and testing methods of this test solution), one test strain was selected as the positive control strain. The positive control test should detect the corresponding test strain.

6. The method for detecting Burkholderia cepacia in fluoxetine hydrochloride oral solution according to claim 1, characterized in that: The S5 negative control test: Use pH 7.0 sodium chloride-peptone buffer solution instead of the test solution for the negative control test. No bacterial growth should be observed in the negative control test.

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