Preparation method of simulated sputum quality control product and application of simulated sputum quality control product in microbiological detection kit

Abstract

The invention discloses a preparation method of a simulated sputum quality control product and application of the simulated sputum quality control product in a microbiological detection kit, and belongs to the technical field of biological detection. The simulation effect on the sputum matrix is good, simulation of sputum color, bubbles and viscosity is achieved, good uniformity, dispersity and stability are achieved, and viscosity and sputum foam samples can still be maintained after the sputum matrix is stood and stored for 7 days at different storage temperatures. After being mixed with a culture, thalli are uniformly wrapped, and the bacterial strain has good biocompatibility and stability, has no influence on the morphology of the thalli, can stably simulate the dispersity of the thalli in sputum, is beneficial to the preparation of reference substances and quality control substances of in-vitro diagnosis related kits, and has a good auxiliary effect on microscopic examination of clinical microorganisms.

Description

Technical Field

[0001] The invention relates to a preparation method of a simulated sputum quality control product and application of the same in a microbiological detection kit, belonging to the technical field of biological detection. Background Art

[0002] Sputum is the secretion of alveoli, bronchi and trachea. Healthy people have very little sputum. Under normal circumstances, bronchial mucosal glands and goblet cells can secrete a small amount of mucus to keep the respiratory mucosa moist. Under pathological conditions, the respiratory mucosa and alveoli are stimulated and the mucosa becomes congested and edematous, serous fluid exudates, and mucus secretion increases. Various cells (red blood cells, white blood cells, macrophages, etc.), fibrin and other exudates and mucus, inhaled dust, and certain tissue necrosis are mixed to form sputum. The composition of sputum is very complex, consisting of 95% water and 5% dust, protein, etc. The purpose of sputum examination is: ① to assist in the diagnosis of certain respiratory diseases, such as bronchial asthma, bronchiectasis, etc.; ② to confirm certain respiratory diseases, such as tuberculosis, lung cancer, paragonimiasis, etc.; ③ to observe the efficacy and prognosis, etc.

[0003] Sputum examination mainly includes general characteristics examination, microscopic examination, immunological examination, microbiological examination, etc. There are many types of microorganisms in sputum, and a small number of them are normal flora of the upper respiratory tract mixed in when coughing up sputum. Most of them are pathogens that appear when there is infection in the bronchi and lungs, such as Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Mycobacterium tuberculosis, etc. In this case, sputum is generally taken for smear staining examination to observe the morphology of microorganisms in sputum.

[0004] In the process of developing in vitro diagnostic kits related to sputum testing, quality control substances are used to evaluate or verify the precision, accuracy, compliance of the developed kits, or analytical deviations caused by changes in reagents or analytical instruments. Quality control substances themselves have high requirements for stability and batch differences. When developing kits, quality control products that can be stably stored, repeated, and traceable should be selected. At present, for the preparation of sputum matrix, due to its poor stability, poor control of batch differences, and poor repeatability, the development of corresponding quality control substances is relatively difficult, and there are very few quality control matrices for related products for sputum testing. Therefore, in order to better simulate the physiological environment of the specimen, the sputum matrix needs to simulate the matrix environment of sputum in the body, such as composition, viscosity, dispersibility, compatibility and other properties, to provide support for the development of the kit, and to monitor the impact of the liquefaction process of sputum samples on the test results. At present, there is an urgent need for an artificial simulated sputum that can be stably and repeatedly prepared and is very similar to the sputum specimen, and is widely used as a quality control substance in the microbial detection of sputum. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for preparing a simulated sputum quality control product and its application in a microbial detection kit, aiming to solve the technical problem of the lack of stability and repeatability of the quality control material of the sputum matrix in the prior art.

[0006] The first technical solution provided by the present invention is a method for preparing a simulated sputum matrix, which includes natural hydrophilic colloid, surfactant, protein, polysaccharide, inorganic salt and water.

[0007] In certain embodiments, the hydrophilic colloid includes one or more of xanthan gum, gum arabic and locust bean gum; and the mass percentage of the hydrophilic colloid is 1% to 5%.

[0008] In certain embodiments, the mass percentage of the hydrophilic colloid may be 1% to 2%.

[0009] In certain embodiments, the hydrocolloid is xanthan gum or gum arabic.

[0010] In certain embodiments, the surfactant includes at least two of polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol and hydroxypropyl methylcellulose; the mass percentage of the surfactant may be 3% to 10%.

[0011] In certain embodiments, the mass percentage of the surfactant may be 4% to 6%.

[0012] In certain embodiments, the surfactant is composed of polyvinyl pyrrolidone and hydroxypropyl methylcellulose, and the mass ratio of polyvinyl pyrrolidone to hydroxypropyl methylcellulose is (1-2.5): (2-3).

[0013] In certain embodiments, the protein includes one or more of casein, mucin and albumin; the mass percentage of the protein may be 0.2% to 2%.

[0014] In certain embodiments, the mass percentage of the protein may be 0.3% to 0.5%.

[0015] In certain embodiments, the protein is casein or albumin.

[0016] In certain embodiments, the polysaccharide includes one or more of carboxymethyl chitosan, dextran and glycosaminoglycan; the mass percentage of the polysaccharide may be 0.3% to 2%.

[0017] In certain embodiments, the mass percentage of the polysaccharide may be 0.5% to 0.8%.

[0018] In certain embodiments, the polysaccharide is carboxymethyl chitosan or dextran.

[0019] In certain embodiments, the inorganic salt is a non-toxic metal ion inorganic salt, including one or more of sodium chloride, potassium chloride, sodium carbonate, potassium carbonate, sodium phosphate and potassium phosphate; the mass percentage of the inorganic salt may be 0.2% to 2%.

[0020] In certain embodiments, the mass percentage of the inorganic salt may be 0.6% to 0.8%.

[0021] In certain embodiments, the inorganic salt is sodium chloride.

[0022] In certain embodiments, the water can be sterile purified water.

[0023] The second technical solution provided by the present invention is a quality control product, which contains the sputum matrix described in the first technical solution.

[0024] In certain embodiments, the quality control product also includes a microbial culture.

[0025] In certain embodiments, the microbial culture includes bacteria, fungi, and cells.

[0026] The third technical solution provided by the present invention is a kit, which contains the sputum matrix described in the first technical solution, or contains the quality control product described in the second technical solution.

[0027] In certain embodiments, the kit further comprises fluorescein, a specific binding protein, and a counterstain.

[0028] Specifically, the specific binding protein in the kit is β-D-glucan binding protein, which can specifically bind to D-glucan on the fungal cell wall to form a complex, emit strong fluorescence under a specific excitation wavelength, and can be distinguished from other substances through a biological (fluorescence) microscope.

[0029] The fourth technical solution provided by the present invention is the use of the sputum matrix described in the first technical solution, or the quality control product described in the second technical solution, or the kit described in the third technical solution in detecting pathogens in a sample.

[0030] In certain embodiments, the test sample includes skin scrapings or sputum.

[0031] The technical effects of the present invention are as follows: The present invention has a good simulation effect on sputum matrix, including simulating sputum color, bubbles, viscosity, and having good uniformity, dispersibility and stability. After being stored at different storage temperatures for 7 days, the viscosity and sputum foam can still be maintained. After mixing with the culture, the bacteria are evenly wrapped, have good biocompatibility and stability, have no effect on the morphology of the bacteria, can stably simulate the dispersibility of the bacteria in sputum, contribute to the preparation of reference products and quality control products for in vitro diagnostic related kits, and have a good auxiliary effect on the microscopic examination of clinical microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 To compare the wet mount method for the application effect of the test kit for simulating sputum. DETAILED DESCRIPTION

[0033] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0034] Test method: 1. The clinical classification of sputum is as follows: Grade I: The sputum is like rice soup or foam, and there is no sputum retained on the inner wall of the glass joint after suction; Grade II: The appearance of sputum is thicker than that of grade I. After suctioning, a small amount of sputum is retained on the inner wall of the glass joint, but it can be easily washed away by water. Grade III: The sputum is obviously sticky and yellow in appearance. The sputum in the suction tube often collapses due to excessive negative pressure. A large amount of sputum is often retained on the inner wall of the glass joint and is not easily washed away by water.

[0035] 2. Viscosity test: Use LVDV-I+ Brookfield viscometer (Brookfield) to test liquid viscosity and operate according to the instrument manual.

[0036] The raw materials used in the embodiment: Candida albicans was sourced from ATCC Biological Standard Resource Center (strain number: ATCC 10231).

[0037] Example 1 A method for preparing simulated sputum is as follows: hydrophilic colloid, surfactant, protein, polysaccharide, and inorganic salt are added to 50 mL of water, and heated and stirred at 60° C.-70° C. for 1-3 hours until uniform.

[0038] Among them, according to the mass percentage (mass is g, volume is mL), the hydrophilic colloid is xanthocolloid, and the mass percentage is 1.0%. The surfactant is polyvinyl pyrrolidone and hydroxypropyl methylcellulose, and the mass percentages are 1% and 3% respectively. The protein is casein, and the mass percentage is 0.3%. The polysaccharide is carboxymethyl chitosan, and the mass percentage is 0.5%. The inorganic salt is sodium chloride, and the mass percentage is 0.6%.

[0039] Example 2 A method for preparing simulated sputum is as follows: hydrophilic colloid, surfactant, protein, polysaccharide, and inorganic salt are added to 50 mL of water, and heated and stirred at 60° C.-70° C. for 1-3 hours until uniform.

[0040] Among them, according to the mass percentage (mass is g, volume is mL), the hydrophilic colloid is xanthocolloid, and the mass percentage is 1.2%. The surfactant is polyvinyl pyrrolidone and hydroxypropyl methylcellulose, and the mass percentages are 1.5% and 2.5% respectively. The protein is casein, and the mass percentage is 0.3%. The polysaccharide is carboxymethyl chitosan, and the mass percentage is 0.5%. The inorganic salt is sodium chloride, and the mass percentage is 0.6%.

[0041] Example 3 A method for preparing simulated sputum is as follows: hydrophilic colloid, surfactant, protein, polysaccharide, and inorganic salt are added to 50 mL of water, and heated and stirred at 60° C.-70° C. for 1-3 hours until uniform.

[0042] Among them, according to the mass percentage (mass is g, volume is mL), the hydrophilic colloid is xanthocolloid, and the mass percentage is 1.4%. The surfactant is polyvinyl pyrrolidone and hydroxypropyl methylcellulose, and the mass percentages are 2.0% and 2.0% respectively. The protein is casein with a mass percentage of 0.4%. The polysaccharide is carboxymethyl chitosan with a mass percentage of 0.6%, and the inorganic salt is sodium chloride with a mass percentage of 0.6%.

[0043] Example 4 A method for preparing simulated sputum is as follows: hydrophilic colloid, surfactant, protein, polysaccharide, and inorganic salt are added to 50 mL of water, and heated and stirred at 60° C.-70° C. for 1-3 hours until uniform.

[0044] Among them, according to the mass percentage (mass is g, volume is mL), the hydrophilic colloid is xanthocolloid, and the mass percentage is 1.6%. The surfactant is polyvinyl pyrrolidone and hydroxypropyl methylcellulose, and the mass percentages are 2.0% and 2.5% respectively. The protein is casein, and the mass percentage is 0.4%. The polysaccharide is carboxymethyl chitosan, and the mass percentage is 0.6%. The inorganic salt is sodium chloride, and the mass percentage is 0.7%.

[0045] Example 5 A method for preparing simulated sputum is as follows: hydrophilic colloid, surfactant, protein, polysaccharide, and inorganic salt are added to 50 mL of water, and heated and stirred at 60° C.-70° C. for 1-3 hours until uniform.

[0046] Among them, according to the mass percentage (mass is g, volume is mL), the hydrophilic colloid is xanthocolloid, and the mass percentage is 1.8%. The surfactant is polyvinyl pyrrolidone and hydroxypropyl methylcellulose, and the mass percentages are 2.5% and 2.0% respectively. The protein is casein with a mass percentage of 0.5%. The polysaccharide is carboxymethyl chitosan with a mass percentage of 0.7%, and the inorganic salt is sodium chloride with a mass percentage of 0.8%.

[0047] Example 6 A method for preparing simulated sputum is as follows: hydrophilic colloid, surfactant, protein, polysaccharide, and inorganic salt are added to 50 mL of water, and heated and stirred at 60° C.-70° C. for 1-3 hours until uniform.

[0048] Among them, according to the mass percentage (mass is g, volume is mL), the hydrophilic colloid is xanthocolloid, and the mass percentage is 2.0%. The surfactant is polyvinyl pyrrolidone and hydroxypropyl methylcellulose, and the mass percentages are 2.5% and 2.5% respectively. The protein is casein with a mass percentage of 0.5%. The polysaccharide is carboxymethyl chitosan with a mass percentage of 0.8%, and the inorganic salt is sodium chloride with a mass percentage of 0.7%.

[0049] Example 7 The xanthocollagen in Example 4 was replaced with gum arabic, and other conditions or parameters were consistent with those in Example 4.

[0050] Example 8 The hydroxypropyl methylcellulose, one of the surfactants in Example 4, was replaced with 2.5% by mass of polyvinyl alcohol, and other conditions or parameters were the same as those in Example 4.

[0051] Example 9 The casein in Example 4 was replaced by albumin, and other conditions or parameters were the same as those in Example 4.

[0052] Example 10 The carboxymethyl chitosan in Example 4 was replaced by dextran, and other conditions or parameters were the same as those in Example 4.

[0053] Comparative Example 1 Artificial Sputum Matrix or Simulated Sputum Matrix (BZ274) is commercially available.

[0054] Comparative Example 2 The hydrophilic colloid in Example 4 was removed, and other conditions or parameters were consistent with those in Example 4.

[0055] Comparative Example 3 The surfactant in Example 4 and other conditions or parameters are consistent with those in Example 4.

[0056] Test Case 1. Evaluation of sputum properties According to the sputum viscosity classification, Examples 1 and 2 belong to Grade I, and are colorless or rice soup-colored foam; Examples 3, 4, 7 to 10 belong to Grade II, and are light yellow transparent foam, which is more viscous; Examples 5 and 6 belong to Grade III, and are viscous yellow. The sputum in Examples 1 to 10 can be completely dissolved.

[0057] Comparative Example 1 uses a commercially available simulated sputum matrix, the state of which tends to be Class II.

[0058] The viscosity of the liquid was tested using a LVDV-I+ Brookfield viscometer (Brookfield Corporation) and the results are shown in Table 1.

[0059] Table 1 Viscosity of sputum matrix in Examples 1 to 10 and Comparative Example 1

[0060] The test viscosity of the sputum matrix of Examples 3 to 10 and Comparative Example 1 is still 10 4 Order of magnitude. When no hydrophilic colloid or surfactant is added, the viscosity of the simulated sputum matrix is ​​relatively low, the simulation degree is not good, and normal research and detection requirements cannot be met. The addition of natural colloids such as xanthan gum and gum arabic of the present invention can better simulate the color and bubbles of sputum, and the addition of protein and the combination of surfactants of the present invention can better maintain the viscosity of sputum, maintain the homogeneity of sputum, and increase the dispersion and protection of bacterial organisms.

[0061] 2. Storage stability After being stored at different storage temperatures for 7 days, the states of the simulated sputum of Examples 1 to 6 and Comparative Example 1 were observed and recorded as shown in Table 2. At the same time, the viscosity of the simulated sputum of Examples 1 to 6 and Comparative Example 1 after being stored for 7 days was tested using a LVDV-I + type Brookfield viscometer (Brookfield Company), as shown in Table 3 below.

[0062] Table 2 Storage stability of simulated sputum

[0063] Table 3 Simulated sputum state viscosity

[0064] As can be seen from Tables 2 and 3, the viscosity and shape of the simulated sputum matrix of the present invention do not change whether it is stored at 4-8°C or 20-25°C; however, for the commercially available simulated sputum base liquid and the embodiments in which the key components are missing, the viscosity and sputum foam cannot be stably guaranteed. The foam is reduced after being stored at 4-8°C for 7 days, and the foam disappears after being stored at 20-25°C for 7 days, the viscosity decreases, and liquid stratification occurs, indicating that the simulated sputum matrix of the present invention is more stable than the commercially available simulated sputum matrix.

[0065] Example 11: Preparation of simulated sputum quality control product The simulated sputum matrices in Examples 1 to 10 and Comparative Example 1 were respectively added with a physiological saline suspension of cultured Candida albicans and mixed evenly to form a simulated sputum quality control product. The specific processing method is shown in Table 4.

[0066] Table 4 Preparation of quality control products

[0067] By adding microorganisms to the simulated sputum matrix provided by the present invention, a uniform state can still be maintained. However, the commercially available simulated sputum cannot stably guarantee the viscosity and sputum foaminess, and after the microbial cells are added, stratification and coagulation are prone to occur during the storage process. At the same time, the lack of colloids and surfactants in the embodiments will also cause uneven distribution of microorganisms, which will have an adverse effect on the microscopic detection of microorganisms.

[0068] Example 12: Application of simulated sputum quality control product in fungal detection kit The quality control product in Example 11 can be directly stained with wet slides. 15ul is drawn and spread on a glass slide in a uniform circle. Two drops of fungal fluorescent staining liquid produced by Jiangsu Meike Medical Technology Co., Ltd. are directly dropped on the sample area. After standing for 1 minute, the cover glass is covered and observed under a biofluorescence microscope (UV band). All the above combinations can achieve good staining effects. The morphological features of fungal hyphae and other morphological features are intact and uniformly colored with bright blue fluorescence. The experimental results are as follows Figure 1 The background coloring of the comparative example was serious after liquefaction, and the hyphae were wrapped by the viscous substances in the simulated sputum, which made it impossible to expose the morphology and affected the observation effect.

[0069] Based on the above example data, it can be seen that the present application uses appropriate hydrophilic colloids and other simulated sputum properties, and the proportions of each component are appropriate, which can form a simulated sputum quality control product with good dispersibility and stable properties, and can maintain the morphological characteristics of the bacteria, with good accuracy.

[0070] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a simulated sputum matrix, characterized in that: Includes natural hydrophilic colloids, surfactants, proteins, polysaccharides, inorganic salts and water.

2. The method according to claim 1, characterized in that: The hydrophilic colloid comprises one or more of xanthan gum, gum arabic and locust bean gum; the mass percentage of the hydrophilic colloid is 1% to 5%, optionally, the mass percentage of the hydrophilic colloid is 1% to 2%, and the hydrophilic colloid is xanthan gum or gum arabic.

3. The method according to claim 1, characterized in that The surfactant comprises at least two of polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol and hydroxypropyl methylcellulose; the mass percentage of the surfactant is 3% to 10%.

4. The method according to claim 1, characterized in that The surfactant includes at least two of polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol and hydroxypropyl methylcellulose; the mass percentage of the surfactant may be 3% to 10%; optionally, the mass percentage of the surfactant is 4% to 6%, and the surfactant is composed of polyvinyl pyrrolidone and hydroxypropyl methylcellulose, and the mass ratio of polyvinyl pyrrolidone to hydroxypropyl methylcellulose is (1 to 2.5): (2 to 3).

5. The method according to claim 1, characterized in that The protein includes one or more of casein, mucin and albumin; the mass percentage of the protein is 0.2% to 2%; optionally, the mass percentage of the protein is 0.3% to 0.5%, and the protein is casein or albumin.

6. The method according to claim 1, characterized in that The polysaccharide comprises one or more of carboxymethyl chitosan, dextran and glycosaminoglycan; the mass percentage of the polysaccharide is 0.3% to 2%; optionally, the mass percentage of the polysaccharide is 0.5% to 0.8%, and the polysaccharide is carboxymethyl chitosan or dextran.

7. The method according to claim 1, characterized in that The inorganic salt is a non-toxic metal ion inorganic salt, including one or more of sodium chloride, potassium chloride, sodium carbonate, potassium carbonate, sodium phosphate and potassium phosphate; the mass percentage of the inorganic salt may be 0.2% to 2%; optionally, the mass percentage of the inorganic salt may be 0.6% to 0.8%, and the inorganic salt is sodium chloride.

8. A quality control product, characterized in that: The quality control product contains the sputum matrix according to any one of claims 1 to 7.

9. A kit, characterized in that: The kit contains the sputum matrix according to any one of claims 1 to 7, or contains the quality control product according to claim 8.

10. Use of the sputum matrix according to any one of claims 1 to 7, or the quality control product according to claim 8, or the kit according to claim 9 in detecting pathogens in a sample.

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