Microorganism detection bottle for sterility test and preparation method thereof

Abstract

The invention relates to a microbiological detection bottle for sterility test and a preparation method thereof, and belongs to the technical field of sterility test. The microbiological detection bottle comprises a bottle body, the inner surface of the bottle bottom of the bottle body is covered with a color developing layer, and the color developing layer is covered with a light reflecting layer; and the developing layer comprises a transparent silica gel layer containing a developing indicator. The detection sensitivity can be effectively improved, false negative results are reduced, and the detection time is shortened.

Description

Technical Field

[0001] The present application relates to the technical field of sterility inspection, and in particular to a microbial detection bottle for sterility inspection and a preparation method thereof. Background Art

[0002] Microbiological testing of drugs is a necessary means to ensure drug quality and reduce clinical adverse reactions. For high-risk sterile preparations, sterility testing is of great significance in their production, circulation and supervision. At present, the pharmacopoeias of various countries use the culture medium turbidity observation method to judge the sterility of drugs. The traditional pharmacopoeia culture method has certain limitations and is time-consuming. It generally takes 14 days of culture time and has low detection efficiency. For short-term sterile preparations, such as some cell and gene therapy products, the current sterility testing method cannot guarantee that the inspection is completed before the product is used, which is not conducive to the rapid release of the product, nor is it conducive to the company to take corrective measures as soon as possible.

[0003] In order to make up for the shortcomings of the traditional pharmacopoeia's sterility test, such as long time consumption, low degree of automation, and large human errors, various rapid microbial detection technologies have emerged on the market, such as nucleic acid amplification technology, flow cytometry, ATP bioluminescence technology, respiratory colorimetric technology, etc., among which respiratory colorimetric detection technology is the most widely used; Chinese Pharmacopoeia 9046 "Guidelines for Microbiological Inspection of Cell Products" and TSHPPA 012-2022 "Technical Requirements for Validation of Rapid Sterility Test Methods for Cell and Gene Therapy Products" both recommend respiratory colorimetric method as a sterility detection method for cell and gene products. Compared with the traditional pharmacopoeia method, the respiratory colorimetric method has a certain shortening of the detection time, but still requires 7 days. In addition, for bacteria with slow growth rate or non-fermenting bacteria with very little carbon dioxide production, their growth curve is too flat, which is not conducive to the instrument's judgment of the positive and negative nature of the curve, resulting in false negative results. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the embodiments of the present application is to provide a microbial detection bottle for sterility inspection and a preparation method thereof, so as to improve the problems of current sterility detection methods such as short detection time, low detection sensitivity, and easy occurrence of false negative results.

[0005] In a first aspect, an embodiment of the present application provides a microbial detection bottle for sterility inspection, comprising: a bottle body, the inner surface of the bottom of the bottle body is covered with a color developing layer, and the color developing layer is covered with a reflective layer; the color developing layer includes a transparent silicone layer containing a color developing indicator.

[0006] The present application covers a color developing layer on the bottom of the bottle body, and a reflective layer on the color developing layer, wherein the color developing layer includes a transparent silicone layer containing a color developing indicator. During initial detection, the color developing layer is transparent blue, and a light source with a wavelength greater than 570nm is irradiated on the bottom of the bottle, and the light can penetrate through the transparent silicone layer containing the color developing indicator. Since the light has a certain optical path when penetrating through the transparent silicone layer containing the color developing indicator, the light can react with and be absorbed by more color developing indicator molecules in the blue state in the color developing layer, so that a small amount of light that is not absorbed is reflected by the reflective layer, so that the reflected light intensity is low, that is, the light intensity value initially detected is low; as the detection time continues, the light intensity value to be tested is increased. The incubation and fermentation of the microorganisms in the sample continuously produces carbon dioxide, which is gradually absorbed by the color indicator in the color development layer, causing the color development layer to eventually turn into transparent yellow. At this time, the color indicator molecules in the yellow state in the color development layer have an extremely low absorption rate for light with a wavelength greater than 570nm, so that a large amount of light that is not absorbed is efficiently reflected by the reflective layer, making the reflected light intensity higher, that is, the light intensity value detected at the endpoint is higher; since the initial detected light intensity value can be lower and the end point detected light intensity value is higher, the difference between the end point light intensity and the initial light intensity is larger, thereby effectively improving the sensitivity of the detection, reducing the occurrence of false negative results, and shortening the detection time.

[0007] In some embodiments of the present application, the total thickness of the color development layer and the reflective layer is no more than 8 mm; wherein the thickness of the color development layer is 2-6 mm.

[0008] The present application sets the color-developing layer to an appropriate thickness to ensure that the endpoint light intensity and the initial light intensity reach an appropriate range, thereby making the difference between the endpoint light intensity and the initial light intensity larger to improve the sensitivity of detection; and controls the total thickness of the color-developing layer and the reflective layer to an appropriate thickness to facilitate the transmission of gases produced by microorganisms, thereby avoiding prolonged detection time.

[0009] In some embodiments of the present application, the color development layer is prepared by mixing a color indicator, a surfactant, an auxiliary agent and silica gel in a weight ratio of 1-1000:100-2000:100-2000:50000-100000.

[0010] The present application can prepare a transparent emulsion containing a color indicator by mixing a color indicator, a surfactant, an auxiliary agent and silica gel in a suitable weight ratio, thereby forming a transparent silica gel layer containing a color indicator.

[0011] In some embodiments of the present application, the color indicator includes at least one of bromophenol blue, thymol blue, bromocresol purple, bromocresol green, bromothymol blue, xylenol blue, m-cresol purple, nitrate yellow, methyl violet, bromophenol blue, cresol red and bromophenol red.

[0012] In some embodiments of the present application, the surfactant includes at least one of Tween 80, Tween 20, Span 80, Span 80, cetearyl alcohol polyoxyethylene ether, fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether phosphate, stearyl alcohol polyether, octanol polyoxyethylene ether, polyoxyethylene stearate, polyoxyethylene oleate, Triton and polyethylene glycol.

[0013] In some embodiments of the present application, the auxiliary agent includes at least one of phenoxyethanol, n-octanol, n-decyl alcohol, lauryl alcohol, myristyl alcohol, ethanol, ethylene glycol, glycerol, n-propanol and propylene glycol butyl ether.

[0014] In some embodiments of the present application, the reflective layer is a silicone layer containing reflective material; the thickness of the reflective layer is 1-2 mm.

[0015] The present application sets the reflective layer to a suitable thickness, which is beneficial to both the transmission of gases produced by microorganisms and the reflection of light.

[0016] In some embodiments of the present application, the reflective material includes at least one of white carbon black, talcum powder, zinc oxide, magnesium oxide and pearl powder.

[0017] The present application adopts reflective materials with a whitening effect to increase the reflectivity of light, thereby improving the sensitivity and accuracy of detection.

[0018] In some embodiments of the present application, the weight ratio of the reflective material to the silica gel is 0.5-500:10000.

[0019] The present application adopts a reflective material and silica gel mixed in a suitable weight ratio to form a silica gel layer containing the reflective material, which is beneficial to the efficient reflection of light.

[0020] In some embodiments of the present application, the reflective layer includes a microbial liquid culture medium; the microbial liquid culture medium is selected from tryptic soytone liquid culture medium, liquid thioglycollate medium, lauryl sulfate tryptone liquid culture medium, R2B liquid culture medium, Middlebrook 7H9 liquid culture medium, enteric bacteria enrichment liquid culture medium, MacConkey liquid culture medium, RV Salmonella liquid culture medium, cetyltrimethylammonium bromide or Sabouraud dextrose liquid culture medium.

[0021] The present application facilitates the detection of corresponding microorganisms by adopting a suitable liquid culture medium.

[0022] In a second aspect, an embodiment of the present application provides a method for preparing a microbial detection bottle for sterility inspection, comprising:

[0023] (1) Mix a color indicator, a surfactant, an auxiliary agent and silica gel according to a weight ratio, add the mixture into the bottle body before solidification, and solidify the mixture at room temperature or by heating to form a color development layer with a thickness of 2-6 mm on the inner surface of the bottom of the bottle body;

[0024] (2) The reflective material and silica gel are mixed in a weight ratio, added into the bottle body before solidification, and solidified at room temperature or by heating to cover the color development layer to form a reflective layer with a thickness of 1-2 mm.

[0025] In some embodiments of the present application, the preparation method further includes: adding a prepared microbial liquid culture medium to the solidified reflective layer, sealing it and sterilizing it according to the sterilization requirements of the microbial liquid culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 A schematic diagram of a microbial detection bottle for sterility testing provided in an embodiment of the present application.

[0028] Figure 2 A schematic diagram of a microbial detection bottle for sterility testing provided as a comparative example of the present application.

[0029] Icon: 1-bottle body; 2-color developing layer; 3-reflective layer; 4-microorganism liquid culture medium; 5-light source; 6-photoelectric sensor.

[0030] Figure 3 A graph of reflected light intensity provided for implementation group 1 of the present application.

[0031] Figure 4 A graph of reflected light intensity provided for implementation group 2 of the present application.

[0032] Figure 5 A graph of reflected light intensity provided for implementation group 3 of the present application.

[0033] Figure 6 A graph of reflected light intensity provided for implementation group 4 of the present application.

[0034] Figure 7 A graph of reflected light intensity provided for implementation group 5 of the present application.

[0035] Figure 8 A graph of reflected light intensity provided for implementation group 6 of the present application. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0037] In order to improve the problems of short detection time, low detection sensitivity, and easy occurrence of false negative results in the current sterility detection method, the present application embodiment provides a microbial detection bottle for sterility inspection, such as Figure 1 As shown, it comprises: a bottle body 1, the inner surface of the bottom of the bottle body 1 is covered with a color developing layer 2, and the color developing layer 2 is covered with a reflective layer 3; the color developing layer 2 comprises a transparent silica gel layer containing a color developing indicator.

[0038] The present application covers a color developing layer 2 on the bottom of the bottle body 1, and a reflective layer 3 is covered on the color developing layer 2. The color developing layer 2 includes a transparent silicone layer containing a color developing indicator. During the initial detection, the color developing layer 2 is transparent blue. When a light source 5 (with a wavelength greater than 570 nm) is irradiated on the bottom of the bottle, the light can penetrate through the transparent silicone layer containing the color developing indicator. Since the light has a certain optical path when penetrating through the transparent silicone layer containing the color developing indicator, the light can react with more color developing indicator molecules in the color developing layer 2 in a blue state and be absorbed by them, so that a small amount of light that is not absorbed is reflected by the reflective layer 3, so that the reflected light intensity is low, that is, the light intensity value initially detected is low; as the detection time continues, the microorganisms in the sample to be tested are detected. Carbon dioxide is continuously produced during incubation and fermentation of the organism, and the carbon dioxide is gradually absorbed by the color indicator in the color layer 2, causing the color layer 2 to eventually turn into transparent yellow. At this time, the color indicator molecules in the yellow state in the color layer 2 have an extremely low absorption rate for light with a wavelength greater than 570 nm, so that a large amount of light that is not absorbed is efficiently reflected by the reflective layer 3, so that the reflected light intensity is higher, that is, the light intensity value detected at the endpoint is higher; a photoelectric sensor 6 is used to collect the reflected light intensity signal, and since the initial detected light intensity value can be lower and the end point detected light intensity value is higher, the difference between the end point light intensity and the initial light intensity is larger, thereby effectively improving the detection sensitivity, reducing the occurrence of false negative results, and shortening the detection time.

[0039] In some embodiments of the present application, the total thickness of the color layer 2 and the reflective layer 3 is not greater than 8 mm; wherein the thickness of the color layer 2 is 2-6 mm. Further, the total thickness of the color layer 2 and the reflective layer 3 is 3-8 mm. As an example, the total thickness of the color layer 2 and the reflective layer 3 may be, but not limited to, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm. Further, the thickness of the color layer 2 is 2-4 mm. The thickness of the color layer 2 may be, but not limited to, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm. The color-developing layer 2 is set to an appropriate thickness to ensure that the endpoint light intensity and the initial light intensity are within an appropriate range, so that the difference between the endpoint light intensity and the initial light intensity is larger to improve the sensitivity of detection; by controlling the total thickness of the color-developing layer 2 and the reflective layer 3 to an appropriate thickness, the transmission of the gas produced by the microorganisms is facilitated to avoid prolonged detection time.

[0040] In some embodiments of the present application, the color developing layer 2 is prepared by mixing a color indicator, a surfactant, an auxiliary agent and silica gel in a weight ratio of 1-1000:100-2000:100-2000:50000-100000. By mixing a color indicator, a surfactant, an auxiliary agent and silica gel in a suitable weight ratio, a transparent emulsion containing a color indicator can be prepared, thereby forming a transparent silica gel layer containing a color indicator.

[0041] In some embodiments of the present application, the color indicator includes but is not limited to at least one of bromophenol blue, thymol blue, bromocresol purple, bromocresol green, bromothymol blue, xylenol blue, m-cresol purple, nitrate yellow, methyl violet, bromophenol blue, cresol red and bromophenol red.

[0042] In some embodiments of the present application, the surfactant includes but is not limited to at least one of Tween 80, Tween 20, Span 80, Span 80, cetearyl alcohol polyoxyethylene ether, fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether phosphate, stearyl alcohol polyether, octanol polyoxyethylene ether, polyoxyethylene stearate, polyoxyethylene oleate, Triton and polyethylene glycol.

[0043] In some embodiments of the present application, the auxiliary agent includes but is not limited to at least one of phenoxyethanol, n-octanol, n-decyl alcohol, lauryl alcohol, myristyl alcohol, ethanol, ethylene glycol, glycerol, n-propanol and propylene glycol butyl ether.

[0044] In some embodiments of the present application, the reflective layer 3 is a silicone layer containing reflective material; the thickness of the reflective layer 3 is 1-2 mm. As an example, the thickness of the reflective layer 3 is 1 mm, 1.5 mm, and 2 mm. Setting the reflective layer 3 to an appropriate thickness is beneficial to both the transmission of the gas produced by the microorganisms and the reflection of light.

[0045] In some embodiments of the present application, the reflective material includes but is not limited to at least one of white carbon black, talcum powder, zinc oxide, magnesium oxide and pearl powder. The use of reflective materials with whitening effect can improve the reflectivity of light, thereby improving the sensitivity and accuracy of detection.

[0046] In some embodiments of the present application, the weight ratio of the reflective material to the silica gel is 0.5-500:10000. The reflective material and the silica gel are mixed in a suitable weight ratio to form a silica gel layer containing the reflective material, which is conducive to efficient reflection of light. As an example, the silica gel can be, but is not limited to, ELASTOSIL RT 625A / B.

[0047] In some embodiments of the present application, the reflective layer 3 is above the microbial liquid culture medium 4; the microbial liquid culture medium 4 can be selected from, but not limited to, trypticase soytone liquid culture medium, liquid thioglycollate culture medium, lauryl sulfate tryptone liquid culture medium, R2B liquid culture medium, Middlebrook 7H9 liquid culture medium, enteric bacteria enrichment liquid culture medium, MacConkey liquid culture medium, RV Salmonella liquid culture medium, hexadecyltrimethylammonium bromide or Sabouraud glucose liquid culture medium. By adopting a suitable liquid culture medium, it is convenient to detect the corresponding microorganisms. It is understandable that after adding a suitable amount of suitable liquid culture medium above the reflective layer in the microbial detection bottle for sterility inspection as needed, the detection can be carried out; it is also possible to directly prepare a microbial detection bottle for sterility inspection containing different liquid culture media, and directly select a microbial detection bottle for sterility inspection containing a suitable liquid culture medium for detection.

[0048] The present application embodiment provides a method for preparing a microbial detection bottle for sterility inspection, comprising:

[0049] (1) Mix a color indicator, a surfactant, an auxiliary agent and silica gel according to a weight ratio, add the mixture into the bottle body before solidification, and solidify the mixture at room temperature or by heating to form a color development layer with a thickness of 2-6 mm on the inner surface of the bottom of the bottle body;

[0050] (2) The reflective material and silica gel are mixed in a weight ratio, added into the bottle body before solidification, and solidified at room temperature or by heating to cover the color development layer to form a reflective layer with a thickness of 1-2 mm.

[0051] In some embodiments of the present application, the method for preparing a microbial detection bottle for sterility inspection also includes: adding a prepared microbial liquid culture medium to the solidified reflective layer, sealing and sterilizing according to the sterilization requirements of the microbial liquid culture medium.

[0052] The embodiment of the present application provides a microbial detection method for sterility inspection, which is applicable to the above-mentioned microbial detection bottle for sterility inspection, including:

[0053] (1) adding the sample to be tested into the microbial liquid culture medium in the microbial test bottle for sterility inspection, and placing the microbial test bottle containing the sample to be tested in the testing instrument for incubation and culture;

[0054] (2) A light source with a wavelength greater than 570 nm is used to irradiate the bottom of the bottle until the incubation or detection is completed, and the electrical signal data of the reflected light is collected multiple times by a photoelectric sensor.

[0055] Using the above-mentioned microbial detection bottle for sterility inspection, the sample to be tested is added to the microbial liquid culture medium in the bottle body, placed in the detection instrument for incubation and culture, and then a light source with a wavelength greater than 570nm is used to irradiate the bottom of the bottle until the incubation or detection is completed. The electrical signal data of the reflected light is collected multiple times at intervals by a photoelectric sensor, and a reflected light intensity curve graph is generated based on the collected electrical signal data of the multiple reflected light rays. The initial light intensity and the end point light intensity of the reflected light can be obtained based on the reflected light intensity curve graph.

[0056] The color developing layer is a blue transparent silica gel layer, and the long wavelength light greater than 570nm is yellow light-red light. During the initial detection, the light source is irradiated to the bottom of the bottle, and the yellow light-red light can penetrate through the blue transparent silica gel layer. Since the yellow light-red light has a certain optical path when penetrating through the blue transparent silica gel layer, the light can react with more blue color indicator molecules in the blue transparent silica gel layer and be absorbed by them, so that a small amount of light that is not absorbed is reflected by the reflective layer, so that the reflected light intensity is low, that is, the light intensity value detected initially is low; as the detection time continues, the microorganisms in the sample to be tested incubate and ferment and continue to produce dioxygen Carbon dioxide is gradually absorbed by the color indicator in the color developing layer to change the color of the color developing layer. The color indicator absorbs carbon dioxide until it reaches saturation until the color developing layer turns completely transparent yellow. The color indicator molecules in the yellow state in the color developing layer have extremely low absorption rate for light with a wavelength greater than 570nm, so that a large amount of light that is not absorbed is reflected by the reflective layer. At the same time, the reflective layer has a high reflectivity for light, so that the reflected light intensity is high, that is, the light intensity value detected at the endpoint is high; the difference between the endpoint light intensity and the initial light intensity is large, thereby effectively improving the sensitivity of the detection, reducing the occurrence of false negative results, and shortening the detection time.

[0057] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0058] All microorganisms used in this application were purchased from Guangdong Huankai Microbiological Technology Co., Ltd.

[0059] Example 1

[0060] This embodiment provides a microbial detection bottle for sterility inspection, comprising: a bottle body, the inner surface of the bottom of the bottle body is covered with a 4mm thick color development layer, and the color development layer is covered with a 1mm thick reflective layer; the color development layer is a transparent silicone layer containing a color development indicator;

[0061] The color development layer is prepared by mixing a color indicator, a surfactant, an auxiliary agent and silica gel in a weight ratio of 10:120:2000:60000; wherein the color indicator is p-xylenol blue; the surfactant is a mixture of Tween 80 and Tween 20 in a weight ratio of 1:3; the auxiliary agent is a mixture of myristyl alcohol and ethylene glycol in a weight ratio of 1:9; and the silica gel is ELASTOSIL RT625A / B;

[0062] The reflective layer is prepared by mixing a reflective material and silica gel at a weight ratio of 1:100; wherein the reflective material is talcum powder; and the silica gel is ELASTOSIL RT 625A / B.

[0063] Example 2

[0064] This embodiment provides a microbial detection bottle for sterility inspection, comprising: a bottle body, the inner surface of the bottom of the bottle body is covered with a 2mm thick color development layer, and the color development layer is covered with a 1mm thick reflective layer; the color development layer is a transparent silicone layer containing a color development indicator;

[0065] The color development layer is prepared by mixing a color indicator, a surfactant, an auxiliary agent and silica gel in a weight ratio of 20:120:2000:60000; wherein the color indicator is bromocresol purple; the surfactant is a mixture of Tween 80 and fatty alcohol polyoxyethylene ether-4 in a weight ratio of 1:1; the auxiliary agent is a mixture of myristyl alcohol and ethylene glycol in a weight ratio of 1:9; and the silica gel is ELASTOSIL RT 625A / B;

[0066] The reflective layer is prepared by mixing a reflective material and silica gel at a weight ratio of 1:100; wherein the reflective material is talcum powder; and the silica gel is ELASTOSIL RT 625A / B.

[0067] Example 3

[0068] This embodiment provides a microbial detection bottle for sterility inspection, comprising: a bottle body, the inner surface of the bottom of the bottle body is covered with a 3mm thick color development layer, and the color development layer is covered with a 1mm thick reflective layer; the color development layer is a transparent silicone layer containing a color development indicator;

[0069] The color development layer is prepared by mixing a color indicator, a surfactant, an auxiliary agent and silica gel in a weight ratio of 25:120:1500:60000; wherein the color indicator is bromothymol blue; the surfactant is a mixture of Tween 80 and Tween 20 in a weight ratio of 1:3; the auxiliary agent is a mixture of myristyl alcohol and glycerol in a weight ratio of 1:9; and the silica gel is ELASTOSIL RT625A / B;

[0070] The reflective layer is prepared by mixing a reflective material and silica gel at a weight ratio of 2:100; wherein the reflective material is talcum powder; and the silica gel is ELASTOSIL RT 625A / B.

[0071] Comparative Example 1

[0072] This comparative example provides a microbial detection bottle for sterility inspection, such as Figure 2 As shown, it is a BioMérieux BACT / ALERT iAST aerobic detection bottle; it comprises: a bottle body 1, the inner surface of the bottom of the bottle body 1 is covered with an opaque color-developing layer 2 with a thickness of 2 mm, and above the opaque color-developing layer 2 is a microbial liquid culture medium 4.

[0073] Implementation Group 1

[0074] The present implementation group provides a microbial detection method for sterility inspection, using the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1 respectively, and the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1 both contain 40 mL of tryptone soytone liquid culture medium TSB, and the detection method comprises:

[0075] (1) 32 CFU of Escherichia coli CMCC(B)44102 were inoculated into the microbial liquid culture medium in the bottles provided in Example 1 and Comparative Example 1, respectively, and the microbial detection bottles containing the samples to be detected were placed in a detection instrument for incubation at 32.5° C. and 30 times / min on a shaking table;

[0076] (2) A light source with a wavelength of 590 nm is used to irradiate the bottom of the bottle until the incubation or detection is completed, and the electrical signal data of the reflected light is collected multiple times every 10 minutes by the photoelectric sensor; the above detection is performed in two groups in parallel, and a reflected light intensity curve graph is generated based on the collected electrical signal data of the multiple reflected lights. The reflected light intensity curve graph is shown in FIG. Figure 3 shown.

[0077] Implementation Group 2

[0078] The present implementation group provides a microbial detection method for sterility inspection, using the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1 respectively, and the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1 both contain 40 mL of tryptone soytone liquid culture medium TSB, and the detection method comprises:

[0079] (1) 58 CFU of Staphylococcus aureus CMCC(B)26003 were inoculated into the microbial liquid culture medium in the bottles provided in Example 1 and Comparative Example 1, respectively, and the microbial detection bottles containing the samples to be detected were placed in a detection instrument for incubation at 32.5° C. and 30 times / min on a shaking table;

[0080] (2) A light source with a wavelength of 590 nm is used to irradiate the bottom of the bottle until the incubation or detection is completed, and the electrical signal data of the reflected light is collected multiple times every 10 minutes by the photoelectric sensor; the above detection is performed in two groups in parallel, and a reflected light intensity curve graph is generated based on the collected electrical signal data of the multiple reflected lights. The reflected light intensity curve graph is shown in FIG. Figure 4 shown.

[0081] Implementation Group 3

[0082] The present implementation group provides a microbial detection method for sterility inspection, using the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1 respectively, and the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1 both contain 40 mL of tryptone soytone liquid culture medium TSB, and the detection method comprises:

[0083] (1) 21 CFU of Candida albicans CMCC(F)98001 were inoculated into the microbial liquid culture medium in the bottles provided in Example 1 and Comparative Example 1, respectively, and the microbial detection bottles containing the samples to be detected were placed in a detection instrument for incubation at 32.5° C. and 30 times / min on a shaking table;

[0084] (2) A light source with a wavelength of 590 nm is used to irradiate the bottom of the bottle until the incubation or detection is completed, and the electrical signal data of the reflected light is collected multiple times every 10 minutes by the photoelectric sensor; the above detection is performed in two groups in parallel, and a reflected light intensity curve graph is generated based on the collected electrical signal data of the multiple reflected lights. The reflected light intensity curve graph is shown in FIG. Figure 5 shown.

[0085] Implementation Group 4

[0086] The present implementation group provides a microbial detection method for sterility inspection, using the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1 respectively, and the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1 both contain 40 mL of tryptone soytone liquid culture medium TSB, and the detection method comprises:

[0087] (1) 73 CFU of Aspergillus niger CMCC(F)98003 were inoculated into the microbial liquid culture medium in the bottles provided in Example 1 and Comparative Example 1, respectively, and the microbial detection bottles containing the samples to be detected were placed in a detection instrument for incubation at 32.5° C. and 30 times / min on a shaking table;

[0088] (2) A light source with a wavelength of 590 nm is used to irradiate the bottom of the bottle until the incubation or detection is completed, and the electrical signal data of the reflected light is collected multiple times every 10 minutes by the photoelectric sensor; the above detection is performed in two groups in parallel, and a reflected light intensity curve graph is generated based on the collected electrical signal data of the multiple reflected lights. The reflected light intensity curve graph is shown in FIG. Figure 6 shown.

[0089] Implementation Group 5

[0090] The present implementation group provides a microbial detection method for sterility inspection, using the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1 respectively, and the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1 both contain 40 mL of tryptone soytone liquid culture medium TSB, and the detection method comprises:

[0091] (1) 2 CFU of Micrococcus luteus CMCC(B)28001 were inoculated into the microbial liquid culture medium in the bottles provided in Example 1 and Comparative Example 1, respectively, and the microbial detection bottles containing the samples to be detected were placed in a detection instrument for incubation at 32.5° C. and 30 times / min on a shaking table;

[0092] (2) A light source with a wavelength of 590 nm is used to irradiate the bottom of the bottle until the incubation or detection is completed, and the electrical signal data of the reflected light is collected multiple times every 10 minutes by the photoelectric sensor; the above detection is performed in two groups in parallel, and a reflected light intensity curve graph is generated based on the collected electrical signal data of the multiple reflected lights. The reflected light intensity curve graph is shown in FIG. Figure 7 shown.

[0093] Implementation Group 6

[0094] The present implementation group provides a microbial detection method for sterility inspection, using the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1 respectively, and the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1 both contain 40 mL of tryptone soytone liquid culture medium TSB, and the detection method comprises:

[0095] (1) 41 CFU of Ralstonia piezoelectrica ATCC275110 were inoculated into the microbial liquid culture medium in the bottles provided in Example 1 and Comparative Example 1, respectively, and the microbial detection bottles containing the samples to be detected were placed in a detection instrument for incubation at 32.5° C. and 30 times / min on a shaking table;

[0096] (2) A light source with a wavelength of 590 nm is used to irradiate the bottom of the bottle until the incubation or detection is completed, and the electrical signal data of the reflected light is collected multiple times every 10 minutes by the photoelectric sensor; the above detection is performed in two groups in parallel, and a reflected light intensity curve graph is generated based on the collected electrical signal data of the multiple reflected lights. The reflected light intensity curve graph is shown in FIG. Figure 8 shown.

[0097] Test Example 1

[0098] In this test example, the reflected light intensity curves obtained by performing two groups of parallel tests on six different microorganisms using the microbial test bottles for sterility testing provided in Example 1 and Comparative Example 1 in implementation groups 1-6 are shown in FIG. Figure 1-8 The TTP value and the initial light intensity and the final light intensity of the reflected light are obtained by using the results of the initial light intensity and the final light intensity of the reflected light. The difference between the initial and final light intensity of the reflected light is calculated. The results are shown in Table 1. Note: The initial values ​​of all reflected light intensity curves are reset to zero.

[0099] Table 1 TTP value and initial and final difference of reflected light intensity

[0100]

[0101] It can be seen from the results in Table 1 that, by comparing the detection time of different microorganisms detected by the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1, the TTP time of different microorganisms detected by the detection bottle provided in Example 1 is shorter, which is 2.9h-31.3h shorter than the detection time of the detection bottle provided in Comparative Example 1, among which the TTP time difference for the detection of Ralstonia pievophyllum is the largest, reaching 31.3h. This indicates that the use of the microbial detection bottle for sterility inspection provided in this application for sterility inspection can effectively shorten the detection time.

[0102] Comparing the difference between the beginning and end of the reflected light intensity of different microorganisms detected by the microbial detection bottles for sterility inspection provided in Example 1 and Comparative Example 1, there are also large differences in the detection results of the two detection bottles provided in Example 1 and Comparative Example 1. The difference between the beginning and end of the highest reflected light intensity detected by the detection bottle provided in Comparative Example 1 is only 1380 (Escherichia coli), and the difference between the beginning and end of the lowest reflected light intensity is only 265 (Aspergillus niger) and 142 (Ralstonia pievophyllum); while the difference between the beginning and end of the highest reflected light intensity detected by the detection bottle provided in Example 1 is all above 1100, among which the difference between the beginning and end of the highest reflected light intensity detected reaches more than 2000 (Escherichia coli), and the difference between the beginning and end of the lowest reflected light intensity is also above 1100 (Ralstonia pievophyllum), which is greatly improved compared with the detection result of the detection bottle provided in Comparative Example 1. It shows that the use of the microbial detection bottle for sterility inspection provided in the present application for sterility detection can effectively improve the sensitivity of the detection and reduce the occurrence of false negative results.

[0103] On the other hand, by comparing the difference between the beginning and the end of the reflected light intensity of Escherichia coli detected by the microbial detection bottles for sterility inspection provided by Example 1 and Comparative Example 1, the detection result of the detection bottle provided by Example 1 is not particularly improved compared with the detection result of the detection bottle provided by Comparative Example 1, and is only improved by about 1.5 times, indicating that the detection result of the detection bottle provided by Example 1 for fast-growing bacteria with large gas production is not particularly improved; and by comparing the difference between the beginning and the end of the reflected light intensity of Aspergillus niger and Ralstonia pievophyllum detected by the microbial detection bottles for sterility inspection provided by Example 1 and Comparative Example 1, the detection result of the detection bottle provided by Example 1 is significantly improved compared with the detection result of the detection bottle provided by Comparative Example 1, reaching 5 times and 7.8 times respectively, indicating that the detection result of the detection bottle provided by Example 1 for bacteria with slow growth and low gas production is significantly improved. It shows that the microbial detection bottle for sterility inspection provided by the present application has high sensitivity for sterility detection, which is more conducive to the detection of bacteria with slow growth and low gas production.

[0104] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. A microbial detection bottle for sterility inspection, characterized in that: include: A bottle body, wherein the inner surface of the bottom of the bottle body is covered with a color developing layer, and the color developing layer is covered with a reflective layer; The color development layer comprises a transparent silica gel layer containing a color development indicator.

2. The microbial detection bottle for sterility inspection according to claim 1, characterized in that: The total thickness of the color development layer and the reflective layer is no more than 8 mm; wherein the thickness of the color development layer is 2-6 mm.

3. The microbial detection bottle for sterility inspection according to claim 2, characterized in that: The color development layer is prepared by mixing a color development indicator, a surfactant, an auxiliary agent and silica gel in a weight ratio of 1-1000:100-2000:100-2000:50000-100000.

4. The microbial detection bottle for sterility inspection according to claim 3, characterized in that: The color indicator comprises at least one of bromophenol blue, thymol blue, bromocresol purple, bromocresol green, bromothymol blue, xylenol blue, metacresol purple, nitrate yellow, methyl violet, bromophenol blue, cresol red and bromophenol red; And / or, the surfactant includes at least one of Tween 80, Tween 20, Span 80, Span 80, cetearyl alcohol polyoxyethylene ether, fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether phosphate, stearyl alcohol polyether, octanol polyoxyethylene ether, stearic acid polyoxyethylene ester, oleic acid polyoxyethylene ester, Triton and polyethylene glycol; And / or, the auxiliary agent includes at least one of phenoxyethanol, n-octanol, n-decanol, lauryl alcohol, myristyl alcohol, ethanol, ethylene glycol, glycerol, n-propanol and propylene glycol butyl ether.

5. The microbial detection bottle for sterility inspection according to claim 1, characterized in that: The reflective layer is a silicone layer containing reflective material; the thickness of the reflective layer is 1-2 mm.

6. The microbial detection bottle for sterility inspection according to claim 5, characterized in that: The reflective material includes at least one of white carbon black, talcum powder, zinc oxide, magnesium oxide and pearl powder.

7. The microbial detection bottle for sterility inspection according to claim 6, characterized in that: The weight ratio of the reflective material to the silica gel is 0.5-500:10000.

8. The microbial detection bottle for sterility inspection according to any one of claims 1 to 7, characterized in that: The reflective layer includes a liquid culture medium for microorganisms; The microbial liquid culture medium is selected from tryptic soy liquid culture medium, liquid thioglycollate culture medium, lauryl sulfate tryptone liquid culture medium, R2B liquid culture medium, Middlebrook7H9 liquid culture medium, enteric bacteria enrichment liquid culture medium, MacConkey liquid culture medium, RV Salmonella liquid culture medium, cetyltrimethylammonium bromide or Sabouraud dextrose liquid culture medium.

9. A method for preparing a microbial detection bottle for sterility inspection according to any one of claims 1 to 8, characterized in that: include: (1) Mixing the color indicator, the surfactant, the auxiliary agent and the silica gel according to a weight ratio, adding the mixture into the bottle body before solidification, and solidifying the mixture at room temperature or by heating to form a color development layer with a thickness of 2-6 mm on the inner surface of the bottom of the bottle body; (2) The reflective material and the silica gel are mixed in a weight ratio, added into the bottle body when not solidified, and solidified at room temperature or by heating to form a reflective layer with a thickness of 1-2 mm on the color developing layer.

10. The preparation method according to claim 9, characterized in that: Also includes: The prepared microbial liquid culture medium is added to the solidified reflective layer, and the layer is sealed and sterilized according to the sterilization requirements of the microbial liquid culture medium.

Images