Microorganism detection kit, detection device and detection method

Abstract

The invention provides a microbiological detection kit, a detection device and a detection method. The microbiological detection kit comprises a detection chamber for storing a culture solution and a gas supply chamber which is communicated with the detection chamber and is used for storing culture gas, an injection port communicated with the detection chamber is formed in the detection chamber; and a hydrophobic breathable film is arranged at the communication part of the detection chamber and the air supply chamber. The microbiological detection efficiency is improved, and meanwhile, the accuracy of a microbiological detection result is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and in particular relates to a microbial detection box, a detection device and a detection method. Background Art

[0002] In order to ensure the safe use of biological drugs by patients, it is crucial to conduct sterility tests on biological drugs. In the existing technology, traditional culture methods rely on the professional skills and experience of operators, and have a large subjective judgment component. Differences in judgment standards among different operators may make it difficult to ensure the accuracy of the test results, and microbial culture usually takes a long time, and the detection efficiency is low. On the one hand, the nucleic acid amplification detection method has limited detection sensitivity. For low-concentration microbial contamination, the nucleic acid amplification signal is weak and may not be detected in a timely and accurate manner; on the other hand, the instruments and equipment used are expensive and the cost is high. The accuracy and specificity of the detection method based on microbial metabolism are easily interfered with. Cells, cell fragments, metabolic intermediates, etc. in the sample may lead to false positive or false negative results. Therefore, the microbial detection in the existing technology cannot take into account both detection efficiency and detection accuracy. Summary of the Invention

[0003] The present invention addresses the technical problems in the prior art of microbial detection that cannot balance detection efficiency and detection accuracy, and provides a microbial detection box, a detection device and a detection method.

[0004] In view of the above technical problems, an embodiment of the present invention provides a microbial detection box, comprising a detection chamber for storing culture fluid, and an air supply chamber connected to the detection chamber and for storing culture gas; the detection chamber is provided with an injection port connected to the detection chamber; and a hydrophobic breathable membrane is provided at the connection between the detection chamber and the air supply chamber.

[0005] A microorganism detection device comprises an optical detection module, a controller connected to the optical detection module, and the microorganism detection box; A transparent window is provided on the microorganism detection box at a position opposite to the detection chamber, and the optical detection module is used to obtain the image to be detected in the detection chamber through the transparent window.

[0006] A microbial detection method, applied to the microbial detection device, comprising: receiving a sample injection signal, controlling the sample liquid to enter the detection chamber from the injection port, so that the sample liquid is mixed with the culture liquid in the detection chamber, and then incubating the mixed liquid under the condition of contact with the culture gas from the gas supply chamber; During the incubation process, the optical detection module regularly acquires the image to be detected in the detection chamber; A microorganism detection result is determined based on the image to be detected.

[0007] The microbial detection box provided by the present invention includes a detection chamber for storing culture fluid, and an air supply chamber connected to the detection chamber and used to store culture gas; the detection chamber is provided with an injection port connected to the detection chamber; and a hydrophobic breathable membrane is provided at the connection between the detection chamber and the air supply chamber.

[0008] The microbial detection kit provided by the present invention stores culture fluid in a detection chamber to provide necessary nutrients for the growth of microorganisms, and stores culture gas in an air supply chamber connected to the detection chamber to maintain the stability of the culture environment in the detection chamber, ensuring that the microorganisms in the detection chamber grow in a suitable gas environment, thereby ensuring that the microbial contamination in the cell therapy product can be accurately detected in a relatively short time, thereby improving the efficiency of microbial detection; at the same time, by providing a hydrophobic breathable membrane at the connection between the detection chamber and the air supply chamber, while ensuring that the culture gas circulates between the detection chamber and the air supply chamber, the liquid in the detection chamber is prevented from entering the air supply chamber and causing leakage and loss, thereby improving the stability of microbial culture and thereby improving the accuracy of the microbial detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The present invention will be further described below with reference to the accompanying drawings and examples.

[0010] Figure 1 It is a structural schematic diagram of a microorganism detection box provided by one embodiment of the present invention.

[0011] Figure 2 It is a structural schematic diagram of a microorganism detection box provided by another embodiment of the present invention.

[0012] Figure 3 It is a structural schematic diagram of a microorganism detection device provided by one embodiment of the present invention.

[0013] Figure 4 This is a flow chart of a microbial detection method provided by one embodiment of the present invention.

[0014] Figure 5 This is an image to be detected when detecting an Escherichia coli sample liquid according to an embodiment of the present invention.

[0015] Figure 6 This is an image to be detected when detecting a Staphylococcus aureus sample liquid according to an embodiment of the present invention.

[0016] Figure 7 This is an image to be detected when detecting a Pseudomonas aeruginosa sample solution according to an embodiment of the present invention.

[0017] Figure 8 This is an image to be detected when detecting a Bacillus subtilis sample liquid according to an embodiment of the present invention.

[0018] Figure 9 This is an image to be detected when detecting a micrococcal sample solution according to an embodiment of the present invention.

[0019] Figure 10 This is an image to be detected when detecting a Streptococcus pyogenes sample liquid according to an embodiment of the present invention.

[0020] Figure 11 This is an image to be detected when detecting a Candida albicans sample liquid according to an embodiment of the present invention.

[0021] Figure 12 This is an image to be detected when detecting an Aspergillus niger sample liquid according to an embodiment of the present invention.

[0022] Figure 13 This is an image to be detected when a negative aerobic bacteria culture medium blank sample solution is detected according to an embodiment of the present invention.

[0023] Figure 14 This is an image to be detected when detecting a Clostridium sporogenes sample solution according to an embodiment of the present invention.

[0024] Figure 15 This is an image to be detected when detecting a Propionibacterium acnes sample solution according to an embodiment of the present invention.

[0025] Figure 16 This is an image to be detected when detecting a negative anaerobic bacteria culture medium blank sample solution according to an embodiment of the present invention.

[0026] Figure 17 This is an image to be detected when detecting a sample solution co-cultured with Candida albicans and Jurkat cells according to an embodiment of the present invention.

[0027] Figure 18 This is an image to be detected when detecting a sample solution co-cultured with Candida albicans and Jurkat cells according to another embodiment of the present invention.

[0028] Figure 19 This is an image to be detected when detecting a sample solution co-cultured with Aspergillus niger and Jurkat cells according to an embodiment of the present invention.

[0029] Figure 20 This is an image to be detected when detecting a sample solution co-cultured with Aspergillus niger and Jurkat cells according to another embodiment of the present invention.

[0030] The reference numerals in the specification are as follows: 100. Microbial detection box; 110. Detection chamber; 111. Injection port; 112. Transparent window; 120. Air supply chamber; 130. Hydrophobic breathable membrane; 140. Three-way connector; 150. First pipette; 160. Second pipette; 200. Optical detection module. DETAILED DESCRIPTION

[0031] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] like Figures 1 to 2 As shown, an embodiment of the present invention provides a microorganism detection box 100, including a detection chamber 110 for storing culture fluid, and an air supply chamber 120 connected to the detection chamber 110 and for storing culture gas; the detection chamber 110 is provided with an injection port 111 connected to the detection chamber 110; the connection between the detection chamber 110 and the air supply chamber 120 is provided with a hydrophobic breathable membrane 130.

[0033] The sample liquid may be a sample liquid of a cell therapy product. The microorganisms may be microorganisms, including aerobic bacteria or anaerobic bacteria. Microorganisms may or may not exist in the sample liquid of a cell therapy product. The microorganisms may be accidentally introduced into the sample liquid during preparation, storage or transportation, or may be carried by the sample liquid itself. The present invention is used to detect microorganisms in the sample liquid, the purpose of which is to ensure the safety and effectiveness of cell therapy products and prevent potential risks to patients from microbial contamination. The culture liquid is used to provide nutrients for the growth of microorganisms. The culture gas is used to maintain a stable culture environment for the growth of microorganisms. The sample liquid can be injected into the detection chamber 110 through the injection port 111. After the sample liquid is injected into the detection chamber 110, the sample liquid is mixed with the culture liquid pre-stored in the detection chamber 110 and incubated under the condition of contact with the culture gas entering the detection chamber 110 from the gas supply chamber 120. The incubated object is the microorganism that may exist in the sample liquid. The hydrophobic gas-permeable membrane 130 allows gas to be exchanged between the detection chamber 110 and the gas supply chamber 120 , while preventing liquid in the detection chamber 110 from entering the gas supply chamber 120 .

[0034] It is understood that the shape and material of the detection chamber 110 or the air supply chamber 120 can be configured based on actual conditions (e.g., the type of microorganism and the culture scale, taking into account factors such as the microorganism's growth characteristics, culture conditions, and experimental requirements). For example, if the microorganism is Escherichia coli, an aerobic bacterium suitable for cultivation and monitoring in small-scale experiments, the length, width, and height of the detection chamber 110 can be 55 mm, 35 mm, and 15 mm, respectively. In this case, the volume of the detection chamber 110 is approximately 29 mL, which meets the requirements of small-scale experiments. The detection chamber 110 or the air supply chamber 120 can be made of transparent, chemically resistant polystyrene to facilitate observation of the microorganism's growth status while avoiding toxicity to the microorganism. If the microorganism is Aspergillus niger, a filamentous fungus, since its hyphae form mycelial balls in the culture medium, a large surface area is required to support mycelial expansion, and it is usually cultured in medium-scale experiments. The length, width and height of the detection chamber 110 can be 70 mm, 50 mm and 15 mm, respectively. At this time, the volume of the detection chamber 110 is approximately 50 mL, which is suitable for medium-scale culture; the material of the detection chamber 110 or the air supply chamber 120 can be polycarbonate, which has good transparency and chemical corrosion resistance, and is suitable for observing mycelial growth.

[0035] The shape of the gas supply chamber 120 can be the same as that of the detection chamber 110, thereby facilitating manufacturing, simplifying the production process, reducing production costs, and ensuring consistent quality between the detection chamber 110 and the gas supply chamber 120. The shape of the gas supply chamber 120 can also be different from that of the detection chamber 110, as long as the culture gas can be uniformly and efficiently passed through the hydrophobic gas-permeable membrane 130 and circulated between the detection chamber 110 and the gas supply chamber 120, so that the microorganisms in the detection chamber 110 can promptly access the culture gas in the gas supply chamber 120.

[0036] The material of the hydrophobic breathable membrane 130 can be adjusted based on practical needs. In one embodiment, the hydrophobic breathable membrane 130 is made of polytetrafluoroethylene, with a pore size of 0.22 μm. The hydrophobic breathable membrane 130 can be installed at the connection between the detection chamber 110 and the gas supply chamber 120 by bonding, snapping, or integral molding. Any method is recommended, as long as it allows gas exchange between the detection chamber 110 and the gas supply chamber 120 while preventing liquid in the detection chamber 110 from entering the gas supply chamber 120.

[0037] The composition of the culture gas can be set according to actual conditions (such as the type of microorganism to be cultured). For example, when culturing aerobic bacteria, the culture gas can be an aerobic mixed gas with a preset oxygen ratio. The preset oxygen ratio can be set according to demand. For example, when the preset oxygen ratio is 9:1, the aerobic mixed gas with the preset oxygen ratio can be 90% oxygen and 10% carbon dioxide. When culturing anaerobic bacteria, the culture gas can be an anaerobic mixed gas with a preset anaerobic ratio. The preset anaerobic ratio can be set according to demand. For example, when the preset anaerobic ratio is 9:1, the anaerobic mixed gas with the preset anaerobic ratio can be 90% nitrogen and 10% carbon dioxide. The culture gas in the gas supply chamber 120 can be pre-stored or injected into the gas supply chamber 120 through the injection port 111 by displacement using a syringe or other device when needed.

[0038] In the above embodiment of the present invention, the culture fluid is stored in the detection chamber 110 to provide necessary nutrients for the growth of microorganisms, and the culture gas is stored in the air supply chamber 120 connected to the detection chamber 110 to maintain the stability of the culture environment in the detection chamber 110, thereby ensuring that the microorganisms in the detection chamber 110 grow in a suitable gas environment, thereby ensuring that the microbial contamination in the cell therapy product is accurately detected in a relatively short time, thereby improving the efficiency of microbial detection; at the same time, by providing a hydrophobic breathable membrane 130 at the connection between the detection chamber 110 and the air supply chamber 120, while ensuring that the culture gas circulates between the detection chamber 110 and the air supply chamber 120, the liquid in the detection chamber 110 is prevented from entering the air supply chamber 120 and causing leakage and loss, thereby improving the stability of microbial culture and thereby improving the accuracy of the microbial detection results.

[0039] like Figure 1 and Figure 2 As shown, in one embodiment, the microbial detection box 100 further includes a three-way connector 140; the first channel of the three-way connector 140 is connected to the injection port 111; the microbial detection box 100 further includes a first pipette 150 for accommodating sample liquid, and a second pipette 160 for accommodating sealing oil; the first pipette 150 and the second pipette 160 are respectively connected to the second channel and the third channel of the three-way connector 140, for injecting sample liquid or sealing oil into the detection chamber 110 when needed.

[0040] It can be understood that the tee joint 140 can be installed after the culture solution is stored in the detection chamber 110 and / or the culture gas is stored in the gas supply chamber 120. The type of the tee joint 140 can be set according to actual conditions, as long as the first pipettor 150 and the second pipettor 160 connected to the second channel and the third channel of the tee joint 140 can inject sample solution or sealing oil into the detection chamber 110 when needed. In an embodiment, the tee joint 140 is a tee plug, so as to facilitate the control of the flow direction and flow rate of the fluid, and the connection and switching of different media or pipelines. The sealing oil is used to be laid on the mixed solution of the culture solution and the sample solution when the microorganism is an anaerobic bacterium, so as to limit the gas exchange between the mixed solution and the outside world, and facilitate the growth of the anaerobic bacterium. The sealing oil can be mineral oil or silicone oil.

[0041] As shown in Figure 3 An embodiment of the present application further provides a microorganism detection device, which comprises an optical detection module 200, a controller connected to the optical detection module 200, and the microorganism detection box 100; a transparent window 112 is arranged on the microorganism detection box 100 at a position opposite to the detection chamber 110, and the optical detection module 200 is used to obtain a to-be-detected image in the detection chamber 110 through the transparent window 112.

[0042] It can be understood that the detection chamber 110 corresponding to the position of the transparent window 112 can be made of polystyrene or other transparent materials with good optical transmittance, so as to facilitate the optical detection through the transparent window 112. The transparent window 112 serves as a monitoring window, which facilitates the real-time observation of the growth state of the microorganism in the detection chamber 110, and the accurate acquisition of the to-be-detected image in the detection chamber 110 through the optical detection module 200, so as to realize the intuitive and real-time online monitoring of the microorganism culture process. In some embodiments, the bottom surface of the entire detection chamber 112 or the entire microorganism detection box 100 or the bottom surface thereof can be made of transparent materials, so as to simplify the manufacturing process. Further, the optical detection module 200 can be a microscopic imaging device. The optical detection module 200 can continuously and dynamically image the microorganism cultured in the detection chamber 110 according to a preset time sequence and imaging parameter, and can accurately capture information such as the morphological change and the number increase and decrease of the microorganism. In an embodiment, the optical detection module 200 comprises a 5-fold or 10-fold objective lens. The controller can process and analyze the image obtained by the optical detection module 200, and obtain key feature information such as the morphological change of the bacterial membrane or the fungal hypha, and the dynamic change of the number.

[0043] In the above-mentioned embodiments of the present application, the culture solution is stored in the detection chamber 110 to provide necessary nutrients for the growth of microorganisms, the culture gas is stored in the gas supply chamber 120 communicated with the detection chamber 110 to maintain the stability of the culture environment in the detection chamber 110 and ensure the growth of microorganisms in the detection chamber 110 in a suitable gas environment, thereby ensuring the accurate detection of the microbial contamination in the cell therapy product in a shorter time and improving the efficiency of microbial detection; at the same time, the hydrophobic gas-permeable membrane 130 arranged at the communication position between the detection chamber 110 and the gas supply chamber 120 can ensure the circulation of culture gas between the detection chamber 110 and the gas supply chamber 120 and avoid the leakage of liquid in the detection chamber 110 into the gas supply chamber 120, thereby improving the stability of microbial culture and the accuracy of microbial detection results. At the same time, through the transparent window 112, the growth state of microorganisms in the detection chamber 110 can be observed in real time, and the image to be detected in the detection chamber 110 can be accurately obtained through the optical detection module 200, thereby realizing the intuitive and real-time online monitoring of the microbial culture process, providing strong support for the accurate judgment of the microbial condition in the cell therapy product, and further improving the efficiency and accuracy of microbial detection.

[0044] As shown in Figure 4 An embodiment of the present application also provides a microbial detection method applied to the microbial detection device, and the microbial detection method comprises steps S100-S300: S100, receiving a sample injection signal, controlling the sample liquid to enter the detection chamber 110 from the injection port 111, so that the sample liquid is mixed with the culture liquid in the detection chamber 110, and then the mixed liquid is incubated under the condition of contact with the culture gas from the gas supply chamber 120. The sample liquid can be a sample liquid of a cell therapy product. The above-mentioned microorganisms refer to microbial bacteria, including aerobic bacteria or anaerobic bacteria. Microorganisms may or may not exist in the sample liquid of a cell therapy product. The above-mentioned microorganisms may be accidentally introduced into the sample liquid during the preparation, storage or transportation process, or may be carried by the sample liquid itself. The purpose of detecting microorganisms in the sample liquid is to ensure the safety and effectiveness of the cell therapy product and prevent microbial contamination from posing potential risks to patients. The sample liquid may include aerobic bacteria or anaerobic bacteria, etc., and when the sample liquid is used as a control group, the sample liquid may be made into a negative blank sample liquid without containing microorganisms such as aerobic bacteria or anaerobic bacteria. The composition of the culture gas can be adjusted based on actual conditions (e.g., the type of microorganism to be cultured). For example, a high-oxygen environment is required for aerobic bacterial culture, while an anaerobic environment is required for anaerobic bacterial culture. For cell-microorganism co-culture, a high-oxygen or anaerobic environment is determined based on the type of microorganism. The microbial concentration in the sample solution can be adjusted as needed. For a positive control test in a sterility test, the amount of bacteria added should be less than 100 CFU. For example, the microbial concentration in the sample solution can be set to less than 50 CFU. The incubation temperature of the mixed solution can be adjusted based on actual conditions, for example, a constant temperature of 33°C to provide optimal temperature conditions for microbial growth.

[0045] In one embodiment, in step S100, before receiving the sample injection signal, the following steps are included: S400. Determine the type of microbial detection, which includes anaerobic bacteria detection, aerobic bacteria detection or cell-microorganism co-culture; the cell-microorganism co-culture includes cell and anaerobic bacteria co-culture and cell and aerobic bacteria co-culture. The aerobic bacteria detection is used to determine the growth of aerobic bacteria in an aerobic environment, such as one or more of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Micrococcus, Streptococcus pyogenes, Candida albicans, Aspergillus niger, Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa. The anaerobic bacteria detection is used to determine the growth of anaerobic bacteria in an anaerobic environment, such as one or more of Clostridium sporogenes or Propionibacterium acnes. The cell co-culture is used to assess whether the presence of cells will affect the observation of microorganisms, such as the co-culture sample solution of Candida albicans and Jurkat cells or the co-culture sample solution of Aspergillus niger and Jurkat cells.

[0046] S500 , when the microbial detection type is anaerobic bacteria detection or co-culture of cells and anaerobic bacteria, injecting an anaerobic mixed gas with a preset anaerobic ratio into the gas supply chamber 120 .

[0047] It is understood that the preset anaerobic ratio can be set according to actual conditions. In one embodiment, the anaerobic mixed gas with the preset anaerobic ratio can be nitrogen and carbon dioxide with the preset anaerobic ratio. For example, when the preset anaerobic ratio is 9:1, the anaerobic mixed gas with the preset anaerobic ratio is 90% nitrogen and 10% carbon dioxide.

[0048] S600 , when the microorganism detection type is aerobic bacteria detection or co-culture of cells and aerobic bacteria, an aerobic mixed gas having a preset oxygen demand ratio is supplied to the gas supply chamber 120 .

[0049] It is understood that the preset oxygen demand ratio can be set according to actual conditions. In one embodiment, the oxygen-demanding mixed gas with the preset oxygen demand ratio can be oxygen and carbon dioxide with the preset oxygen demand ratio. For example, when the preset oxygen demand ratio is 9:1, the oxygen-demanding mixed gas with the preset oxygen demand ratio can be 90% oxygen and 10% carbon dioxide.

[0050] In one embodiment, in step S100, before incubating the mixed solution in contact with the culture gas from the gas supply chamber 120, the process further includes: S700 , when the microorganism detection type is anaerobic bacteria detection or co-culture of cells and anaerobic bacteria, inject sealing oil into the detection chamber 110 through the injection port 111 .

[0051] It can be understood that when the microorganisms are anaerobic bacteria, the sealing oil is laid on the mixture of culture solution and sample solution, thereby limiting the gas exchange between the mixture and the outside world, which is conducive to the growth of anaerobic bacteria.

[0052] S200. During the incubation process, the optical detection module 200 periodically acquires images of the detection chamber 110 to be detected. The images of the detection chamber 110 to be detected can be acquired periodically based on the growth status (e.g., growth rate, reproduction cycle, etc.) of the aerobic, anaerobic, or cellular microbial co-culture in the detection chamber 110 and detection requirements (e.g., detection accuracy, detection frequency, etc.). For example, for rapidly growing microorganisms, the imaging interval can be appropriately shortened to more promptly capture their growth changes. For detections requiring high image clarity, imaging parameters need to be optimized to obtain high-quality images. In one embodiment, images of the detection chamber 110 to be detected can be acquired once every day to capture changes in microbial growth. The optical detection module 200 can adjust the resolution and focal length to acquire clear and accurate images of the detection chamber 110, providing a reliable basis for determining the microbial detection results in the subsequent step S300.

[0053] S300: Determine a microorganism detection result based on the image to be detected. The image to be detected may be a plurality of images to be detected in the detection chamber 110 that are periodically acquired by the optical detection module 200.

[0054] It is understood that when the microbial detection is anaerobic or aerobic, the microbial detection result can be determined based on the morphological changes of the biofilm or hyphae of the aerobic or anaerobic bacteria. When the microbial detection is cell-microorganism co-culture, the microbial detection result can be determined based on the morphological changes of the biofilm or hyphae of the aerobic or anaerobic bacteria and their interaction with the host cells.

[0055] In the above embodiment of the present invention, the culture fluid is stored in the detection chamber 110 to provide necessary nutrients for the growth of microorganisms, and the culture gas is stored in the air supply chamber 120 connected to the detection chamber 110 to maintain the stability of the culture environment in the detection chamber 110, thereby ensuring that the microorganisms in the detection chamber 110 grow in a suitable gas environment, thereby ensuring that the microbial contamination in the cell therapy product is accurately detected in a relatively short time, thereby improving the efficiency of microbial detection; at the same time, by providing a hydrophobic breathable membrane 130 at the connection between the detection chamber 110 and the air supply chamber 120, while ensuring that the culture gas circulates between the detection chamber 110 and the air supply chamber 120, the liquid in the detection chamber 110 is prevented from entering the air supply chamber 120 and causing leakage and loss, thereby improving the stability of microbial culture and thereby improving the accuracy of the microbial detection results. In addition, through the transparent window 112, the growth status of microorganisms in the detection chamber 110 can be observed in real time, and the image to be detected in the detection chamber 110 can be accurately obtained through the optical detection module 200, thereby realizing intuitive and real-time online monitoring of the microbial culture process, providing strong support for subsequent accurate judgment of the microbial status in cell therapy products, and further improving the efficiency and accuracy of microbial detection.

[0056] In one embodiment, the step S300 of determining the microorganism detection result based on the image to be detected includes steps S310-S320: S310: Perform image recognition on the image to be detected to obtain a recognition result corresponding to the image to be detected.

[0057] As can be understood, image recognition is used to extract key features from the image to be tested, such as microbial morphological characteristics (e.g., shape, texture, and edge features), as well as information such as changes in microbial populations. Accurately extracting these key features provides quantitative data for microbial detection results.

[0058] In one embodiment, the step S310 of performing image recognition on the image to be detected to obtain a recognition result corresponding to the image to be detected includes: S311: Preprocess the image to be detected to obtain a target image, wherein the preprocessing includes but is not limited to image classification and labeling, image data cropping, normalization, and target area recognition based on dynamic grayscale thresholds.

[0059] In one embodiment, the step S311 of preprocessing the image to be detected to obtain a target image includes: S3111: Crop the image to be detected to a preset size. That is, crop the image to be detected to a preset size.

[0060] S3112: Perform pixel normalization on the image to be detected so that the pixel values ​​of the image to be detected are within a preset pixel range. In other words, the pixel values ​​of the image to be detected are normalized to unify the pixel values ​​of different images within the same interval (preset pixel range), eliminating interference caused by differences in images or shooting conditions.

[0061] S3113. After pixel normalization, the image to be detected is subjected to image region segmentation and region threshold adjustment based on a dynamic grayscale threshold to obtain a target image. Specifically, the image to be detected is segmented, and different thresholds are applied to different segmented subregions. Specifically, an image segmentation method that adapts to local grayscale value variations is used to divide the image to be detected into multiple subregions, and thresholds are calculated and adjusted separately within each subregion. When the brightness of a subregion is below a preset brightness threshold and the pixel contrast within the subregion is high (for example, at the edge of a microorganism, where pixel differences are large, the pixel contrast is high), the threshold is adjusted lower to avoid missing the target microorganism during detection. When the brightness of a subregion is greater than or equal to the preset brightness threshold and the pixel contrast is low (for example, at the background, where pixel differences are small, the pixel contrast is low), the threshold is adjusted higher to avoid misidentification.

[0062] S312: extracting feature data from the target image through a preset image recognition model, and performing dimensionality reduction processing on the feature data based on a principal component analysis method to obtain target feature information.

[0063] It is understood that the feature data refers to graphical features that capture changes in microbial growth, including but not limited to shape, texture, or edge features during microbial growth. The preset image recognition models include but are not limited to local binary patterns (LBP), histogram of oriented gradients (HOG), Canny edge detection, or convolutional neural networks (CNN). The principal component analysis method, which performs a linear transformation on the pixel matrix, retains the principal components, i.e., microbial features such as color, shape, and texture. Prior to principal component analysis, image scaling (i.e., reducing the image to a lower resolution) and color space compression (i.e., converting an RGB three-channel image to a single grayscale channel) can also be used to preserve the data's key information and reduce computational complexity.

[0064] S313 , classifying and identifying the target feature information using a preset microbial classification model to obtain an identification result corresponding to the image to be detected.

[0065] It is understood that the preset microbial classification model can be trained based on a support vector machine (SVM), a random forest method (RF), or a K-Nearest Neighbors (KNN) algorithm. Specifically, a sample image containing microorganisms is first obtained, and the microorganisms are circled along the edges of the sample image using a software tool. The sample type can then be labeled based on the microorganism's shape, texture, and edge features. After the sample image is processed as in steps S311 and S312, classification training is performed based on the target feature information corresponding to the sample image and the sample type corresponding to the sample image using a support vector machine (SVM), a random forest method (RF), or a K-Nearest Neighbors (KNN) algorithm, thereby training to obtain the preset microbial classification model, thereby further improving the accuracy of the recognition results.

[0066] S320: Determine the microbial detection result based on all the identification results within a preset incubation period. The preset incubation period refers to the incubation stage corresponding to rapid microbial growth. The preset incubation period can be set based on the microbial growth conditions (e.g., growth rate, reproduction cycle, etc.) to ensure that the presence of microorganisms in the sample liquid can be identified within the preset incubation period.

[0067] Understandably, in the present invention, all of the recognition results within the preset incubation time can be probability arrays used to characterize the presence of one or more microorganisms. In the present invention, in step S320, the microorganism detection result is determined based on all of the recognition results within the preset incubation time. Specifically, the arrays corresponding to all of the recognition results are combined to form a matrix, and then the distribution characteristics of the matrix are confirmed to obtain a microorganism detection result corresponding to one of the microorganisms corresponding to the aforementioned recognition results. This microorganism detection result can indicate the presence of the microorganism in the detection image.

[0068] In one embodiment, the step S320 of determining the microorganism detection result based on all the identification results within a preset incubation time includes: S321: When the actual incubation time does not exceed the preset incubation time and the recognition result indicates that microorganisms exist in the image to be detected, confirm that the microorganism detection result is that microorganisms exist in the sample liquid.

[0069] It is understood that if microorganisms are present in the sample liquid, then within the preset incubation period, the microorganism monitoring method can be used to detect microorganisms in the image to be detected, and the recognition result can then indicate the presence of microorganisms in the image to be detected. That is, in this embodiment, as long as any recognition result within the actual incubation period indicates the presence of microorganisms in the image to be detected, the presence of microorganisms in the sample liquid can be determined.

[0070] S322: When the actual incubation time exceeds the preset incubation time and all the recognition results indicate that no microorganisms exist in the image to be detected, confirm that the microorganism detection result is that no microorganisms exist in the sample liquid.

[0071] Understandably, if the sample liquid contains no microorganisms, then no microorganisms will be detected in the image to be tested within the preset incubation time. In other words, all recognition results indicate that the image to be tested contains no microorganisms. Therefore, after the actual incubation time exceeds the preset incubation time, and all recognition results indicate that the image to be tested contains no microorganisms, monitoring can be stopped, confirming that the microorganism detection result indicates that the sample liquid contains no microorganisms.

[0072] In one embodiment, in step S321, after confirming that the microorganism detection result indicates that microorganisms are present in the sample liquid, the step further includes: Obtaining all the recognition results within a preset incubation time, and comparing the target feature information corresponding to all the recognition results; When all the target characteristic information indicates that the microorganisms in the sample liquid have a preset growth change trend, confirming that the growth of the microorganisms is normal; When all the target characteristic information indicates that the microorganisms in the sample liquid do not produce a preset growth change trend, it is determined that the microorganism growth is abnormal.

[0073] It is understandable that when the type of microbial detection is cell-microorganism co-culture, it is necessary to confirm whether the microorganisms produce a preset growth trend. The preset growth trend refers to the corresponding growth trend when the microorganisms are cultured alone in an environment that does not contain cells. If the growth trend of the microorganisms during cell-microorganism co-culture basically matches the preset growth trend, it means that the microorganisms in the sample liquid produce the preset growth trend. At this time, it can be assumed that the presence of cells will not affect the growth and observation of the microorganisms, and the microorganisms can be confirmed to be growing normally. Conversely, when the growth trend of the microorganisms deviates significantly from the preset growth trend, it means that the microorganisms in the sample liquid do not produce the preset growth trend. At this time, it can be assumed that the presence of cells has adversely affected the growth and observation of the microorganisms, and abnormal microbial growth can be confirmed.

[0074] like Figure 5As shown, when aerobic bacteria detection is performed using the microbial detection kit (or microbial detection device) of the present invention, when the microorganism in the sample liquid is Escherichia coli, the number of Escherichia coli can be seen to gradually increase in the image to be detected obtained after 1 day of incubation, and the colony morphology is initially revealed; the image to be detected obtained after 2 days of incubation shows that Escherichia coli has multiplied in large numbers, the colony morphology is clearer, and the distribution is more dense.

[0075] like Figure 6 As shown, when aerobic bacteria detection is performed using the microbial detection kit (or microbial detection device) of the present invention, when the microorganism in the sample liquid is Staphylococcus aureus, the image to be detected obtained after one day of incubation shows a significant increase in the number of Staphylococcus aureus, and tiny colonies begin to aggregate and form; the image to be detected obtained after two days of incubation shows significant growth characteristics of Staphylococcus aureus, and the biofilm completely covers the surface of the chamber, showing a uniform distribution state.

[0076] like Figure 7 As shown, when aerobic bacteria detection is performed using the microbial detection kit (or microbial detection device) of the present invention, when the microorganism in the sample liquid is Pseudomonas aeruginosa, the number of Pseudomonas aeruginosa can be seen to increase significantly in the image to be detected obtained after 1 day of incubation, and the Pseudomonas aeruginosa is spread evenly on the surface of the chamber; the image to be detected obtained after 2 days of incubation shows that the Pseudomonas aeruginosa colonies are evenly distributed, covering the entire surface of the chamber.

[0077] like Figure 8 As shown, when aerobic bacteria detection is performed using the microbial detection kit (or microbial detection device) of the present invention, when the microorganism in the sample liquid is Bacillus subtilis, the image to be detected obtained after one day of incubation shows that the number of Bacillus subtilis increases and a thin biofilm begins to form; the image to be detected obtained after two days of incubation shows that the Bacillus subtilis biofilm gradually thickens, expands in area, and becomes more dense and obvious.

[0078] like Figure 9 As shown, when aerobic bacteria detection is performed using the microbial detection kit (or microbial detection device) of the present invention, when the microorganisms in the sample liquid are micrococci, the image to be detected obtained after one day of incubation shows that the number of micrococci has increased significantly, and small, tightly packed colonies have begun to form; the image to be detected obtained after two days of incubation shows that the micrococcal colonies continue to expand and merge with each other, and eventually the biofilm completely covers the surface of the chamber, showing a uniform distribution state.

[0079] like Figure 10As shown, when aerobic bacteria detection is performed using the microbial detection kit (or microbial detection device) of the present invention, when the microorganism in the sample liquid is Streptococcus pyogenes, the image to be detected obtained after 1 day of incubation shows an increase in the number of Streptococcus pyogenes and the initial appearance of a biofilm morphology; the image to be detected obtained after 2 days of incubation shows a continuous increase in the number of Streptococcus pyogenes and the biofilm gradually covers the entire surface of the incubation detection chamber.

[0080] like Figure 11 As shown, when aerobic bacteria detection is performed using the microbial detection kit (or microbial detection device) of the present invention, when the microorganism in the sample liquid is Candida albicans, the image to be detected obtained after 1 day of incubation shows an increase in the number of Candida albicans, and a small amount of dispersed bacteria and initial budding begin to appear; the image to be detected obtained after 2 days of incubation shows that Candida albicans multiplies in large numbers, budding reproduction becomes more frequent, and the biofilm gradually covers the entire surface of the chamber, forming a dense covering layer.

[0081] like Figure 12 As shown, when aerobic bacteria detection is performed using the microbial detection kit (or microbial detection device) of the present invention, when the microorganism in the sample liquid is Aspergillus niger, the image to be detected obtained after 1 day of incubation shows that the number of Aspergillus niger increases and sparse, short hyphae begin to grow; the image to be detected obtained after 2 days of incubation shows that Aspergillus niger hyphae grow rapidly, intertwine with each other, and are densely distributed in the entire incubation detection chamber, and conidial heads are also formed in large quantities, and the color gradually becomes darker.

[0082] like Figure 13 As shown, when aerobic bacteria detection is performed using the microbial detection kit (or microbial detection device) of the present invention, when the sample liquid is a negative aerobic bacteria culture medium blank sample liquid, no growth characteristics of any microorganisms can be found in the image to be detected after incubation for 1 day and 2 days, and the culture medium remains clear and free of foreign matter.

[0083] It can be seen that the microbial detection device provided in this embodiment has high sensitivity for detecting aerobic bacteria, with a detection limit of less than 50 CFU, and the detection time is within 3 days, with high detection efficiency.

[0084] like Figure 14 As shown, when anaerobic bacteria detection is performed using the microorganism detection kit (or microorganism detection device) of the present invention, when the microorganism in the sample liquid is Clostridium sporogenes sample liquid, the images to be detected are obtained after incubation for 1 day and 2 days.

[0085] like Figure 15As shown in the figure, the microbial detection box (or microbial detection device) of the present application is used for anaerobic bacteria detection. When the microorganism in the sample liquid is Propionibacterium acnes, the Propionibacterium acnes begins to form a small colony in the image to be detected obtained after 3 days of incubation. The Propionibacterium acnes colony gradually increases and the morphology is more clear and the color is darker in the image to be detected obtained after 4 days of incubation.

[0086] As shown in the figure, the microbial detection box (or microbial detection device) of the present application is used for anaerobic bacteria detection. When the microorganism in the sample liquid is Propionibacterium acnes, the Propionibacterium acnes begins to form a small colony in the image to be detected obtained after 3 days of incubation. The Propionibacterium acnes colony gradually increases and the morphology is more clear and the color is darker in the image to be detected obtained after 4 days of incubation. Figure 16

[0087] It can be seen that the microbial detection device provided in the embodiment has high sensitivity for anaerobic bacteria detection, the detection limit is less than 50 CFU, and the detection time is within 3 days, so the detection efficiency is high.

[0088] As shown in the figure, the microbial detection box (or microbial detection device) of the present application is used for cell-microorganism co-culture. When the microorganism in the sample liquid is Candida albicans and Jurkat cells, the number of Candida albicans begins to increase in the image to be detected obtained after 1 day of incubation. Figure 17 As shown in the figure, the number of Candida albicans significantly increases and densely covers the bottom surface of the chamber in the image to be detected obtained after 4 days of incubation. Through image analysis and judgment, the presence of Jurkat cells does not interfere with the detection result of Candida albicans, and the growth characteristics of Candida albicans can be clearly distinguished. Figure 18

[0089] As shown in the figure, the microbial detection box (or microbial detection device) of the present application is used for cell-microorganism co-culture. When the microorganism in the sample liquid is Candida albicans and Jurkat cells, the number of Candida albicans begins to increase in the image to be detected obtained after 1 day of incubation. Figure 19 As shown in the figure, the number of Candida albicans significantly increases and densely covers the bottom surface of the chamber in the image to be detected obtained after 4 days of incubation. Through image analysis and judgment, the presence of Jurkat cells does not interfere with the detection result of Candida albicans, and the growth characteristics of Candida albicans can be clearly distinguished. Figure 20

[0090] It can be seen that the microbial detection device provided in the embodiment has good anti-interference ability in complex sample liquid detection and can accurately identify the growth state of microorganisms.

[0091] ​​​It should be understood that the size of the serial number of each step in the above embodiment does not mean the order of execution, and the execution order of each process should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiment of the application.

[0092] The above merely describes the embodiments of the microbial detection kit, detection device and detection method of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A microbial detection kit, characterized in that: The invention comprises a detection chamber for storing culture fluid and an air supply chamber connected to the detection chamber and for storing culture gas; the detection chamber is provided with an injection port connected to the detection chamber; and a hydrophobic breathable membrane is provided at the connection between the detection chamber and the air supply chamber.

2. The microorganism detection kit according to claim 1, characterized in that: The microbial detection box also includes a three-way connector; the first channel of the three-way connector is connected to the injection port; the microbial detection box also includes a first pipette for accommodating sample liquid and a second pipette for accommodating sealing oil; the first pipette and the second pipette are respectively connected to the second channel and the third channel of the three-way connector, and are used to inject sample liquid or sealing oil into the detection chamber when needed.

3. A microorganism detection device, characterized in that: comprising an optical detection module, a controller connected to the optical detection module, and the microorganism detection box according to claim 1 or 2; A transparent window is provided on the microorganism detection box at a position opposite to the detection chamber, and the optical detection module is used to obtain the image to be detected in the detection chamber through the transparent window.

4. A microbial detection method, characterized in that: Applied to the microbial detection device according to claim 3, the microbial detection method comprises: receiving a sample injection signal, controlling the sample liquid to enter the detection chamber from the injection port, so that the sample liquid is mixed with the culture liquid in the detection chamber, and then incubating the mixed liquid under the condition of contact with the culture gas from the gas supply chamber; During the incubation process, the optical detection module regularly acquires the image to be detected in the detection chamber; A microorganism detection result is determined based on the image to be detected.

5. The microorganism detection method according to claim 4, characterized in that: Determining the microorganism detection result according to the image to be detected includes: Performing image recognition on the image to be detected to obtain a recognition result corresponding to the image to be detected; The microorganism detection result is determined based on all the identification results within a preset incubation time.

6. The microorganism detection method according to claim 5, characterized in that: The performing image recognition on the image to be detected to obtain a recognition result corresponding to the image to be detected includes: Preprocessing the image to be detected to obtain a target image; Extracting feature data from the target image using a preset image recognition model, and performing dimensionality reduction processing on the feature data based on a principal component analysis method to obtain target feature information; The target feature information is classified and identified using a preset microbial classification model to obtain an identification result corresponding to the image to be detected.

7. The microorganism detection method according to claim 6, characterized in that: The preprocessing of the image to be detected to obtain a target image includes: Cropping the image to be detected into a preset size; Performing pixel normalization processing on the image to be detected so that the pixel values ​​of the image to be detected are within a preset pixel range; The target image is obtained by performing image region segmentation and region threshold adjustment on the image to be detected after pixel normalization processing based on the dynamic grayscale threshold.

8. The microorganism detection method according to claim 6, characterized in that: Before receiving the sample injection signal, the method includes: Determining the type of microbial detection, wherein the microbial detection type includes anaerobic bacteria detection, aerobic bacteria detection, or cell-microbial co-culture; the cell-microbial co-culture includes cell-anaerobic bacteria co-culture and cell-aerobic bacteria co-culture; When the microbial detection type is anaerobic bacteria detection or co-culture of cells and anaerobic bacteria, injecting an anaerobic mixed gas with a preset anaerobic ratio into the gas supply chamber; When the microbial detection type is aerobic bacteria detection or co-culture of cells and aerobic bacteria, an aerobic mixed gas having a preset oxygen demand ratio is supplied to the gas supply chamber; Before the mixed solution is incubated under the condition of contacting with the culture gas from the gas supply chamber, the method further comprises: When the microorganism detection type is anaerobic bacteria detection or co-culture of cells and anaerobic bacteria, sealing oil is injected into the detection chamber through the injection port.

9. The microorganism detection method according to claim 6, characterized in that: Determining the microorganism detection result based on all the identification results within a preset incubation time includes: When the actual incubation time does not exceed the preset incubation time and the recognition result indicates that microorganisms exist in the image to be detected, confirming the microorganism detection result as the presence of microorganisms in the sample liquid; When the actual incubation time exceeds the preset incubation time and all the recognition results indicate that no microorganisms exist in the image to be detected, the microorganism detection result is confirmed to be that no microorganisms exist in the sample liquid.

10. The microorganism detection method according to claim 9, characterized in that: After confirming that the microorganism detection result indicates that microorganisms are present in the sample liquid, the method further includes: Obtaining all the identification results within a preset incubation time; Compare the target feature information corresponding to all the identification results. When all the target feature information indicates that the microorganisms in the sample liquid produce a preset growth change trend, confirm that the microorganism growth is normal; when all the target feature information indicates that the microorganisms in the sample liquid do not produce the preset growth change trend, confirm that the microorganism growth is abnormal.

Images