Three-dimensional fluorescence detection method and system for identifying escherichia coli in bile

Abstract

The invention relates to the technical field of visible light absorption spectrum detection, in particular to a three-dimensional fluorescence detection method and system for identifying escherichia coli in bile, and the method comprises the following steps: generating a light source through a light source generation module; adjusting the intensity of the light source by using a light splitting module, and dividing the light source into two paths of light so as to obtain sample detection light and a reference spectrum; acquiring a background spectrum and a bile detection spectrum by utilizing a sample detection module based on the sample detection light; according to the reference spectrum, the background spectrum and the bile detection spectrum, the upper computer uses a spectrum detection model to identify escherichia coli in the bile sample. Based on the detection principle of three-dimensional fluorescence, the intelligent degree and efficiency of detection of escherichia coli in bile can be improved, and the detection cost can be reduced.

Description

Technical Field

[0001] This invention relates to the field of visible light absorption spectroscopy detection technology, and in particular to a three-dimensional fluorescence detection method and system for identifying Escherichia coli in bile. Background Technology

[0002] Biliary tract infection is one of the most common infectious diseases in clinical practice. As an infectious disease, the occurrence and development of biliary tract infection are often inseparable from changes in pathogens. Although bile itself is sterile, the bile duct is connected to the intestine, so the types of bacteria that cause biliary tract infection roughly correspond to the Escherichia coli spectrum in the intestine. Treatment of this infection inevitably involves the use of antibiotics. Therefore, it is crucial to accurately identify the type of biliary tract infection and find antibiotics that are sensitive to it.

[0003] However, existing methods for identifying bacterial colony types primarily employ bacterial culture. A certain amount of sample is placed on a test card in a constant-temperature incubator, and then various biochemical reactions are used to detect the bacterial types in the sample using colorimetric or turbidimetric methods. Although several devices have integrated and automated this process, and mature commercial devices are available on the market, detection devices based on these methods still generally suffer from the following limitations: 1. The detection response is slow, the detection cycle is long, and the detection methods are complex. Because bacterial culture methods require culturing live bacteria, the culturing process generally takes 18-24 hours, and specific results can only be obtained after manual diagnosis and review. This cannot meet the needs of some clinical scenarios that require rapid monitoring.

[0004] 2. Bacterial culture carries a certain possibility of failure. Because bacterial culture is a complex biochemical process, it is necessary to select an appropriate test card when inoculating bacteria to begin the culture, and to ensure the absence of contaminants and other microorganisms throughout the entire culture process. However, these conditions are quite demanding.

[0005] 3. Current testing equipment is expensive and consumables are costly. Existing equipment requires stable hardware peripherals and robust and precise control equipment because it needs to maintain sealed, constant temperature, and sterile conditions for a long time.

[0006] Therefore, given the existing problems in the clinical diagnosis and treatment of biliary tract infections, and to provide multi-level support for the accurate identification of biliary tract lesions, there is an urgent need for a rapid, adaptable, and low-cost Escherichia coli detection method to improve the application capabilities of detection equipment. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a three-dimensional fluorescence detection method and system for identifying Escherichia coli in bile.

[0008] To achieve the above objectives, in a first aspect, the present invention provides a three-dimensional fluorescence detection method for identifying *E. coli* in bile. The method includes the following steps: generating a light source using a light source generation module; adjusting the intensity of the light source using a spectrometer module and splitting the light source into two paths to obtain a sample detection light and a reference spectrum; acquiring a background spectrum and a bile detection spectrum using a sample detection module based on the sample detection light; and using a host computer to identify *E. coli* in the bile sample based on the reference spectrum, the background spectrum, and the bile detection spectrum, according to a spectral detection model. This invention, based on the detection principle of three-dimensional fluorescence, not only improves the intelligence and efficiency of *E. coli* detection in bile but also reduces detection costs.

[0009] Optionally, the light source generating module includes a xenon lamp and a reflector.

[0010] Optionally, the spectroscopic module includes a monochromator, an adjustable slit, a beam splitter, and a reference detection spectrometer.

[0011] Optionally, the step of using a spectrophotometer to adjust the intensity of the light source and split the light source into two paths to obtain the sample detection light and the reference spectrum includes the following steps: The beam splitting module adjusts the light source to different intensities through the monochromator and the adjustable slit, and then uses a beam splitter to split the light source into two paths, including a reference detection light and a sample detection light; The spectrophotometer is used to detect the reference light to obtain the reference spectrum.

[0012] Optionally, the wavelength of the light source can be adjusted using the monochromator according to a set wavelength step value, with the wavelength adjustment range being 250nm-550nm and the wavelength step value being 5nm.

[0013] Optionally, the sample detection module includes a sample chamber, a quartz cuvette, and an emission detection spectrometer, with the quartz cuvette placed inside the sample chamber.

[0014] Optionally, the step of acquiring the background spectrum and bile detection spectrum using the sample detection module based on the sample detection light includes the following steps: A blank sample is placed in the quartz cuvette, and the sample detection light is used to illuminate the quartz cuvette to obtain a first excitation light. The first excitation light is collected by an emission detection spectrometer to obtain the background spectrum. A bile sample is placed in the quartz cuvette, and the sample detection light is irradiated into the quartz cuvette to obtain a second excitation light. The second excitation light is collected by an emission detection spectrometer to obtain the bile detection spectrum.

[0015] Optionally, the step of identifying E. coli in a bile sample using a spectral detection model based on the reference spectrum, the background spectrum, and the bile detection spectrum includes the following steps: The host computer uses the reference spectrum, the background spectrum, and the bile detection spectrum to acquire the sample spectrum; Construct the spectral detection model; The host computer obtains Escherichia coli in the bile sample based on the sample spectrum and the spectral detection model.

[0016] Optionally, constructing the spectral detection model includes the following steps: The host computer collects and stores the sample spectra of different bacteria, and then constructs a bile lesion dataset; Based on the bile lesion dataset, the host computer uses the K-nearest neighbor algorithm to construct the spectral detection model.

[0017] Secondly, the present invention provides a three-dimensional fluorescence detection system for identifying Escherichia coli in bile. This system utilizes a three-dimensional fluorescence detection method for identifying Escherichia coli in bile provided by the present invention. The system includes: a light source generation module for generating a light source; a spectrometer module for adjusting the intensity of the light source and splitting it into two paths to obtain a sample detection light and a reference spectrum; a sample detection module for acquiring a background spectrum and a bile detection spectrum based on the sample detection light; and a host computer for identifying Escherichia coli in a bile sample using a spectral detection model based on the reference spectrum, the background spectrum, and the bile detection spectrum.

[0018] The present invention has at least the following beneficial effects: 1. Traditional methods for detecting Escherichia coli in bile are costly. This method, based on the detection principle of three-dimensional fluorescence, identifies bacteria, which is not only low-cost but also enables rapid identification of bacterial species.

[0019] 2. This method has a certain degree of scalability. It can not only identify the bacterial species in the database, but also expand the database by adding calibrated bacterial species in the later stage, thereby realizing the detection of different bacterial species. Moreover, traditional detection methods require the consumption of test cards, while this method does not require the consumption of test cards and other consumables.

[0020] 3. This system has a simple structure and is easy to operate, overcoming the limitations of current commercially available instruments that lack intelligence in monitoring, and improving the practicality and promotion of this method. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic flowchart of a three-dimensional fluorescence detection method for identifying Escherichia coli in bile according to an embodiment of the present invention; Figure 2 This is a schematic diagram illustrating the spectral acquisition principle of an embodiment of the present invention; Figure 3 This is a schematic diagram illustrating the process of preparing the bile lesion dataset according to an embodiment of the present invention; Figure 4 This is a schematic diagram of the framework of a three-dimensional fluorescence detection system for identifying Escherichia coli in bile, according to an embodiment of the present invention. Detailed Implementation

[0023] Specific embodiments of the present invention will now be described in detail. It should be noted that the embodiments described herein are for illustrative purposes only and are not intended to limit the invention. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other instances, well-known circuits, software, or methods have not been specifically described to avoid obscuring the invention.

[0024] Throughout this specification, references to "an embodiment," "an embodiment," "an example," or "an example" mean that a particular feature, structure, or characteristic described in connection with that embodiment or example is included in at least one embodiment of the invention. Therefore, the phrases "in an embodiment," "in an embodiment," "an example," or "an example" appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments or examples in any suitable combination and / or sub-combination. Moreover, those skilled in the art will understand that the illustrations provided herein are for illustrative purposes and are not necessarily drawn to scale.

[0025] It should be noted in advance that, in one alternative embodiment, apart from being described independently, the same symbols or letters appearing in all formulas have the same meaning and value.

[0026] In one optional embodiment, please refer to Figure 1 and Figure 2This invention provides a three-dimensional fluorescence detection method for identifying Escherichia coli in bile, the method comprising the following steps: S1. Generate a light source through the light source generation module.

[0027] Specifically, in this embodiment, the light source generation module includes a xenon lamp and a reflector. The light source is generated by the xenon lamp, focused by the reflector, and then processed by a matching lens. Figure 2 (Not shown in the image) After collimation, it enters the beam splitting module.

[0028] In other alternative embodiments, the light source generation module may also include a cooling fan.

[0029] S2. Use a spectrometer to adjust the intensity of the light source and split the light source into two paths to obtain the sample detection light and the reference spectrum.

[0030] The spectroscopic module includes a monochromator, an adjustable slit, a beam splitter, and a reference detection spectrometer. The reference detection spectrometer is an Ocean Optics Maya2000 spectrometer.

[0031] Step S2 specifically includes the following steps: S21. The beam splitting module adjusts the light source to different intensities through the monochromator and the adjustable slit, and then uses a beam splitter to split the light source into two paths, including a reference detection light and a sample detection light.

[0032] Specifically, in this embodiment, after the light source enters the beam splitting module, it passes sequentially through the monochromator and the adjustable slit within the module. Specifically, the monochromator in the beam splitting module is controlled by the host computer to adjust the wavelength of the light source according to a set wavelength step value, and the spectral range of the light source is controlled by changing the width of the adjustable slit. The wavelength adjustment range is 220nm-800nm, and the wavelength step value is 5nm.

[0033] Furthermore, after passing through the adjustable slit, the light source is split into two beams by a beam splitter, one of which is the reference detection light and the other is the sample detection light.

[0034] S22. The spectrophotometer is used to detect the reference light to obtain the reference spectrum.

[0035] Specifically, in this embodiment, a reference detection spectrometer is used to detect the reference detection light to obtain the reference spectrum, while the sample detection light enters the sample chamber.

[0036] S3. Based on the sample detection light, the background spectrum and bile detection spectrum are obtained using the sample detection module.

[0037] The sample detection module includes a sample compartment, quartz cuvettes, and an emission detection spectrometer. The emission detection spectrometer is an Ocean Optics Maya2000 spectrometer. The sample compartment is a stainless steel box with perforations, and its interior contains a cuvette holder on which the quartz cuvettes are placed.

[0038] Step S3 specifically includes the following steps: S31. Place a blank sample in the quartz cuvette, irradiate the quartz cuvette with the sample detection light to obtain the first excitation light, and collect the first excitation light by an emission detection spectrometer to obtain the background spectrum.

[0039] Specifically, in this embodiment, through the door on the sample chamber ( Figure 2 (Not shown) Pure water is placed in a quartz cuvette as a blank sample. Sample detection light enters through an entrance on one side of the sample chamber and illuminates the quartz cuvette, exciting the blank sample to produce the first excitation light. The first excitation light exits the sample chamber through an exit port perpendicular to the incident light, and the emitted light is collected by an emission detection spectrometer attached to the outside of the sample chamber to obtain the background spectrum.

[0040] S32. Place a bile sample in the quartz cuvette, irradiate the quartz cuvette with the sample detection light to obtain a second excitation light, and collect the second excitation light with an emission detection spectrometer to obtain the bile detection spectrum.

[0041] Specifically, in this embodiment, similar to step S31, a blank sample is taken out from the sample chamber and a bile sample is placed in a quartz cuvette to obtain a second excitation light, thereby obtaining the bile detection spectrum.

[0042] S4. Based on the reference spectrum, the background spectrum, and the bile detection spectrum, the host computer uses a spectral detection model to identify Escherichia coli in the bile sample.

[0043] Step S4 specifically includes the following steps: S41. The host computer uses the reference spectrum, the background spectrum, and the bile detection spectrum to obtain the sample spectrum.

[0044] Specifically, in this embodiment, the host computer receives and stores the reference spectrum, background spectrum, and bile detection spectrum, and obtains the sample spectrum according to the following relationship: Where P is the sample spectrum, For bile detection spectrum, For reference spectrum, This is the background spectrum.

[0045] S42. Construct the spectral detection model.

[0046] Specifically, step S42 includes the following steps: S421. The host computer collects and stores the sample spectra of different bacterial species, and then constructs a bile lesion dataset.

[0047] Specifically, in this embodiment, please refer to Figure 3 First, suspensions of different bacterial strains are prepared. Then, equal volumes of healthy bile are added to the suspensions of different bacterial strains to obtain experimental samples, and the sample spectra of each experimental sample are acquired, along with the strain numbers. Finally, the host computer receives and stores the sample spectra and strain numbers corresponding to different strains. In addition, the sample spectra of healthy bile also need to be acquired and stored in the host computer. Taking *E. coli* as an example, the process for preparing the suspension is as follows: 1. Pick a single colony from the solid culture medium of Escherichia coli and inoculate it into 5 ml of LB liquid medium (Luria-Bertani broth). Place the inoculated medium in a shaker at 37°C and incubate at 200 rpm for 12-16 hours (overnight) to allow the bacteria to reach the logarithmic growth phase.

[0048] 2. Take 1 ml of the overnight cultured bacterial solution from the LB liquid medium in step 1, add it to 9 ml of fresh LB liquid medium, mix well, and continue to culture in a shaker at 37°C for 2-3 hours until the OD600 value of the bacterial solution reaches 0.4-0.6 (mid-logarithmic growth phase).

[0049] 3. Transfer the bacterial culture from step 2 to a sterile centrifuge tube, centrifuge at 5000 rpm for 10 minutes at 4°C, discard the supernatant, gently resuspend the bacterial pellet with sterile physiological saline, and repeat the centrifugation once to remove any remaining culture medium.

[0050] 4. Resuspend the bacterial precipitate in an appropriate amount of sterile physiological saline to prepare a homogeneous E. coli suspension. Measure its OD600 value using a spectrophotometer, and then apply the result according to the standard curve or empirical formula. Adjust the bacterial concentration to the required value (e.g.) or ).

[0051] 5. The prepared E. coli suspension can be used immediately for experiments or stored for a short period at 4°C (not exceeding 24 hours). For long-term storage, the bacterial suspension can be mixed with glycerol in a 1:1 ratio, dispensed, and frozen at -80°C. Before the experiment, the lyophilized bacteria should be thawed and cultured to prepare an E. coli suspension.

[0052] Note that strict aseptic technique must be maintained throughout the entire process to avoid contamination by other microorganisms. The bacterial concentration should be adjusted according to experimental requirements; too high or too low a concentration may affect the experimental results. When resuspending the bacterial cells after centrifugation, handle them gently to avoid damaging the bacterial structure.

[0053] For each sample spectrum, since the wavelength of the light source is adjusted according to wavelength steps, the host computer can obtain the fluorescence intensity of the corresponding suspension at different wavelengths based on the sample spectrum. By using the fluorescence intensity of one strain at different wavelengths as a data sample, a dataset of biliary lesions can be constructed using the fluorescence intensities of different strains at different wavelengths.

[0054] S422. Based on the bile lesion dataset, the host computer uses the K-nearest neighbor algorithm to construct the spectral detection model.

[0055] Specifically, in this embodiment, the host computer first performs Z-score normalization on the fluorescence intensity in the bile lesion dataset. Then, the bile lesion dataset is divided into a training set and a test set in a 7:3 ratio to train and test the K-nearest neighbor algorithm, resulting in a spectral detection model. The model input is the normalized fluorescence intensity, and the output is the bacterial strain category. The optimal K value is selected through cross-validation or grid search, and classification accuracy, precision, recall, and F1-score are used as model evaluation metrics.

[0056] S43. The host computer obtains Escherichia coli in the bile sample based on the sample spectrum and the spectral detection model.

[0057] Specifically, in this embodiment, after the host computer obtains the sample spectrum of the bile sample to be detected, it first obtains the fluorescence intensity at different wavelengths, and then inputs the obtained fluorescence intensity into the spectral detection model, that is, it identifies Escherichia coli in the bile sample.

[0058] It should be noted that in some cases, the actions described in the specification can be performed in different orders and still achieve the desired results. In this embodiment, the order of steps is given only to make the embodiment clearer and easier to explain, and not to limit it.

[0059] In one optional embodiment, please refer to Figure 4 The present invention also provides a three-dimensional fluorescence detection system for identifying Escherichia coli in bile, the system comprising a light source generation module A1, a spectrophotometer module A2, a sample detection module A3, and a host computer A4.

[0060] The light source generation module A1 is used to generate a light source; The beam splitting module A2 is used to adjust the intensity of the light source and split the light source into two paths to obtain the sample detection light and the reference spectrum; The sample detection module A3 acquires the background spectrum and bile detection spectrum based on the sample detection light; The host computer A4 is used to identify Escherichia coli in bile samples using a spectral detection model based on the reference spectrum, the background spectrum, and the bile detection spectrum.

[0061] The present invention has at least the following beneficial effects: 1. Traditional methods for detecting E. coli in bile require culturing live bacteria, resulting in slow response times, long detection cycles, complex procedures, and high costs. This method, based on the principle of three-dimensional fluorescence detection, not only improves the intelligence and efficiency of E. coli detection in bile but also reduces detection costs.

[0062] 2. This method has a certain degree of scalability. It can not only identify the bacterial species in the database, but also expand the database by adding calibrated bacterial species in the later stage, thereby realizing the detection of different bacterial species. Moreover, traditional detection methods require the consumption of test cards, while this method does not require the consumption of test cards and other consumables.

[0063] 3. A system adapted to the method is provided. This system has a simple structure and is easy to operate, which makes up for the limitations of the current market instruments in monitoring that are not intelligent, and can improve the practicality of this method and facilitate its promotion.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A three-dimensional fluorescence detection method for identifying Escherichia coli in bile, characterized in that, Includes the following steps: A light source is generated using the light source generation module; The intensity of the light source is adjusted using a spectrometer, and the light source is split into two paths to obtain the sample detection light and the reference spectrum. Based on the sample detection light, the background spectrum and bile detection spectrum are obtained using the sample detection module; Based on the reference spectrum, the background spectrum, and the bile detection spectrum, the host computer uses a spectral detection model to identify Escherichia coli in the bile sample.

2. The three-dimensional fluorescence detection method for identifying Escherichia coli in bile according to claim 1, characterized in that: The light source generation module includes a xenon lamp and a reflector.

3. The three-dimensional fluorescence detection method for identifying Escherichia coli in bile according to claim 1, characterized in that: The spectroscopic module includes a monochromator, an adjustable slit, a beam splitter, and a reference detection spectrometer.

4. The three-dimensional fluorescence detection method for identifying Escherichia coli in bile according to claim 3, characterized in that, The step of using a spectrophotometer to adjust the intensity of the light source and split the light source into two paths to obtain the sample detection light and the reference spectrum includes the following steps: The beam splitting module adjusts the light source to different intensities through the monochromator and the adjustable slit, and then uses a beam splitter to split the light source into two paths, including a reference detection light and a sample detection light; The spectrophotometer is used to detect the reference light to obtain the reference spectrum.

5. The three-dimensional fluorescence detection method for identifying Escherichia coli in bile according to claim 3, characterized in that: The wavelength of the light source is adjusted using the monochromator according to the set wavelength step value. The wavelength adjustment range is 250nm-550nm, and the wavelength step value is 5nm.

6. The three-dimensional fluorescence detection method for identifying Escherichia coli in bile according to claim 1, characterized in that: The sample detection module includes a sample chamber, a quartz cuvette, and an emission detection spectrometer, with the quartz cuvette placed inside the sample chamber.

7. The three-dimensional fluorescence detection method for identifying Escherichia coli in bile according to claim 6, characterized in that, The step of acquiring the background spectrum and bile detection spectrum using the sample detection module based on the sample detection light includes the following steps: A blank sample is placed in the quartz cuvette, and the sample detection light is used to illuminate the quartz cuvette to obtain a first excitation light. The first excitation light is collected by an emission detection spectrometer to obtain the background spectrum. A bile sample is placed in the quartz cuvette, and the sample detection light is irradiated into the quartz cuvette to obtain a second excitation light. The second excitation light is collected by an emission detection spectrometer to obtain the bile detection spectrum.

8. The three-dimensional fluorescence detection method for identifying Escherichia coli in bile according to claim 1, characterized in that, The step of identifying E. coli in a bile sample using a spectral detection model based on the reference spectrum, the background spectrum, and the bile detection spectrum includes the following steps: The host computer uses the reference spectrum, the background spectrum, and the bile detection spectrum to acquire the sample spectrum; Construct the spectral detection model; The host computer obtains Escherichia coli in the bile sample based on the sample spectrum and the spectral detection model.

9. The three-dimensional fluorescence detection method for identifying Escherichia coli in bile according to claim 8, characterized in that, The construction of the spectral detection model includes the following steps: The host computer collects and stores the sample spectra of different bacteria, and then constructs a bile lesion dataset; Based on the bile lesion dataset, the host computer uses the K-nearest neighbor algorithm to construct the spectral detection model.

10. A three-dimensional fluorescence detection system for identifying Escherichia coli in bile, wherein the three-dimensional fluorescence detection system for identifying Escherichia coli in bile uses the three-dimensional fluorescence detection method for identifying Escherichia coli in bile according to any one of claims 1-9, the system comprising: A light source generation module, wherein the light source generation module is used to generate a light source; The beam splitting module is used to adjust the intensity of the light source and split the light source into two paths to obtain the sample detection light and the reference spectrum. The sample detection module acquires the background spectrum and bile detection spectrum based on the sample detection light; The host computer is used to identify Escherichia coli in a bile sample using a spectral detection model based on the reference spectrum, the background spectrum, and the bile detection spectrum.

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