Portable fluorescence detection device and method

Abstract

The invention discloses a portable fluorescence detection device and method, and relates to the field of fluorescence detection.The device comprises an excitation light source module, a sample reaction module and a mobile terminal; the excitation light source module is used for emitting laser to the sample reaction module, so that a microorganism sample to be detected generates a fluorescence signal; the mobile terminal is used for acquiring the image of the fluorescence signal generated by the microorganism sample to be detected and determining the fluorescence intensity of the fluorescence signal according to the image of the fluorescence signal generated by the microorganism sample to be detected, and the portable fluorescence detection device is low in cost, short in consumed time and simple in operation and structure.

Description

Technical Field

[0001] The present application relates to the field of fluorescence detection, and in particular to a portable fluorescence detection device and method. Background Art

[0002] Fluorescence detection has always been a very common detection method in biochemical testing. However, traditional fluorescence detection technology usually requires the use of large and expensive optical detection equipment in a laboratory environment. For example, the nucleic acid molecules in the sample are first amplified through PCR amplification technology to amplify the biological signal. The sample is then excited by fluorescence using large optical excitation equipment. Finally, the fluorescence intensity is detected and analyzed using professional optical equipment such as spectrometers, photomultiplier tubes, or CCD and CMOS cameras. Due to the high cost and complex operation of professional optical equipment, the detection process is time-consuming and its application scenarios are limited to laboratories. It is not suitable for rapid and immediate detection of biochemical samples in the field or in resource-scarce areas.

[0003] In view of the shortcomings of the above-mentioned technologies, such as high cost, long time consumption and complex structure, there is an urgent need for a portable fluorescence detection device with low cost, short time consumption and simple operation and structure. Summary of the Invention

[0004] The purpose of this application is to provide a portable fluorescence detection device and method, which can achieve the effects of low cost, short time consumption and simple structure and operation.

[0005] To achieve the above objectives, this application provides the following solutions:

[0006] In a first aspect, the present application provides a portable fluorescence detection device, comprising:

[0007] An excitation light source module, a sample reaction module and a mobile terminal; the excitation light source module is used to emit laser light to the sample reaction module, so that the microbial sample to be detected generates a fluorescent signal; the mobile terminal is used to obtain an image of the fluorescent signal generated by the microbial sample to be detected, and determine the fluorescence intensity of the fluorescent signal based on the image of the fluorescent signal generated by the microbial sample to be detected.

[0008] In a second aspect, the present application provides a portable fluorescence detection method, which is applied to the portable fluorescence detection device described above. The portable fluorescence detection method includes:

[0009] Acquire an image of the fluorescence signal generated by the microbial sample to be detected at the current moment;

[0010] Performing target area recognition on the image of the fluorescence signal generated by the microbial sample to be detected at the current moment to obtain a fluorescence image of the target area;

[0011] Converting the fluorescent image of the target area into a YUV color space to obtain a converted image;

[0012] The fluorescence intensity of the fluorescence signal at the current moment is calculated according to the converted image.

[0013] According to the specific embodiments provided in this application, this application has the following technical effects:

[0014] The present application provides a portable fluorescence detection device and method, which can achieve rapid and accurate detection of fluorescence intensity by simply acquiring and analyzing fluorescence signal images through an integrated mobile terminal. After testing and calibration, there is no need to use large-scale laboratory optical equipment to analyze the fluorescence intensity. It is low-cost, short in time, simple in operation and structure, and easy to carry. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 A side view of a portable fluorescence detection device provided in one embodiment of the present application;

[0017] Figure 2 The detection result of fluorescence detection using the portable fluorescence detection device provided in one embodiment of the present application;

[0018] Figure 3 This is the fluorescent image of the target area when there are four experimental test tubes. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0020] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the present application is further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0021] In an exemplary embodiment, Figure 1As shown, a portable fluorescence detection device is provided, comprising: an excitation light source module, a sample reaction module and a mobile terminal; the excitation light source module is used to emit laser light to the sample reaction module, so that the microbial sample to be detected generates a fluorescence signal; the mobile terminal is used to obtain an image of the fluorescence signal generated by the microbial sample to be detected, and determine the fluorescence intensity of the fluorescence signal based on the image of the fluorescence signal generated by the microbial sample to be detected.

[0022] In another exemplary embodiment of the present application, the sample reaction module includes: multiple experimental centrifuge tubes 6; a mixed reaction substrate is set in each experimental centrifuge tube 6; the mixed reaction substrate includes a microbial sample to be detected, an RPA-CRISPR / Cas12a reaction system reagent and a FAM fluorescent group; the excitation light source module is used to emit laser to the mixed reaction substrate in each experimental centrifuge tube 6, so that the microbial sample to be detected produces a fluorescent signal.

[0023] In another exemplary embodiment of the present application, the portable fluorescence detection device also includes: a temperature control module, which is used to control the temperature of the mixed reaction substrate in each experimental centrifuge tube 6 so as to perform isothermal amplification of nucleic acid on the microbial sample to be detected in each experimental centrifuge tube 6.

[0024] In another exemplary embodiment of the present application, the portable fluorescence detection device is further equipped with a mobile power supply 14 to ensure power supply to the temperature control module and the excitation light source module. This eliminates the need for an external power source, enabling the entire system to operate independently outdoors. The mobile power supply 14 is a large-capacity power bank.

[0025] In another exemplary embodiment of the present application, the excitation light source module includes a laser 12 and an optical fiber 10; the optical fiber 10 is used to irradiate the laser light emitted by the laser 12 into the mixed reaction substrate in each experimental centrifuge tube 6. The laser 12 is a 488nm laser generator that can effectively excite the FAM fluorescent group in the reaction.

[0026] In another exemplary embodiment of the present application, the temperature control module includes: a reference centrifuge tube, a heating pad 11, a temperature probe and a temperature controller 4; double distilled water is set in the reference centrifuge tube; the reference centrifuge tube and the experimental centrifuge tube 6 are both set on the heating pad 11, and the heating pad 11 is used to heat the mixed reaction substrate in each experimental centrifuge tube 6 and the double distilled water in the reference centrifuge tube; the temperature probe is set in the reference centrifuge tube, and the temperature probe is used to measure the temperature of the double distilled water in the reference centrifuge tube; the temperature controller 4 is connected to the temperature probe and the heating pad 11, and the temperature controller 4 is used to control the heating pad 11 according to the temperature measured by the temperature probe, so that the temperature of the mixed reaction substrate in each experimental centrifuge tube 6 reaches a preset temperature. The temperature controller 4 is connected to the temperature probe via a temperature control probe line 5. The temperature probe is a high-precision temperature probe.

[0027] In another exemplary embodiment of the present application, the mobile terminal is a smartphone 3; the smartphone 3 includes a camera and a chip; the chip has a built-in fluorescence intensity detection module; a filter 1 is disposed between the camera and the sample reaction module, the camera being configured to capture an image of the fluorescence signal generated by the microbial sample to be detected through the filter 1; and the fluorescence intensity detection module is configured to determine the fluorescence intensity of the fluorescence signal based on the image of the fluorescence signal generated by the microbial sample to be detected. The filter 1 has a central wavelength of 520 nm, which can filter out stray light from the emitted light and only allow fluorescence signal bands of specific wavelengths to pass through, thereby effectively reducing signal interference from background stray light and ensuring accurate detection of the fluorescence signal.

[0028] In another exemplary embodiment of the present application, the portable fluorescence detection device also includes a centrifuge tube rack 7 for placing experimental centrifuge tubes 6 and reference centrifuge tubes, and holes are provided on the centrifuge tube rack 7. There are 4 experimental centrifuge tubes 6 and 1 reference centrifuge tube, and there are 5 holes in total. Both the experimental centrifuge tubes 6 and the reference centrifuge tubes are 1.5mL centrifuge tubes, and the position and size of each hole are accurately calculated. The hole size is 11mm, and the centrifuge tube rack 7 width is 15mm. The hole center distance is 7mm from the edge of the centrifuge tube rack 7 on one side of the heating pad 11 to ensure that the experimental centrifuge tube 6 and the reference centrifuge tube can be firmly placed and maintain the best light receiving angle. This multi-channel design enables the device to detect 4 reaction samples simultaneously to ensure that the detection reaction is carried out efficiently.

[0029] In another exemplary embodiment of the present application, a laser generator is connected to four optical fibers 10 via SMA connectors, with each optical fiber 10 corresponding to a centrifuge tube 6. After the laser generator emits excitation light, it is transmitted along the optical fibers 10 to the bottom of the centrifuge tube 6 in the reaction zone, stimulating the reaction system to produce fluorescence.

[0030] In another exemplary embodiment of the present application, the portable fluorescence detection device further includes a remote sensing controller 13. The remote sensing controller 13 is connected to a mobile power supply 14, a laser 12 and a temperature controller 4 via wires 9.

[0031] In another exemplary embodiment of the present application, the portable fluorescence detection device also includes: a shell, an excitation light source module, a sample reaction module, a remote sensing controller 13 and a temperature control module are all encapsulated inside the shell, and the shell is made of photosensitive resin through light curing 3D printing, which is sturdy and resistant to falling, wherein the remote sensing controller 13 is placed on the bottom surface of the shell and is close to the inner side surface and will not slide, and an independent control switch is set on the outside of the shell, and the remote sensing controller 13 is connected to the control switch by wireless. The control switch is operated by the user, and the switch of the remote sensing controller 13 is controlled by the control switch, thereby controlling the start and stop of the mobile power supply 14, and thereby controlling the disconnection of the entire system. The mobile power supply 14 is also placed at the bottom of the shell, which has a standard 5V2A output and a capacity of 13200mAh. It is also close to the side wall and will not slide. The high-performance heating pad 11 is fixed to the inner wall of the rear side of the shell by double-sided tape, close to the experimental centrifuge tube 6 and the reference centrifuge tube in the reaction zone, and can effectively conduct heat to achieve heating and temperature control of the reaction system. Thermostat 4 is a single-chip microcomputer control system with a digital display and control buttons. If the temperature detected by the temperature probe is above the set upper limit, the thermostat 4 turns off the heating pad 11, stopping heating. If the temperature is below the set lower limit, the heating pad 11 is turned on, starting heating. This ensures that the mixed reaction substrates remain within the set temperature range, with a temperature control accuracy of 0.1°C. The thermostat 4 snaps into the side of the housing through a reserved square slot, with the digital display and control buttons visible to the user for easy viewing and control.

[0032] In another exemplary embodiment of the present application, the portable fluorescence detection device also includes: a mobile phone holder 8, the left and right widths of the mobile phone holder 8 can be extended to accommodate mobile phones of different sizes, and it is fixed to the front of the shell by M6 metal screws. By adjusting the mobile phone camera to face the camera lens barrel, the filtered emitted fluorescence can be captured, and the detection results can be analyzed and visualized in real time through the fluorescence intensity detection module in the smart phone 3.

[0033] In another exemplary embodiment of the present application, the portable fluorescence detection device further includes an upper cover 2, which is installed and secured via a slot provided in the upper end of the housing, facilitating user access to or placement of internal components. When the upper cover 2 is in place, a darkroom is formed within the housing, thereby preventing interference from ambient light noise on the quantitative fluorescence analysis being performed.

[0034] The process of fluorescence intensity detection using the portable fluorescence intensity detection device provided in this application is as follows:

[0035] Step 1: Sample preparation and loading.

[0036] Mix the microbial sample to be tested with the RPA-CRISPR / Cas12a reaction system reagent, add the substrate containing the FAM fluorescent group, put the prepared reaction mixture into a 1.5 mL experimental centrifuge tube 6, and place the experimental centrifuge tube 6 into the hole.

[0037] Step 2: Reaction temperature control.

[0038] The temperature probe in the temperature control module detects the temperature of the reaction system in real time and transmits the temperature data to the temperature display. According to the reading on the temperature display, the working state of the heating pad 11 is controlled to maintain the temperature of the reaction system at the optimal temperature required for the RPA-CRISPR / Cas12a reaction (usually 37-42°C), providing suitable conditions for subsequent reactions.

[0039] Step 3: Isothermal nucleic acid amplification and CRISPR / Cas12a reaction.

[0040] At an optimal temperature maintained by the temperature control module, the reaction system undergoes isothermal nucleic acid amplification. If the target microorganism is present in the sample, its specific nucleic acid sequence will be amplified. Subsequently, CRISPR / Cas12a recognizes and binds to the amplified product, activating its nonspecific cleavage activity, cleaving the substrate containing the FAM fluorescent group and releasing a fluorescent signal.

[0041] Step 4: Fluorescence signal excitation.

[0042] The 488nm laser generator in the excitation light source module is connected to four optical fibers 10 via SMA connectors. Laser light is transmitted along these fibers to the bottom of each experimental centrifuge tube 6, stimulating the FAM fluorescent group in the reaction system. If the target microorganism is present in the reaction system in step 3, the released FAM fluorescent group will produce a fluorescent signal with a wavelength of 520nm.

[0043] Step 5: Fluorescence signal filtering.

[0044] The filter 1 with a central wavelength of 520 nm filters the fluorescent signal generated in step 4, removes non-specific background light and stray light, and only allows the FAM fluorescent signal of a specific wavelength to pass through, thereby improving the signal specificity and signal-to-noise ratio.

[0045] Step 6: Fluorescence signal acquisition.

[0046] The smart phone 3 is fixed on the mobile phone holder 8, and its camera captures the filtered fluorescence signal in step 5 through the camera barrel and converts it into digital image data.

[0047] Step 7: Fluorescence signal analysis and result visualization.

[0048] The fluorescence intensity detection software pre-installed in the smartphone 3, namely the above-mentioned fluorescence intensity detection module, performs real-time analysis and processing on the acquired fluorescence image data, calculates the fluorescence intensity, and visualizes the results in an intuitive manner to generate a detection result report, so that the operator can intuitively determine whether the target microorganisms are present in the sample.

[0049] In another exemplary embodiment of the present application, the internal processing flow of the fluorescence intensity detection software is specifically as follows:

[0050] Step 7.1. Image acquisition and sequence processing.

[0051] The system captures a sequence of fluorescence images, extracts time stamps from the file names, and creates a time-series dataset. These images are arranged chronologically, providing the basis for subsequent trend analysis. Each image enters the real-time analysis pipeline immediately after acquisition. The fluorescence image sequence includes the original image corresponding to each moment, which represents the fluorescence signal generated by the microbial sample being tested.

[0052] Subsequent operations are performed on the original image at the current moment.

[0053] Step 7.2. Image enhancement preprocessing.

[0054] To ensure that the fluorescence signal is accurately captured, multi-level enhancement processing is performed on the original image.

[0055] Step 7.2.1: Contrast and brightness adjustment: Optimize image visibility through the conversion function, which is expressed as:

[0056] I enhanced =α·I original +β

[0057] Among them, α=2 is the contrast amplification coefficient, β=-20 is the brightness adjustment value, I original represents the original image, I enhanced This parameter configuration can effectively reduce background noise and highlight the fluorescence signal.

[0058] Step 7.2.2: Color space conversion: Convert the RGB image to the HSV color space to facilitate the subsequent identification of highlighted fluorescent spots.

[0059] Step 7.2.3: Morphological noise removal: Apply an opening operation to remove small noise points and retain the main fluorescent area information.

[0060] Step 7.3. Intelligent region detection.

[0061] The system identifies bright fluorescent spot areas in the HSV color space, significantly reducing ambient light interference.

[0062] Step 7.3.1: Fluorescence threshold definition: Set the HSV range of the white fluorescence area to [0,0,180] to [255,255,255] and create a binary mask.

[0063] Step 7.3.2: Contour extraction and filtering: Detect the contours of connected regions based on a binary mask, and apply an area threshold (10 pixels) to filter out tiny noise points. Delete connected regions with an area greater than or equal to 10 pixels to ensure that only valid fluorescent point areas, i.e., bright fluorescent points, are retained.

[0064] Step 7.3.3: Spatial position constraint: Based on the effective fluorescent spot area, additional vertical position constraints (1500 ≤ y ≤ 2100 pixels) are applied to exclude interference from non-target areas.

[0065] Step 7.4. Accumulate multi-frame position data.

[0066] In response to possible detection instability, the system has developed an adaptive accumulation algorithm for the position of fluorescent spots.

[0067] Historical data accumulation: When the number of highlighted fluorescent points at a certain moment in history is identified to be ≥2, the system will add the positions of these highlighted fluorescent points to the global position library and save them.

[0068] Intelligent position recovery: If some highlighted fluorescent spots fail to be detected in step 7.3.2, the system will automatically use the historical accumulated data to infer the position coordinates of the highlighted fluorescent spots that have not been identified at the current moment. Obtain the coordinate set of the highlighted fluorescent points, specifically as shown in Formula 1 and Formula 2, to ensure detection continuity.

[0069]

[0070] Among them, n r is the number of historically accumulated highlighted fluorescent points, x i is the horizontal coordinate of the highlighted fluorescent point at the i-th historical moment, y i is the vertical coordinate of the highlighted fluorescent point at the i-th historical moment.

[0071] Step 7.5. Image rotation and correction.

[0072] To ensure measurement consistency, the system applies intelligent image correction algorithms.

[0073] Step 7.5.1: Rotation angle calculation: Based on the coordinates of the leftmost highlighted fluorescent point obtained at the current moment (x left ,y left ) and the rightmost highlighted fluorescent spot coordinates (x right ,yright ), calculate the angle θ with the horizontal line, as shown in Formula 3:

[0074]

[0075] Step 7.5.2: Affine transformation: Rectify the image obtained in step 7.3.3 by applying a rotation matrix according to θ around the center of the image, maintaining the horizontal alignment of the fluorescent area.

[0076] Step 7.6. Calculation of Normalized Fluorescence Area: Define a normalized monitoring area for each detection area.

[0077] Step 7.6.1: Standardize the monitoring area: Define a 300×300 pixel square area centered on the location of the identified highlight fluorescent spot as the target area for fluorescence intensity calculation. This application can dynamically adjust the monitoring area based on the location of the detected highlight fluorescent spot to ensure that the target area is always within the calculation range.

[0078] Step 7.6.2: Vertical axis uniformity: Use Figure 3 The average ordinate of the areas corresponding to 2 and 3 shown is used as the standard ordinate for all areas to ensure horizontal alignment. Figure 3 Groups 1, 2, and 3 are experimental groups, and group 4 is the control group.

[0079] Step 7.7. Multi-level fluorescence intensity calculation: Detection sensitivity can be improved by analyzing fluorescence intensity at multiple levels.

[0080] Step 7.7.1: Fluorescence intensity quantification: Convert each extracted target area into YUV color space, and calculate the fluorescence intensity I of each target area using the Y channel (brightness component) abs , as shown in Formula 4:

[0081]

[0082] Where w and h are the width and height of the target area, respectively, and Y(i, j) is the brightness value at the coordinate (i, j) of the target area.

[0083] Step 7.7.2: The relative intensity calculation steps for each target area are: the current intensity of the target area is compared with the initial intensity I initial (According to the first frame image) the relative intensity I of the target area is obtained by comparison rel :

[0084]

[0085] Step 7.7.3: The steps for calculating the normalized fluorescence intensity of each target area are: Calculate the normalized fluorescence intensity I of each target area according to formula 6r :

[0086]

[0087] This indicator can intuitively reflect the relative change trend of fluorescence intensity.

[0088] Step 7.8. Time series data analysis and reporting: After the system completes processing of all images, it generates a comprehensive analysis report.

[0089] Step 7.8.1: Time series trend recording: Arrange the three fluorescence intensity indicators of each target area at different time points in a time series.

[0090] Step 7.8.2: Excel report generation: Generate a data report containing time, region number, absolute fluorescence intensity, relative fluorescence intensity, and normalized fluorescence intensity.

[0091] Step 7.8.3: Microbial determination basis: Determine whether the target microorganism exists in the sample by observing the trend of fluorescence intensity changes, especially the growth pattern of normalized fluorescence intensity.

[0092] This application also provides a specific embodiment of using the above-mentioned device to perform RPA-CRISPR / Cas12a detection.

[0093] After setting up the experimental samples, the reaction samples and the reaction process were placed in the portable fluorescence detection device. During the reaction process, a picture of the reaction sample was taken every 5 minutes, and the fluorescence intensity of the sample in the picture was normalized. The test results are as follows: Figure 2 As shown, Figure 2 Part A is a bar graph of the normalized fluorescence intensity of the experimental group (Phytophthora infestans) and the control group (double-distilled water) when the reaction reaches the end point (normalized fluorescence intensity remains basically unchanged). Figure 2 Part B is the image actually taken at the end point of the reaction. Figure 2 Part C in the middle is the response curve of the normalized fluorescence intensity of the experimental group and the control group changing with time.

[0094] The portable fluorescence detection device provided in this application has a length, width and height of 20 cm, 12 cm and 17.4 cm respectively, and weighs only 450 g. It is compact and portable, equipped with an independent power supply, does not rely on a fixed application scenario, and can be used even in a field environment.

[0095] RPA-CRISPR / Cas12a technology can be used for the detection of microorganisms, and the detection method uses fluorescent signals to characterize the test results. Although conventional qPCR instruments, microplate readers and other fluorescence intensity detection instruments can accurately measure the fluorescent signals generated during the reaction. However, due to the high cost, complicated operation and inconvenience of professional detection instruments, current application scenarios can only be limited to laboratories and cannot be extended to field detection in the field. The portable fluorescence detection device provided by the application is small, light and portable, low in cost, and simple to operate. It can be extended to the field for early sensitive diagnosis of crop diseases. The entire process of nucleic acid isothermal amplification, CRISPR / Cas12a reaction and fluorescence signal detection can be achieved in the device, and 4 reaction samples can be detected simultaneously to improve detection efficiency. The test results can also be visualized by the fluorescence intensity detection software in a smart phone, so that even ordinary agricultural practitioners without professional training can quickly get started and use it.

[0096] This application enables rapid testing of a variety of interchangeable samples using a mobile terminal. A laser beam emitted by a laser generator excites fluorescence at the bottom of an experimental centrifuge tube through an optical fiber. This allows for convenient and rapid acquisition of functional group-specific fluorescence signals with minimal dissipation. Simultaneously, developed fluorescence detection software is used to rapidly acquire the sample's fluorescence intensity, thereby providing biochemical information.

[0097] Existing portable conversion fluorescence detectors: use image recognition technology to detect different types of samples in the sample chamber module, and use a wireless module to transmit specific light spots to the PC or mobile terminal for analysis. The disadvantage of this device is that the internal optical path design is relatively complex, and an additional wireless transmission module is still required to transmit the light spot, which is then detected on the PC or mobile terminal, which takes a long time. The present application uses optical fiber to conduct the excitation light beam, avoiding the complex design of the optical path and the influence of external stray light. At the same time, a smartphone-based fluorescence detection software has been developed to perform image processing and fluorescence signal analysis on the fluorescence signal pictures directly collected by the mobile phone camera, so that users can perform instant testing operations based on biosensors under field conditions, and the diagnostic accuracy and efficiency are relatively high. The image is taken by the mobile phone camera and then analyzed by the built-in fluorescence detection software without the need for wireless transmission, which reduces processing time and delays.

[0098] In an exemplary embodiment, a portable fluorescence detection method is provided for use with the portable fluorescence detection device described above. The portable fluorescence detection method includes:

[0099] Acquire an image of the fluorescence signal generated by the sample at the current moment;

[0100] Perform target area recognition on the image of the fluorescence signal generated by the sample at the current moment to obtain a fluorescence image of the target area;

[0101] Converting the fluorescent image of the target area into a YUV color space to obtain a converted image;

[0102] The fluorescence intensity of the fluorescence signal at the current moment is calculated according to the converted image.

[0103] As an optional embodiment, the image of the fluorescent signal generated by the microbial sample to be detected includes the image of the fluorescent signal generated by the microbial sample to be detected in all experimental centrifuge tubes; a mixed reaction substrate is set in each experimental centrifuge tube; the mixed reaction substrate includes the microbial sample to be detected, the RPA-CRISPR / Cas12a reaction system reagent and the FAM fluorescent group, and the target area is identified on the image of the fluorescent signal generated by the microbial sample to be detected at the current moment to obtain a fluorescent image of the target area, specifically including:

[0104] Performing enhancement preprocessing on the image of the fluorescence signal generated by the microbial sample to be detected at the current moment to obtain a preprocessed image;

[0105] Connected region detection is performed on the preprocessed image to obtain a set of highlighted fluorescent point coordinates at the current moment. The image of the fluorescence signal generated by the microbial sample to be tested in one experimental centrifuge tube contains a highlighted fluorescent point, and the highlighted fluorescent point coordinate set includes the coordinates of all highlighted fluorescent points in the fluorescence signal images generated by the microbial samples to be tested in all experimental centrifuge tubes. A highlighted fluorescent point is the brightest fluorescent point, which has a higher fluorescence intensity and is therefore more convenient for target region extraction, image rotation, and correction.

[0106] Using each highlighted fluorescent point in the current highlight fluorescent point coordinate set as a reference point, the image of the fluorescent signal generated by the microbial sample to be detected at the current moment is rotated and corrected to obtain a corrected image;

[0107] Determine each target area in the correction image according to the set of coordinates of the highlighted fluorescent points at the current moment; a target area contains a highlighted fluorescent point in the set of coordinates of the highlighted fluorescent points;

[0108] A fluorescence image of the target area is obtained according to each target area in the correction image.

[0109] As an optional implementation, connected region detection is performed on the preprocessed image to obtain a set of highlighted fluorescent point coordinates at the current moment, specifically including:

[0110] Performing connected region detection on the preprocessed image to obtain an initial set of highlighted fluorescent point coordinates;

[0111] If the number of highlighted fluorescent points in the initial highlighted fluorescent point coordinate set is less than the number of experimental centrifuge tubes, the target fluorescent point coordinates in the image of the fluorescent signal generated by the microbial sample to be detected at the historical moment are obtained; the target fluorescent point is a highlighted fluorescent point that is not detected in the preprocessed image.

[0112] Determine that the target fluorescent point coordinates in the image of the fluorescent signal generated by the microorganism sample to be detected at the current moment and the initial highlight fluorescent point coordinate set constitute the highlight fluorescent point coordinate set at the current moment.

[0113] As an optional implementation manner, after calculating the fluorescence intensity of the fluorescence signal at the current moment according to the converted image, the method further includes:

[0114] The relative intensity is obtained according to the fluorescence intensity of the fluorescence signal at the initial moment and the fluorescence intensity of the fluorescence signal at the current moment.

[0115] The fluorescence intensity of the fluorescence signal at the initial moment is used to normalize the fluorescence intensity of the fluorescence signal at the current moment to obtain the normalized fluorescence intensity at the current moment.

[0116] It should be noted that the user information (including but not limited to user device information, user personal information, etc.) and data (including but not limited to data used for analysis, stored data, displayed data, etc.) involved in this application are all information and data authorized by the user or fully authorized by all parties, and the collection, use and processing of relevant data must comply with relevant regulations.

[0117] The technical features of the above embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0118] This document uses specific examples to illustrate the principles and implementation methods of this application. The description of the above examples is only intended to help understand the method and core concept of this application. At the same time, for those skilled in the art, based on the concept of this application, there may be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting this application.

Claims

1. A portable fluorescence detection device, characterized in that: The portable fluorescence detection device comprises: An excitation light source module, a sample reaction module and a mobile terminal; the excitation light source module is used to emit laser light to the sample reaction module, so that the microbial sample to be detected generates a fluorescent signal; the mobile terminal is used to obtain an image of the fluorescent signal generated by the microbial sample to be detected, and determine the fluorescence intensity of the fluorescent signal based on the image of the fluorescent signal generated by the microbial sample to be detected.

2. The portable fluorescence detection device according to claim 1, characterized in that: The sample reaction module includes: multiple experimental centrifuge tubes; a mixed reaction substrate is set in each experimental centrifuge tube; the mixed reaction substrate includes a microbial sample to be detected, an RPA-CRISPR / Cas12a reaction system reagent and a FAM fluorescent group; the excitation light source module is used to emit laser light to the mixed reaction substrate in each experimental centrifuge tube, so that the microbial sample to be detected produces a fluorescent signal.

3. The portable fluorescence detection device according to claim 2, characterized in that: The portable fluorescence detection device further comprises a temperature control module, which is used to control the temperature of the mixed reaction substrate in each experimental centrifuge tube so as to perform isothermal amplification of nucleic acid on the microbial sample to be detected in each experimental centrifuge tube.

4. The portable fluorescence detection device according to claim 2, characterized in that: The excitation light source module includes a laser and an optical fiber; the optical fiber is used to irradiate the laser light emitted by the laser into the mixed reaction substrate in each experimental centrifuge tube.

5. The portable fluorescence detection device according to claim 3, characterized in that: The temperature control module includes: a reference centrifuge tube, a heating pad, a temperature probe and a temperature controller; double distilled water is set in the reference centrifuge tube; the reference centrifuge tube and the experimental centrifuge tube are both placed on the heating pad, and the heating pad is used to heat the mixed reaction substrate in each experimental centrifuge tube and the double distilled water in the reference centrifuge tube; the temperature probe is set in the reference centrifuge tube, and the temperature probe is used to measure the temperature of the double distilled water in the reference centrifuge tube; the temperature controller is connected to the temperature probe and the heating pad, and the temperature controller is used to control the heating pad according to the temperature measured by the temperature probe, so that the temperature of the mixed reaction substrate in each experimental centrifuge tube reaches a preset temperature.

6. The portable fluorescence detection device according to claim 1, characterized in that: The mobile terminal is a smart phone; the smart phone includes a camera and a chip; the chip has a built-in fluorescence intensity detection module; a filter is arranged between the camera and the sample reaction module, and the camera is used to obtain an image of the fluorescence signal generated by the microbial sample to be detected through the filter; the fluorescence intensity detection module is used to determine the fluorescence intensity of the fluorescence signal based on the image of the fluorescence signal generated by the microbial sample to be detected.

7. A portable fluorescence detection method, characterized in that: The portable fluorescence detection device according to any one of claims 1 to 6, wherein the portable fluorescence detection method comprises: Acquire an image of the fluorescence signal generated by the microbial sample to be detected at the current moment; Performing target area recognition on the image of the fluorescence signal generated by the microbial sample to be detected at the current moment to obtain a fluorescence image of the target area; Converting the fluorescent image of the target area into a YUV color space to obtain a converted image; The fluorescence intensity of the fluorescence signal at the current moment is calculated according to the converted image.

8. The portable fluorescence detection method according to claim 7, characterized in that: The image of the fluorescent signal generated by the microbial sample to be detected includes the image of the fluorescent signal generated by the microbial sample to be detected in all experimental centrifuge tubes; a mixed reaction substrate is set in each experimental centrifuge tube; the mixed reaction substrate includes the microbial sample to be detected, the RPA-CRISPR / Cas12a reaction system reagent and the FAM fluorescent group, and the target area is identified in the image of the fluorescent signal generated by the microbial sample to be detected at the current moment to obtain a fluorescent image of the target area, specifically including: Performing enhancement preprocessing on the image of the fluorescence signal generated by the microbial sample to be detected at the current moment to obtain a preprocessed image; Performing connected region detection on the preprocessed image to obtain a set of highlighted fluorescent point coordinates at the current moment; wherein an image of a fluorescent signal generated by a microbial sample to be detected in one experimental centrifuge tube has a highlighted fluorescent point, and the set of highlighted fluorescent point coordinates includes the coordinates of the highlighted fluorescent points in the images of the fluorescent signals generated by the microbial samples to be detected in all experimental centrifuge tubes; Using each highlighted fluorescent point in the current highlight fluorescent point coordinate set as a reference point, the image of the fluorescent signal generated by the microbial sample to be detected at the current moment is rotated and corrected to obtain a corrected image; Determine each target area in the correction image according to the set of coordinates of the highlighted fluorescent points at the current moment; a target area contains a highlighted fluorescent point in the set of coordinates of the highlighted fluorescent points; A fluorescence image of the target area is obtained according to each target area in the correction image.

9. The portable fluorescence detection method according to claim 8, characterized in that: Connected region detection is performed on the preprocessed image to obtain a set of coordinates of the highlighted fluorescent points at the current moment, specifically including: Performing connected region detection on the preprocessed image to obtain an initial set of highlighted fluorescent point coordinates; If the number of highlighted fluorescent points in the initial set of highlighted fluorescent point coordinates is less than the number of experimental centrifuge tubes, the target fluorescent point coordinates in the image of the fluorescent signal generated by the microbial sample to be detected at the current moment are obtained based on the target fluorescent point coordinates in the image of the fluorescent signal generated by the microbial sample to be detected at the previous moment; the target fluorescent point is a highlighted fluorescent point that is not detected in the preprocessed image; Determine that the target fluorescent point coordinates in the image of the fluorescent signal generated by the microorganism sample to be detected at the current moment and the initial highlight fluorescent point coordinate set constitute the highlight fluorescent point coordinate set at the current moment.

10. The portable fluorescence detection method according to claim 7, characterized in that: After calculating the fluorescence intensity of the fluorescence signal at the current moment according to the converted image, the method further includes: Obtaining a relative intensity according to the fluorescence intensity of the fluorescence signal at the initial moment and the fluorescence intensity of the fluorescence signal at the current moment; The fluorescence intensity of the fluorescence signal at the initial moment is used to normalize the fluorescence intensity of the fluorescence signal at the current moment to obtain the normalized fluorescence intensity at the current moment.

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