A raw milk somatic cell counter and its counting method

Abstract

The present invention provides a milk somatic cell counter and counting method, belonging to the field of analytical detection technology. To use the counter, simply place a test card containing a stained milk sample into a defined position on the instrument. The counter then automatically completes the test and obtains the test results. The counter is simple to operate, portable, and provides accurate and reliable counting results. The counter also allows for direct observation of milk somatic cell morphology through fluorescence imaging, addressing existing gaps in the application of milk somatic cell detection.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a milk somatic cell counter and a counting method thereof. Background Art

[0002] Somatic cells in raw milk typically consist of macrophages, lymphocytes, polymorphonuclear neutrophils, and a small number of mammary epithelial cells. The total somatic cell count (SSC) per milliliter of raw milk is a measure of its quality. For example, in bovine raw milk, under normal conditions, there are approximately 20,000 to 200,000 somatic cells per milliliter. However, when the lactating system is infected and damaged by bacteria, the number of somatic cells can increase significantly, reaching 500,000 to 1,000,000 per milliliter, or even higher. The concentration of somatic cells is also closely related to livestock management and food safety. In animal husbandry, it directly reflects the health of the cow's udder and its potential milk production; in food safety, it is closely related to the composition, quality, and flavor of the milk. Therefore, rapid and accurate somatic cell counts in raw milk are crucial for livestock health management and milk quality monitoring.

[0003] Existing technologies for measuring total somatic cell counts primarily target bovine somatic cells and include both indirect and direct methods. Indirect methods primarily detect changes in the physical and chemical properties of milk caused by an increase in somatic cells, indirectly reflecting the total somatic cell count; direct methods directly detect and count cells. Common indirect methods include the California Cell Count (CMT), the Wisconsin Mastitis Test (WMT), and viscometry. These methods utilize surfactants to release DNA from cells, then measure DNA aggregates, micelles, and viscosity. The primary drawback of these methods is that the physical and chemical characteristics described are sub-optimal, leading to significant errors in milk that is not fresh, has a high fat content, or experiences significant temperature fluctuations. The most classic direct method is manual microscopy, but due to its heavy reliance on manual labor, it has been gradually replaced by flow cytometry. Flow cytometry involves diluting and driving pre-fluorescently stained cells in a sheath fluid, allowing them to be accurately counted one by one through an optical detection system. As the most advanced cell detection technology currently, it has fast detection speed and high accuracy, but it also has many defects, such as complex operation procedures, high unit price and usage cost of equipment, and the inability to observe cell morphology. Summary of the Invention

[0004] In view of one or more problems existing in the prior art, one aspect of the present invention provides a milk somatic cell counter, comprising:

[0005] case;

[0006] a main control board, which is disposed in the housing;

[0007] a photoelectric detection unit, which is disposed in the housing and electrically connected to the main control board, and includes a first photoelectric detection module and a second photoelectric detection module, each of which includes an image sensor for capturing images;

[0008] a data processing unit, integrated into the main control board, for processing and analyzing images captured by the image sensor;

[0009] a human-computer interaction unit, electrically connected to the main control board; and

[0010] The detection card can be installed in a card insertion slot provided on the housing for installing the detection card, and comprises a first detection piece and a second detection piece, which are respectively adapted to the first photoelectric detection module and the second photoelectric detection module.

[0011] The above-mentioned test card also includes a card tray, the first test piece and the second test piece are arranged on the card tray, the test piece is a slit microfluidic chip structure, including a base and a sample injection detection cavity provided on the base, the sample injection detection cavity is a semi-open cavity formed by two parallel chip side walls with a certain gap, and has one or more analysis areas, a sample injection port, and a drainage groove area connecting the sample injection port and the analysis area. The thickness of the analysis area is H 分 Less than the thickness H of the drainage groove area 引 , further, superimpose any one or more of the following technical means:

[0012] Method 1: The injection port is located at the upper edge opening of the two chip side walls of the injection detection cavity, wherein the upper edge of one of the two chamber side walls located at the injection port is provided with an injection notch, so that the raw milk somatic cell sample can be injected through the injection notch;

[0013] Method 2: A notch is provided at the injection port at one closed end of the injection detection cavity as a venting groove, and the bottom of the venting groove is not lower than the innermost end of the notch of the injection notch.

[0014] Means three: At least one exhaust hole is provided on the sampling detection chamber, and the exhaust hole is a through hole connecting the interior of the sampling detection chamber with the outside atmosphere, which passes through one side wall of the analysis area or the drainage trough area or symmetrically passes through the two side walls of the analysis area or the drainage trough area; preferably, the exhaust hole is an inverted cone-shaped through hole, with the small end of the cone opening facing the interior of the analysis area or the drainage trough area, and the large end of the cone opening facing the outside atmosphere.

[0015] The total area of the analysis area on one side accounts for 50%-90% of the total area of the injection detection cavity on one side, and the thickness of the analysis area is less than the thickness of the drainage groove area H 引 , the thickness of the analysis area H 分 The range is 50μm-400μm, and the thickness of the drainage groove area H 引The range is 120 μm to 500 μm; the analysis area is a region of a single thickness, or two spatially independent but connected partitions of equal or unequal thickness.

[0016] The injection port is in a concave arc shape, and the angle α between the tangent of the downward arc and the horizontal reference plane of the injection port is in the range of 15° to 85°, preferably 45° to 85°, and more preferably 80°.

[0017] The card holder of the above-mentioned test card is also provided with a receiving cavity for storing reagents and serving as a mixing container for reagents and raw milk samples; preferably, the receiving cavity is pre-packaged with reagents; the reagents include cell fluorescent dyes, wherein the cell fluorescent dyes are one or more of SYTO9 dye, propidium iodide, ethidium bromide, acridine orange, Hoechst dye, DAPI dye, Cy3, and Cy5.

[0018] The first photoelectric detection module and the second photoelectric detection module also include:

[0019] a light source for emitting light;

[0020] a first optical filter, configured to transmit light of a fixed wavelength range in the light emitted by the light source;

[0021] A reflector, used to reflect the fluorescence emitted by the test card onto the imaging lens;

[0022] An imaging lens for imaging the fluorescence emitted by the test card; and

[0023] The second filter is used to transmit light of a fixed wavelength range in the imaging fluorescence.

[0024] The first filter is arranged between the detection card and the light source, the detection card is arranged between the first filter and the reflector, the imaging lens is arranged at a position where it can receive the fluorescence reflected by the reflector, and the second filter is arranged between the image sensor and the imaging lens.

[0025] The above-mentioned counter also includes:

[0026] a printer, which is disposed in the housing and electrically connected to the human-computer interaction unit via the main control board, and is used to print out the results of the image processing and analysis performed by the data processing unit; and / or

[0027] The human-computer interaction unit includes a display screen, function keys and a switch button; and / or

[0028] The counter further comprises one or more of a card inserting base plate, a silicone pad, a USB interface, a power interface, a heat dissipation port, a battery compartment and a heat dissipation fan, wherein the card inserting base plate is used for placing the detection card.

[0029] Another aspect of the present invention provides a method for counting somatic cells in milk, comprising the following steps:

[0030] 1) Sample production

[0031] Add freshly sampled raw milk sample or diluted raw milk sample into the accommodating cavity of the test card to react with the reagent, and take the reaction solution and add it into the sample injection detection cavity of the two detection pieces of the test card;

[0032] 2) Imaging detection

[0033] Insert the test card containing the reaction solution into the card slot of the raw milk somatic cell counter, start the detection program to perform imaging detection, and use the image sensors of the first photoelectric detection module and the second photoelectric detection module to respectively collect fluorescence images of the sample in the corresponding analysis area;

[0034] 3) Image processing

[0035] The data processing unit processes the fluorescence image collected by the image sensor to obtain the detection result;

[0036] 4) Result output

[0037] The detection results after image processing are displayed and output in the manual interaction unit and / or printed out through a printer.

[0038] The specific method of imaging detection in the above step 2) is:

[0039] For the first photoelectric detection module: the main control board drives the light source to light up. The light emitted by the light source first passes through the first filter, exciting the analysis area on the detection card to emit fluorescence. The fluorescence is reflected by the reflector and polarized 90 degrees. It is then focused by the imaging lens and the second filter and imaged onto the image sensor to obtain a fluorescence image.

[0040] For the second photoelectric detection module: the method is the same as the imaging detection method of the first photoelectric detection module;

[0041] The first photoelectric detection module and the second photoelectric detection module collect fluorescence images synchronously or sequentially.

[0042] The specific method of image processing in step 3) above is:

[0043] For the fluorescence image collected by the first photoelectric detection module: before processing, the magnification of the imaging light path is pre-calibrated with a graticule; then, based on the image, the number of fluorescent bright spots within the sample detection range is calculated, and then the cavity area of the detection card analysis area corresponding to the detection range is calculated based on the magnification. Based on the cavity thickness of the detection card analysis area, the solution volume corresponding to the detection range is calculated, and the milk somatic cell concentration MilkNum1 is obtained using the following calculation formula:

[0044]

[0045] Where: MilkNum is the total somatic cell concentration of raw milk; N is the number of fluorescent bright spots; a is the dilution ratio of the raw milk sample; S is the cavity area of the analysis area of the test card corresponding to the detection range, unit is mm 2 ; d is the chamber thickness, unit: μm;

[0046] For the fluorescence image collected by the second photoelectric detection module: the processing method is the same as the processing method of the fluorescence image collected by the first photoelectric detection module to obtain its milk somatic cell concentration MilkNum2;

[0047] The final somatic cell concentration of the raw milk sample is (MilkNum1+MilkNum2) / 2.

[0048] In the above step 1), the freshly sampled raw milk sample is diluted 2-20 times with a buffer solution containing PO4 3- 、Cl - 、CO3 2- One or more of Na + , K + an aqueous solution of one or more of .

[0049] The raw milk somatic cell counter and counting method provided by the above technical solution have the following beneficial effects compared with the existing technology:

[0050] 1) The counter uses a test card that can be pre-fixed with staining reagents such as cell fluorescent dyes. When in use, the raw milk sample only needs to be mixed on the test card. During testing, the test card containing the raw milk sample stained with the cell fluorescent dye only needs to be placed in a designated position on the instrument, and the counter will automatically complete the test and obtain the test results. It is easy to operate, and within the concentration range of 5,000 cells / ml to 4,717,000 cells / ml of raw milk somatic cells tested, the consistency with the detection results of the flow cytometer is as high as 0.9776, and the counting results are accurate and reliable.

[0051] 2) Due to the low concentration of somatic cells in raw milk, using a single detection plate to count too few cells would result in large errors in the test results, making it difficult to meet practical requirements. The detection card of the counter of the present invention is provided with two detection plates, each with a unique structural design and a separate analysis area. Correspondingly, the counter is also provided with two sets of photoelectric detection modules, which can expand the analysis area of raw milk somatic cells and improve the accuracy of detection.

[0052] 3) The counter can directly observe the morphology of lactogenic somatic cells through fluorescence images, filling the gap in the existing application field of lactogenic somatic cell detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 A schematic diagram of the system structure of the milk somatic cell counter provided by the present invention;

[0054] Figure 2 A schematic diagram of the internal structure of the milk somatic cell counter provided by the present invention;

[0055] Figure 3 Panels A and B in the middle are schematic diagrams of the external structure of the milk somatic cell counter provided by the present invention;

[0056] Figure 4 A schematic structural diagram of a detection card for a milk somatic cell counter provided by the present invention;

[0057] Figure 5A Schematic diagram of the front structure of the test piece;

[0058] Figure 5B Schematic diagram of the side structure of the detection piece;

[0059] Figure 5C Schematic diagram of the three-dimensional structure of the detection piece;

[0060] Figure 5D for Figure 5A Schematic diagram of the cross-section structure taken along line AA;

[0061] Figure 6 This is a schematic structural diagram of the photoelectric detection unit of the milk somatic cell counter provided by the present invention;

[0062] Figure 7 The detection sample image of Example 3;

[0063] Figure 8 This is the accuracy analysis result of the milk somatic cell counter provided by the present invention. DETAILED DESCRIPTION

[0064] In view of the defects of the existing technology in detecting lactogenic somatic cells, such as the complex operating procedures and the inability to directly observe the morphology of lactogenic somatic cells, the purpose of the present invention is to provide a lactogenic somatic cell counter that is simple to operate and can directly observe the morphology of lactogenic somatic cells.

[0065] like Figure 1Figure 1 shows a schematic diagram of the system architecture of the lactogenic somatic cell counter provided by the present invention, which includes a test card, a photoelectric detection unit, a data processing unit, and a human-computer interface unit. The test card serves as a carrier for quantitative lactogenic somatic cell detection samples and has two built-in detection plates to increase the sample volume and analysis area, thereby improving detection accuracy and precision. The photoelectric detection unit is composed of two sets of parallel photoelectric detection modules, including a first photoelectric detection module and a second photoelectric detection module, which are used to synchronously detect the analysis areas of the two parallel detection plates on the test card, thereby avoiding the need for a mobile mechanism. Each set of photoelectric detection modules includes a light source, an imaging optical path, and an image sensor, wherein the image sensor is used to capture a fluorescent image of the corresponding analysis area. The data processing unit primarily processes and analyzes the fluorescent images captured by the image sensor, and obtains the total lactogenic somatic cell count by counting all fluorescent bright spots in the fluorescent image. The human-computer interface unit primarily includes a display screen (which may be a touch screen), function keys, a switch button, and a data interface, which complete the detection process control and result output. The photoelectric detection unit, data processing unit, and human-computer interface unit are electrically connected to each other via a main control board.

[0066] like Figure 2 The figure shows a schematic diagram of the main internal structure of the raw milk somatic cell counter provided by the present invention, including: a main control board 101, a photoelectric detection unit 102, a card insertion base plate 103, a heat dissipation fan 104 and a battery compartment 105 arranged in the instrument housing 100. The data processing unit is integrated on the main control board 101, and the photoelectric detection unit 102 includes two photoelectric detection modules, both of which are electrically connected to the main control board 101; the card insertion base plate 103 constitutes a limited position for the detection card, for placing the detection card; the heat dissipation fan 104 is used to reduce the ambient temperature of the counter during operation; and the battery compartment 105 is used to place the power supply to provide power for the counter. Figure 3 Middle A and Figure 3As shown in panel B, a schematic diagram of the external structure of the raw milk somatic cell counter provided by the present invention is shown, including: a housing 100, a display screen 106 (which can be a touch screen including function keys), a switch button 107, a card slot 108, a silicone pad 109, a printer 110, a heat dissipation vent 111, a USB interface 112 and a power interface 113. The display screen 106 is arranged on the shell 100, which can be used to display the final test results and is electrically connected to the main control board 101; the card insertion port 108 is arranged on the shell 100, corresponding to the card insertion base plate 103, and the test card is inserted into the limited position on the card insertion base plate 103 in the instrument through the card insertion port 108 for testing; the silicone pad 109 can play a shock-proof effect on the instrument to help protect the counter; the printer 110 is arranged in the shell 100, and can be electrically connected to the human-computer interaction unit through the main control board 101, and can be used to print out the test results; the heat dissipation port 111 corresponds to the installation of the cooling fan 104, which is used to reduce the ambient temperature of the counter when it is working; the USB interface 112 can be used to transmit data, and the power interface 113 is connected to the battery compartment 105 to provide a power source in the battery compartment 105.

[0067] like Figure 4Figure 4 shows a test card 4 for a lactogenic somatic cell counter provided by the present invention. The card includes a tray 4c and two parallel test strips mounted on the tray 4c: a test strip 4a and a test strip 4b. The front ends of the two test strips protruding from the tray 4c comprise a lactogenic somatic cell analysis area. To facilitate pre-processing of lactogenic somatic cell samples, a receiving cavity 4d is provided on the tray 4c. The receiving cavity 4d is cylindrical, frustum-conical, or hemispherical, with a volume of 30 μL to 100 μL and a depth of 5 mm to 14 mm. Reagents reactive with lactogenic somatic cell samples can be pre-fixed within the receiving cavity 4d. The reagents can be freeze-dried or dried to form a cell fluorescent dye. The reagents can be selected from one or more fluorescent dyes such as SYTO9, propidium iodide, ethidium bromide, acridine orange, Hoechst, DAPI, Cy3, and Cy5. The raw milk sample to be introduced into the analysis area of the test strip can be pre-reacted with the reagents in the accommodating cavity 4d to stain the raw milk somatic cells. The card holder 4c is also provided with at least two engaging portions 4e for engaging the test strips 4a and 4b. The test card 4 provided by the present invention is provided with two identical test strips 4a and 4b. This allows for accurate verification of the test results of each test strip (i.e., the two test strips are tested sequentially, and the difference between the two test results is considered valid if it does not exceed a preset threshold, with the sum or mean of the two test results being taken as the final test result). Furthermore, due to the weak fluorescence of raw milk somatic cells, the test results of multiple test strips can be superimposed to ensure accuracy. To facilitate assembly and engagement with the engaging portion 4e of the card holder 4c, the test strips 4a and 4b are further provided with internal positioning grooves 4f. Accordingly, the card holder 4c is provided with protrusions that mate with the internal positioning grooves 4f to ensure accurate assembly and engagement of the test strips on the card holder 4c. In order to facilitate the use of the detection card 4 in the raw milk somatic cell counter, the detection card 4 is further provided with an external positioning groove 4g. The external positioning groove 4g is arranged on the bottom surface of the tail end of the card holder 4c and is used to position the detection card 4 on the card insertion base plate 103.

[0068] like Figures 5A-5D As shown, a schematic diagram of the structure of the detection pieces 4a and 4b is shown, wherein Figure 5A The diagram shows the front structure of the detection piece. Figure 5B The figure shows a side structural diagram of the detection piece. Figure 5C What is shown is a schematic diagram of the three-dimensional structure of the detection piece, Figure 5D Shown is Figure 5AThe cross-sectional structure diagram taken along line AA in FIG. The detection chip provided by the present invention is a slit microfluidic chip structure, comprising a base 21 and a sample injection detection cavity 22 extending from the front end of the base 21 with an arc-shaped edge; the sample injection detection cavity 22 is a semi-open cavity formed by two parallel chip sidewalls 23 with a certain gap, including a sample injection port 25, a sample injection notch 28, an analysis area 24, a vent 26, and a drainage channel area 27 connecting the sample injection port 25 and the analysis area 24, wherein:

[0069] like Figures 5A-5C As shown, the sampling port 25 is located at the upper edge opening of the two chip side walls 23 of the sampling and detection chamber 22. The upper edges of the two chip side walls 23 at the sampling port 25 are provided with a sampling notch 28, which facilitates positioning of the raw milk sample pipette. This allows injection of the sample through the sampling notch 28. Under the combined action of the injection force of the pipette and capillary force, the raw milk somatic cell sample smoothly enters the sampling and detection chamber 22 through the flow path formed by the drainage groove area 27 (the detailed structure of which is described below) and then enters the analysis area 24. To prevent large bubbles in the raw milk somatic cell sample from entering the sampling and detection chamber 22, a notch is provided on the side of the sampling port 25 at the closed end of the sampling and detection chamber 22, near the base 21 (the unopened side). This notch serves as a vent 29. The thickness of the vent 29 is consistent with that of the drainage groove area 27, and the bottom of the vent 29 does not extend beyond the innermost end of the notch of the sampling notch 28. When the raw milk sample in the pipette is injected from the sampling notch 28 , larger bubbles in the raw milk sample escape from the venting groove 29 on one side of the sampling inlet 25 , thereby preventing the bubbles from entering the sampling detection chamber 22 .

[0070] like Figure 5A and 5C As shown, the injection port 25 is concave arc-shaped, and its downward arc ( Figure 5A The angle α between the tangent of the sample (the left arc in the figure) and the horizontal reference plane of the sample inlet 25 determines the direction of flow of the raw milk sample into the drainage trough area 27. This ensures that the raw milk sample injected into the gap 28 flows spontaneously into the analysis area 24 in a predetermined manner and fills the analysis area 24. Angle α ranges from 15° to 85°. Given the high fat and protein content and high viscosity of raw milk samples, angle α is preferably selected from 45° to 85°, with 80° being the most preferred. A larger angle α results in a faster flow rate for the injected sample. However, the faster the flow rate, the greater the chance of deformation of the front surface and the generation of bubbles. Therefore, an angle of 80° is preferred.

[0071] like Figure 5A and Figure 5C As shown, the analysis area 24 is located in the sample injection detection cavity 22. The shape of the analysis area 24 can be a rectangle, a square, a trapezoid, a circle, or a combination of arcs and other shapes, and each shape can have rounded corners, right angles, or a combination of rounded corners and right angles. The present invention does not limit the specific shape of the analysis area 24; the analysis area 24 can have a single thickness H分 The raw milk sample enters the analysis zone 24, forming a detection surface with a thickness generally ranging from 50μm to 400μm. The analysis zone 24 can also be divided into multiple interconnected zones of equal or different thicknesses. For example, two interconnected zones of equal or different thicknesses can be used for comparative analysis of raw milk somatic cell samples on the same test strip. The total area of the analysis zone 24 on one side accounts for 50% to 90% of the total area of the sample injection and detection cavity 22 on one side. This large area ratio of the analysis zone 24 can simultaneously increase the raw milk cell carrying capacity and spreading area, balancing accurate overall cell counting with precise single-cell analysis. Furthermore, the chip sidewalls 23 in the analysis zone 24 are relatively thick, making deformation of the larger area of the chip sidewall 23 less likely, thereby ensuring a uniform thickness in the analysis zone 24.

[0072] like Figure 5A and Figure 5D As shown, the drainage groove area 27 is located in the sample injection detection cavity 22 and is connected to the sample injection port 25 and the analysis area 24. The thickness H of the drainage groove area 27 is 引 The range is generally 120 μm to 500 μm. The thickness H of the analysis area 24 分 Less than the thickness H of the drainage groove area 7 引 The milk somatic cell sample enters the flow path formed by the drainage groove area 27 from the sample inlet 25 and can be evenly and quickly introduced into the analysis area 24 and filled. The thickness of the analysis area 24 is H 分 Less than the thickness H of the drainage groove area 27 引 , which is conducive to the discharge of bubbles. To ensure that the milk somatic cell sample can flow continuously from the drainage groove area 27 into the analysis area 24 under the action of capillary force and fill it, the capillary pressure is required to be greater than zero. Capillary force and the thickness H of the analysis area 24 分 , the thickness H of the drainage groove area 27 引 , the surface tension of the liquid being tested, and the contact angle of the liquid being tested on the surface of the material of the drainage groove area 27 have the following relationship:

[0073]

[0074] From the above formula, we can know that by designing the thickness H of the drainage groove area 27 引 and the thickness H of the analysis region 24 分 , it is possible to adjust different liquid flow rates and liquid laminar flow characteristics, thereby avoiding the formation of bubbles.

[0075] like Figure 5A and Figure 5DAs shown, to further prevent the generation of bubbles, at least one exhaust hole 26 is provided on the sampling and detection chamber 22. The exhaust hole 26 is a through hole connecting the interior of the sampling and detection chamber 22 with the outside atmosphere. The exhaust hole 26 can be located on one side or both sides of the analysis area 24 or the drainage trough area 27. It can be a symmetrical or asymmetrical through hole, that is, the exhaust hole 26 passes through one side wall of the analysis area 24 or the drainage trough area 27 or symmetrically passes through both side walls of the analysis area 24 or the drainage trough area 27. Preferably, the exhaust hole 26 is a symmetrical inverted conical through hole, that is, the small end of the cone opens toward the interior of the drainage trough area 27, and the large end of the cone opens toward the outside atmosphere. The advantages of the inverted cone-shaped exhaust hole are that, first, it utilizes the surface tension between the raw milk sample to be tested and the gas to make it easier to discharge bubbles. The exhaust method based on the inverted cone hole proposed in the present invention is not affected by the sampling angle and distance, and can effectively discharge bubbles. When the reagent needs to be pre-packaged in the sampling detection chamber 22, since the contact area between the reagent and the external environment is very small, when the reagent is added to the sampling detection chamber 22, its drying process is long. The design of the exhaust hole 26 can increase the contact area between the reagent and the external environment, accelerate the drying and uniform distribution of the reagent, thereby avoiding the generation of bubbles in various situations; it helps to accurately control the sample amount. After the raw milk sample to be tested enters the exhaust hole 26, it is not easy to overflow due to the action of surface tension; the exhaust hole 26 is set on one side or both sides of the sampling detection chamber 22. After the sampling is completed, only the side of the chip needs to be wiped to avoid the loss of liquid sample due to wiping the sampling port; at the same time, the inverted cone hole structure further reduces the possibility of sample loss by wiping.

[0076] like Figure 5D As shown, to prevent the raw milk sample from flowing out due to gravity when the microfluidic chip is moved, the inner edges of the chip sidewalls 23 are provided with transition fillets 210. The radius R of the transition fillets ranges from 0.2mm to 1.5mm. When the microfluidic chip is loaded, the raw milk sample forms a stable liquid bridge surface 211 at the transition fillets 210 between the two chip sidewalls 23, effectively balancing the gravity of the liquid to be tested and preventing it from flowing out.

[0077] like Figure 6The figure shows a schematic structural diagram of the photoelectric detection unit of the raw milk somatic cell counter provided by the present invention. The photoelectric detection unit and the detection card 4 constitute the photoelectric detection device of the raw milk somatic cell counter. The photoelectric detection unit is composed of two groups of identical, symmetrically placed photoelectric detection modules, which can simultaneously perform imaging detection on the analysis areas of the detection plates 4a and 4b on the detection card 4, avoiding the use of a moving mechanism. The photoelectric detection unit mainly includes a light source board 1, a filter 2, a filter 3, a reflector 5, a reflector 9, an imaging lens 6, an imaging lens 10, a filter 7, a filter 11, an image sensor 8, and an image sensor 12. The light source board 1 is provided with two independent light sources, light source 1a and light source 1b. The light source 1a, filter 2, reflector 5, imaging lens 6, filter 7 and image sensor 8 constitute the first photoelectric detection module, and the light source 1b, filter 3, reflector 9, imaging lens 10, filter 11 and image sensor 12 constitute the second photoelectric detection module. These two groups of photoelectric detection modules have the same structure and perform detection on the analysis areas of the two detection sheets on the detection card 4 respectively. Taking the first photoelectric detection module as an example, its operating principle is as follows: light source 1a is an LED light source, located directly below one of the analysis areas of the detection card 4, at a distance of 10-30 mm. The main control board 101 drives light source 1a to emit light, which first passes through filter 2. The center wavelength of the transmission spectrum of filter 2 coincides with the center wavelength of the emission of light source 1a. Filter 2 is located between the detection card 4 and light source 1a. The signal light emitted by somatic cells in the raw milk sample under the illumination of light source 1a is reflected by reflector 5 and polarized 90°. It is then focused by imaging lens 6 and filter 7 and imaged onto image sensor 8 (image sensor 8 is an area array image sensor with a resolution greater than 3 million pixels). The image is then detected and imaged. The image captured by image sensor 8 is finally processed by the data processing unit to obtain the detection result. The operating principle of the second photoelectric detection module is similar to that of the first photoelectric detection module and will not be repeated here.

[0078] The data processing unit processes the fluorescence images captured by image sensors 8 and 12, counting the number of fluorescent bright spots in each corresponding fluorescence image. Each bright spot represents a lactogenic somatic cell, and the total lactogenic somatic cell concentration in each analysis area is obtained. The statistical results of the two analysis areas are then integrated to obtain the final total lactogenic somatic cell count. The morphology of the lactogenic somatic cells can also be directly observed based on the fluorescence images.

[0079] Based on the above milk somatic cell counter, the present invention also provides a milk somatic cell counting method, comprising the following steps:

[0080] 1) Sample production

[0081] Freshly sampled raw milk can be tested directly or with a buffer solution (e.g. containing PO4 3- 、Cl- 、CO3 2- , Na+, K+, etc.) before testing. Add 50μl-100μl of the raw milk sample to the test card's chamber and mix thoroughly by pipetting. After the reaction with the reaction reagents (including cell fluorescent dyes) in the chamber is complete, obtain the reaction solution. Pipette 40μl-90μl of the reaction solution and add it to the analysis areas of the two test pieces of the test card respectively, until the analysis areas are filled.

[0082] 2) Imaging detection

[0083] When performing sample imaging detection, the user clicks the detection function button, which will prompt the user to insert the test sample. Place the detection card containing the reaction solution obtained in step 1) into the defined position of the counter, and then click the confirmation button to start the detection program.

[0084] The main control board activates the light sources corresponding to detection chips 4a and 4b, and then controls the image sensors of the two sets of photoelectric detection modules to capture fluorescent images of the samples in the corresponding analysis areas. The first photoelectric detection module and the second photoelectric detection module capture fluorescent images synchronously or sequentially.

[0085] 3) Image processing

[0086] The data processing unit processes the fluorescence images captured by the image sensors of the two sets of photoelectric detection modules. Before processing, the magnification of the imaging optical path is pre-calibrated using a graticule. The number of characteristic points within the sample detection range (i.e., the number of fluorescent bright spots, representing the number of lactogenic somatic cells) is then calculated. The area of the analysis chamber of the detection card corresponding to the detection range is then calculated based on the magnification. The solution volume corresponding to the detection range is then calculated based on the known thickness (the thickness of the analysis chamber of the detection card). The lactogenic somatic cell concentrations (MilkNum1 and MilkNum2) based on the fluorescence images captured by the first and second photoelectric detection modules are calculated using the following formulas:

[0087]

[0088] Where: MilkNum is the total somatic cell concentration of raw milk; N is the number of characteristic points; a is the dilution ratio of the raw milk sample; S is the imaging analysis area (the area of the analysis area chamber of the test card corresponding to the detection range), unit: mm 2 ; d is the chamber thickness, unit: μm;

[0089] The total somatic cell count in the raw milk sample is (MilkNum1+MilkNum2) / 2.

[0090] 4) Result output

[0091] The image-processed test results are displayed on the screen. At the same time, the user can click the print button and the main control board controls the printer to print out the test results.

[0092] The following is a more detailed description of the present invention with reference to specific examples, and further elaboration of the present invention. However, these examples are by no means intended to limit the present invention.

[0093] Example 1:

[0094] This embodiment provides a raw milk somatic cell counter, comprising a housing, a main control board, a photoelectric detection unit, a data processing unit, a human-computer interaction unit, and a detection card. The housing, the main control board, the data processing unit, and the human-computer interaction unit are as described above in conjunction with the accompanying drawings and are not described in detail here.

[0095] The photoelectric detection unit includes a first photoelectric detection module and a second photoelectric detection module. The system principle is as follows: Figure 6 As shown, light sources 1a and 1b are LEDs with a central wavelength of 480 nm. Filter 2 has a central wavelength of 480 nm and a bandwidth of 30 nm. Under the illumination of the light sources, somatic cells within one analysis area of the test card 4 emit fluorescence. This fluorescence signal is reflected by a reflector 5 and deflected 90° before being imaged by an imaging lens 6 and a filter 7 onto an image sensor 8. Filter 7 is a filter with a central wavelength of 540 nm, with a transmittance greater than 80% for wavelengths between 520 nm and 550 nm and less than 0.01% for wavelengths less than 500 nm. Therefore, filter 7 can eliminate the effects of illumination from light sources 1a and 1b on the sample fluorescence imaging. Fluorescence signals from somatic cells within another analysis area of the test card 4 are reflected by a reflector 9 and deflected 90° before being imaged by an imaging lens 10 and a filter 11 onto an image sensor 12.

[0096] Example 2:

[0097] like Figure 4 and Figures 5A-5D As shown, this embodiment provides a test card 4 for use with the milk somatic cell counter provided in Example 1, comprising a card tray 4c and a receiving cavity 4d disposed on the card tray 4c, test strips 4a and 4b, a snap-fit portion 4e, and an external positioning groove 4g for positioning on the card insertion base 103. The test strips 4a and 4b are slit microfluidic chip structures, comprising a base 21 and a sample injection and detection cavity 22 extending from the front end of the base 21 with an arc-shaped edge. The sample injection and detection cavity 22 is a semi-open cavity formed by two parallel chip sidewalls 23 with a certain gap, and includes a sample injection port 25, a sample injection notch 28, a vent 29, an analysis area 24, a vent 26, and a drainage channel area 27 connecting the sample injection port 25 and the analysis area 24. The structures of the sample injection port 25, the sample injection notch 28, and the vent 29 are as described above and will not be repeated here.

[0098] In this embodiment, the total area of the analysis area 24 on one side accounts for 80% of the total area of the sample injection detection chamber 22 on one side. It has a uniform thickness of 300 μm. The drainage groove area 7 is located in the sample injection detection chamber 22 and is connected to the injection port 5 and the analysis area 4. Its thickness H 引 The angle α between the tangent of the downward arc of the injection port 5 and the horizontal reference plane of the injection port 5 is 80°. There are two exhaust holes 26, one located on either side of the drainage groove area 7 and extending through both side walls of the drainage groove area 7. The radius R of the transition fillet 210 of the inner edge of the chip sidewall 23 is 1.0 mm. Other structures are described above in conjunction with the accompanying drawings and will not be repeated here.

[0099] Example 3:

[0100] This example is based on the raw milk somatic cell counter provided in Example 1 and the detection card provided in Example 2 to detect fresh raw milk samples with a count of 5,000 cells / ml to 4,717,000 cells / ml. The total number of raw milk somatic cells is detected using fluorescent staining.

[0101] The method for counting somatic cells in milk in this embodiment comprises the following steps:

[0102] 1) Sample production

[0103] Freshly sampled raw milk can be tested directly or diluted 2-20 times with buffer (same as above) before testing. Add 50μl-100μl of the raw milk sample to the test card's holding chamber (pre-fixed with SYT09 dye and / or acridine orange, somatic cells are fluorescently stained with an excitation wavelength of 480nm and an emission wavelength of 530nm) and pipette to mix. After the reaction is complete, obtain the reaction solution and pipette 40μl-90μl of the reaction solution into the analysis area of the test card until the analysis area is full.

[0104] 2) Imaging detection

[0105] When performing sample imaging detection, the user clicks the detection function button, which will prompt the user to put in the test sample. Place the detection card containing the reaction solution obtained in step 1) into the defined position of the counter, and then click the confirmation button to start the detection program.

[0106] The main control board drives the corresponding light sources of the detection piece 4a and the detection piece 4b synchronously or sequentially, and then controls the corresponding image sensors of the two groups of photoelectric detection modules to respectively collect the fluorescence images of the samples in the corresponding analysis areas. Figure 7 Panels A and B in the middle show fluorescent images of raw milk samples detected by two groups of photoelectric detection modules in this embodiment, and each fluorescent bright spot in the image represents a somatic cell.

[0107] 3) Image processing

[0108] The data processing unit processes the fluorescence images captured by the image sensors of the two sets of photoelectric detection modules. Before processing, the magnification of the imaging optical path is pre-calibrated using a graticule. The number of characteristic points within the sample detection range (i.e., the number of fluorescent bright spots, representing the number of lactogenic somatic cells) is then calculated. The area of the analysis chamber of the detection card corresponding to the detection range is then calculated based on the magnification. Based on the known thickness (the thickness of the analysis chamber of the detection card), the volume of the solution corresponding to the detection range is calculated. The lactogenic somatic cell concentrations (MilkNum1 and MilkNum2) based on the fluorescence images captured by the first and second photoelectric detection modules are calculated using the following formulas:

[0109]

[0110] Where: MilkNum is the total somatic cell concentration of raw milk; N is the number of characteristic points; a is the dilution ratio of the raw milk sample; S is the imaging analysis area (the area of the analysis area chamber of the test card corresponding to the detection range), unit: mm 2 ; d is the chamber thickness, unit: μm;

[0111] The final total somatic cell count of the raw milk sample is: (MilkNum1+MilkNum2) / 2.

[0112] 4) Result output

[0113] The image-processed test results are displayed on the screen. At the same time, the user can click the print button and the main control board controls the printer to print out the test results.

[0114] Comparative Example 1:

[0115] This comparative example used a flow cytometer (Foss BacSomatic) to analyze fresh raw milk samples ranging from 5,000 cells / ml to 4,717,000 cells / ml. The specific procedure was as follows: 100 μL of raw milk sample was aspirated into a centrifuge tube, followed by 1 mL of the flow cytometer's processing reagent, and thoroughly vortexed to mix. Finally, the mixed sample was placed into the flow cytometer's sample port. The scattered light and fluorescence of the particles in the sample were individually detected to identify and count the raw milk somatic cells.

[0116] Based on the test results of Example 3 and Comparative Example 1 above, Figure 8As shown, the comparison results of the detection results obtained using the raw milk somatic cell counter provided by the present invention and the detection results obtained using a flow cytometer are shown. It can be seen that the raw milk somatic cell counter provided by the present invention has accurate quantitative capability between 5,000 cells / ml and 4,717,000 cells / ml, and has a consistency of 0.9776 with the flow cytometer. However, the raw milk somatic cell counter provided by the present invention has many advantages over the flow cytometer. It can not only directly observe the morphology of raw milk somatic cells through fluorescent images, but also is simple to operate, easy to carry, accurate in counting, and time-consuming, filling the gap in the existing application field of raw milk somatic cell detection.

[0117] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A milk somatic cell counter, characterized in that include: case; a main control board, which is disposed in the housing; a photoelectric detection unit, which is disposed in the housing and electrically connected to the main control board, and includes a first photoelectric detection module and a second photoelectric detection module, each of which includes an image sensor for capturing images; a data processing unit, integrated into the main control board, for processing and analyzing images captured by the image sensor; A human-computer interaction unit, electrically connected to the main control board; and A detection card, which can be installed in a card insertion slot provided on the housing for installing the detection card, includes a first detection piece and a second detection piece, which are respectively adapted to the first photoelectric detection module and the second photoelectric detection module; The test card also includes a tray, the first test piece and the second test piece are arranged on the tray, the test piece is a slit microfluidic chip structure, including a base and a sample injection detection cavity arranged on the base, the sample injection detection cavity is a semi-open cavity formed by two parallel chip side walls with a certain gap, and has one or more analysis areas, a sample injection port, and a drainage groove area connecting the sample injection port and the analysis area. The thickness of the analysis area is Smaller than the thickness of the drainage groove area , further, superimpose the following three technical means: Method 1: The injection port is located at the upper edge opening of the two chip side walls of the injection detection cavity, wherein the upper edge of one of the two chamber side walls located at the injection port is provided with an injection notch, so that the raw milk somatic cell sample can be injected through the injection notch; Method 2: A notch is provided at the closed end of the injection port of the injection detection chamber as a venting groove, and the bottom of the venting groove is not lower than the innermost end of the notch of the injection port; Means three: The sampling and detection chamber is provided with at least one exhaust hole, which is a through hole connecting the interior of the sampling and detection chamber with the outside atmosphere, and the exhaust hole penetrates one side wall of the analysis area or the drainage trough area or symmetrically penetrates both sides of the analysis area or the drainage trough area; the exhaust hole is an inverted conical through hole, with the small end of the cone opening facing the interior of the analysis area or the drainage trough area and the large end of the cone opening facing the outside atmosphere; The injection port is in a concave arc shape, and the angle α between the tangent of the downward arc and the horizontal reference plane of the injection port is in the range of 15°-85°.

2. The milk somatic cell counter according to claim 1, characterized in that The total area of the analysis zone on one side accounts for 50%-90% of the total area of the injection detection cavity on one side, and the thickness of the analysis zone is less than the thickness of the drainage groove zone. , the thickness of the analysis area The range is 50μm-400μm, the thickness of the drainage groove area The range is 120 μm-500 μm; the analysis area is a region of a single thickness, or two spatially independent but connected partitions of equal or unequal thickness.

3. The milk somatic cell counter according to claim 1, characterized in that The card holder is also provided with a receiving cavity for storing reagents and serving as a mixing container for reagents and raw milk samples; the receiving cavity is pre-packaged with reagents; the reagents include cell fluorescent dyes, wherein the cell fluorescent dyes are one or more of SYTO9 dye, propidium iodide, ethidium bromide, acridine orange, Hoechst dye, DAPI dye, Cy3, and Cy5.

4. The milk somatic cell counter according to claim 1, characterized in that The first photoelectric detection module and the second photoelectric detection module each further include: a light source for emitting light; a first optical filter, configured to transmit light of a fixed wavelength range in the light emitted by the light source; A reflector, used to reflect the fluorescence emitted by the test card onto the imaging lens; An imaging lens for imaging the fluorescence emitted by the test card; and a second filter for transmitting light of a fixed wavelength range in the imaging fluorescence; The first filter is arranged between the detection card and the light source, the detection card is arranged between the first filter and the reflector, the imaging lens is arranged at a position where it can receive the fluorescence reflected by the reflector, and the second filter is arranged between the image sensor and the imaging lens.

5. A method for counting milk somatic cells using the milk somatic cell counter according to any one of claims 1 to 4, characterized in that: The following steps are involved: 1) Sample production Add freshly sampled raw milk sample or diluted raw milk sample into the accommodating cavity of the test card to react with the reagent, and take the reaction solution and add it into the sample injection detection cavity of the two detection pieces of the test card; 2) Imaging detection Insert the test card containing the reaction solution into the card slot of the raw milk somatic cell counter, start the detection program to perform imaging detection, and use the image sensors of the first photoelectric detection module and the second photoelectric detection module to respectively collect fluorescence images of the sample in the corresponding analysis area; 3) Image processing The data processing unit processes the fluorescence image collected by the image sensor to obtain the detection result; 4) Result output The detection results after image processing are displayed and output in the manual interaction unit and / or printed out through a printer.

6. The method according to claim 5, characterized in that The specific method of imaging detection in step 2) is: For the first photoelectric detection module: the main control board drives the light source to light up. The light emitted by the light source first passes through the first filter, exciting the analysis area on the detection card to emit fluorescence. The fluorescence is reflected by the reflector and polarized 90 degrees. It is then focused by the imaging lens and the second filter and imaged onto the image sensor to obtain a fluorescence image. For the second photoelectric detection module: the method is the same as the imaging detection method of the first photoelectric detection module; The first photoelectric detection module and the second photoelectric detection module collect fluorescence images synchronously or sequentially.

7. The method according to claim 5, characterized in that The specific method of image processing in step 3) is: For the fluorescence image collected by the first photoelectric detection module: before processing, the magnification of the imaging light path is pre-calibrated with a graticule; then, based on the image, the number of fluorescent bright spots within the sample detection range is calculated, and then the cavity area of the detection card analysis area corresponding to the detection range is calculated based on the magnification, and based on the cavity thickness of the detection card analysis area, the solution volume corresponding to the detection range is calculated, and the milk somatic cell concentration is obtained by the following calculation formula MilkNum1 : in: MilkNum is the total concentration of lactogenic somatic cells; N is the number of fluorescent bright spots; is the dilution ratio of the raw milk sample; S is the cavity area of the analysis area of the test card corresponding to the detection range, unit is mm 2 ; d is the chamber thickness, unit: μm; For the fluorescence image collected by the second photoelectric detection module: the processing method is the same as the processing method of the fluorescence image collected by the first photoelectric detection module to obtain its milk somatic cell concentration MilkNum2 ; The final somatic cell concentration of the raw milk sample is ( MilkNum1 + MilkNum2 ) / 2.

8. The method according to claim 5, characterized in that In step 1), the freshly sampled raw milk sample is diluted 2-20 times with a buffer solution containing PO4 3- 、Cl - 、CO3 2- One or more of Na + , K + an aqueous solution of one or more of .

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