CN210514035U

CN210514035U - Lactogenesis somatic cell counter

Info

Publication number: CN210514035U
Application number: CN201920661071.4U
Authority: CN
Inventors: 李悦琴
Original assignee: Pioneer Precision Beijing Biotechnology Co Ltd
Current assignee: Suzhou Zhongke Sujing Biotechnology Co.,Ltd.
Priority date: 2019-05-09
Filing date: 2019-05-09
Publication date: 2020-05-12
Anticipated expiration: 2029-05-09

Abstract

The utility model provides a lactogenesis somatic cell count appearance belongs to analysis and detection technical field. The provided counting instrument only needs to put the detection card loaded with the dyed raw milk sample into the limit position of the instrument when in use, and the counting instrument can automatically complete detection to obtain a detection result. The counting instrument can also directly observe the form of the breast somatic cells through fluorescence images, and makes up the vacancy of the existing application field of the breast somatic cell detection.

Description

Lactogenesis somatic cell counter

Technical Field

The utility model belongs to the technical field of the analysis detects, concretely relates to lactogenesis somatic cell count appearance.

Background

Somatic cells in raw milk generally consist of macrophages, lymphocytes, polymorphonuclear neutrophils, a small number of mammary tissue epithelial cells and the like, and the total number of somatic cells (SSC) contained in each milliliter of raw milk is a measure of the quality of raw milk. Taking bovine raw milk as an example, about 2 to 20 million individual cells are normally present in each milliliter of bovine raw milk. However, when the lactation system is infected and damaged by bacterial invasion, the number of leukocytes is greatly increased to 50 to 100 ten thousand per ml or even higher. The concentration of the somatic cells is also closely related to livestock management, food safety and the like, and the health degree of breasts, potential milk production capacity and the like of cows are directly reflected in the aspect of livestock raising; in the aspect of food safety, the milk flavor is closely related to the ingredients, quality and flavor of milk. Therefore, the method can rapidly and accurately count the cells of the raw milk bodies, and is very important for the health management of livestock and the quality monitoring of milk products.

The existing somatic cell total number detection technology mainly aims at bovine somatic cells and comprises an indirect method and a direct method. The indirect method is mainly used for detecting the change of the physical and chemical properties of the milk caused by the increase of somatic cells, thereby indirectly reflecting the total number of the somatic cells of the cattle; the direct rule is to perform detection counting directly on cells. Common indirect methods include a California cell number determination method (CMT), a Wisconsin mastitis test method (WMT), a viscosity method and the like, all of which utilize a surfactant to release DNA in cells, and further measure the DNA aggregation lumps, micelle colloid, viscosity and the like. The most classical of the direct methods is manual microscopy, but the manual microscopy is gradually replaced by flow cytometry because the manual microscopy is too dependent on manual operation. The flow cytometry technique is to dilute and drive cells which are subjected to fluorescent staining in advance under the wrapping of sheath fluid, so that one cell can be accurately counted by an optical detection system. As the most advanced cell detection technology at present, the detection speed is high, the accuracy is high, but the defects exist, such as complex operation flow, high equipment unit price and use cost, and the cell morphology cannot be observed.

SUMMERY OF THE UTILITY MODEL

To one or more of the problems that exist among the prior art, the utility model provides a lactogenesis somatic cell count appearance, include:

a housing;

a main control board disposed within the housing;

the photoelectric detection unit is arranged in the shell, is electrically connected with the main control board, and comprises a first photoelectric detection module and a second photoelectric detection module, wherein each photoelectric detection module comprises an image sensor and is used for collecting images;

the data processing unit is integrated with the main control board and is used for processing and analyzing the image acquired by the image sensor;

the human-computer interaction unit is electrically connected with the main control panel; and

the detection card can be installed in a card insertion port which is arranged on the shell and used for installing the detection card, comprises a first detection sheet and a second detection sheet, and is respectively matched with the first photoelectric detection module and the second photoelectric detection module.

The detection card also comprises a card support, the first detection piece and the second detection piece are arranged on the card support, the detection piece is of a slit micro-fluidic chip structure and comprises a base and a sample detection cavity arranged on the base, the sample detection cavity is a semi-open cavity formed by two parallel chip side walls with a certain gap, and is provided with one or more analysis areas, sample inlets and a drainage groove area communicated with the sample inlets and the analysis areas, and the thickness H of the analysis areas_{Is divided into}Is less than the thickness H of the drainage groove area_{Guiding device}Further, any one or more of the following technical means are superposed:

the first means is as follows: the sample inlet is positioned at the upper end edge opening of the two chip side walls of the sample detection cavity, wherein the upper end edge of one of the two cavity side walls, which is positioned at the sample inlet, is provided with a sample inlet notch so as to inject a living milk somatic cell sample through the sample inlet notch;

the second means: a notch is arranged on the sample inlet at the closed end of the sample injection detection cavity and serves as a vent groove, and the bottom of the vent groove is not lower than the innermost end of the opening of the sample injection notch.

The third means: the sample detection cavity is provided with at least one vent hole, the vent hole is a through hole for communicating the interior of the sample detection cavity with the outside atmosphere and penetrates through the side wall of one side of the analysis region or the drainage groove region or symmetrically penetrates through the two side walls of the analysis region or the drainage groove region; preferably, the exhaust hole is an inverted cone-shaped through hole, the opening of the conical small end faces the inside of the analysis area or the drainage groove area, and the opening of the conical large end faces the outside atmosphere.

The total area of the single side of the analysis area accounts for 50-90% of the total area of the single side of the sample introduction detection cavity, and the thickness of the analysis area is smaller than the thickness H of the drainage groove area_{Guiding device}Thickness H of the analysis zone_{Is divided into}In the range of 50-400 μm, the thickness H of the drainage channel region_{Guiding device}The range is 120-500 mu m; the analysis zone is a zone of a single thickness, or two spatially separate but interconnected zones of equal or unequal thickness.

The sample inlet is in a concave arc shape, and the value range of an included angle α between the tangent line of the downward sliding arc line of the sample inlet and the horizontal reference plane of the sample inlet is 15-85 degrees, preferably 45-85 degrees, and more preferably 80 degrees.

The card holder of the detection card is also provided with an accommodating cavity for storing a reagent and serving as a mixing container for the reagent and a raw milk sample; preferably, reagents are pre-packaged in the accommodating cavity; the reagent comprises a cytofluorescent dye, wherein the cytofluorescent dye is one or more of SYTO9 dye, propidium iodide, ethidium bromide, acridine orange, Hoechst dye, DAPI dye, Cy3 and Cy 5.

The first photoelectric detection module and the second photoelectric detection module each further include:

a light source for emitting light;

a first optical filter for transmitting light of a fixed wavelength range among the light emitted from the light source;

the reflecting mirror is used for reflecting the fluorescence emitted by the detection card to the imaging lens;

the imaging lens is used for imaging the fluorescence emitted by the detection card; and

and the second optical filter is used for transmitting light with a fixed wavelength range in the imaging fluorescence.

The first optical filter is arranged between the detection card and the light source, the detection card is arranged between the first optical filter and the reflector, the imaging lens is arranged at a position capable of receiving the fluorescence reflected by the reflector, and the second optical filter is arranged between the image sensor and the imaging lens.

The counter further comprises:

the printer is arranged in the shell, is electrically connected with the human-computer interaction unit through the main control board and is used for printing and outputting the result of the data processing unit for processing and analyzing the image; and/or

The human-computer interaction unit comprises a display screen, a function key and a switch button; and/or

The counter further comprises one or more of a card insertion baseplate, a silica gel pad, a USB interface, a power interface, a heat dissipation port, a battery cabin and a heat dissipation fan, wherein the card insertion baseplate is used for placing the detection card.

Compared with the prior art, the raw milk somatic cell counting instrument and the raw milk somatic cell counting method based on the technical scheme have the following beneficial effects:

1) the detection card used by the counter can be fixed with reaction reagents for dyeing such as cell fluorescent dye in advance, and the raw milk sample is only required to be mixed on the detection card when in use; during detection, the detection card carrying the raw milk sample dyed by the cell fluorescent dye is placed at the limited position of the instrument, so that the detection can be automatically completed by the counter to obtain a detection result, the operation is convenient, the consistency with the detection result of the flow cytometer is up to 0.9776 in the concentration range of 5-4717-thousand cells/ml of raw milk cells to be detected, and the counting result is accurate and reliable.

2) Because somatic cell concentration is low in the raw milk, adopts one to detect piece statistics cell quantity too few, can lead to the testing result error big, is difficult to satisfy practical requirement, the utility model discloses be provided with two on the detection card of count appearance and detect the piece, every detects piece structural design unique, is provided with the analysis area alone on it, also is provided with two sets of photoelectric detection modules on the corresponding count appearance, can enlarge the analysis region of raw milk somatic cell, improves the precision that detects.

3) The counting instrument can directly observe the form of the breast somatic cells through fluorescence images, and makes up for the vacancy of the existing application field of the breast somatic cell detection.

Drawings

Fig. 1 is a schematic view of a system structure of a raw milk somatic cell counter provided by the present invention;

fig. 2 is a schematic view of the internal structure of the raw milk somatic cell counter provided by the present invention;

in fig. 3, a and B are the external structural intentions of the raw milk somatic cell counter provided by the present invention;

fig. 4 is a schematic structural diagram of a detection card of the raw milk somatic cell counter provided by the present invention;

FIG. 5A is a schematic front view of the test strip;

FIG. 5B is a schematic side view of the test strip;

FIG. 5C is a schematic perspective view of the test strip;

FIG. 5D is a schematic cross-sectional view taken along line A-A of FIG. 5A;

fig. 6 is a schematic structural view of a photoelectric detection unit of the raw milk somatic cell counter provided by the present invention;

fig. 7 shows the result of the accuracy analysis of the raw milk somatic cell counter provided by the present invention.

Detailed Description

The utility model aims at providing a simple operation and can direct observation raw milk somatic cell form raw milk somatic cell count appearance to the defect etc. that the operation flow that detects among the prior art is complicated and can not direct observation raw milk somatic cell form.

As shown in fig. 1, the schematic system structure diagram of the raw milk somatic cell counter provided by the present invention is shown, which includes: the device comprises a detection card, a photoelectric detection unit, a data processing unit and a human-computer interaction unit. The detection card is a carrier for quantitatively detecting a sample for breast somatic cells, and 2 detection sheets are arranged in the detection card so as to increase the volume of the detected sample and the area of an analysis area and improve the detection accuracy and precision. The photoelectric detection unit comprises 2 groups of photoelectric detection modules arranged in parallel, comprises a first photoelectric detection module and a second photoelectric detection module, is used for synchronously detecting 2 analysis areas of parallel detection sheets on the detection card, can avoid using a moving mechanism, and comprises a light source, an imaging light path and an image sensor, wherein the image sensor is used for acquiring fluorescence images of the corresponding analysis areas. The data processing unit is mainly used for processing the fluorescence image acquired by the image sensor and analyzing the result, and the total number of the raw milk somatic cells is obtained by counting the number of all the fluorescence bright spots in the fluorescence image. The man-machine interaction unit mainly comprises a display screen (which can be a touch screen), a function key, a switch button, a data interface and the like, and completes detection flow control and result output. The photoelectric detection unit, the data processing unit and the man-machine interaction unit can be electrically connected with each other through the main control board.

As shown in fig. 2, the schematic diagram of the main structure of the inside of the raw milk somatic cell counter provided by the present invention is shown, including: the device comprises a main control board 101, a photoelectric detection unit 102, a card-inserting bottom board 103, a heat-radiating fan 104 and a battery compartment 105 which are arranged in an instrument shell 100, wherein a data processing unit is integrated on the main control board 101, and the photoelectric detection unit 102 comprises two photoelectric detection modules which are electrically connected with the main control board 101; the card insertion bottom plate 103 forms a limit position of a detection card and is used for placing the detection card; the heat dissipation fan 104 is used for reducing the ambient temperature of the counter during working; the battery compartment 105 is used for placing a power supply and providing power for the operation of the counter. As shown in a of fig. 3 and B of fig. 3, the external structure diagram of the raw milk somatic cell counter provided by the present invention is shown, including: the portable electronic device comprises a housing 100, a display screen 106 (which may be a touch screen and includes function keys), a switch button 107, a card slot 108, a silicone pad 109, a printer 110, a heat sink 111, a USB interface 112 and a power interface 113. The display screen 106 is disposed on the housing 100, can be used for displaying a final detection result, and is electrically connected to the main control board 101; the card slot 108 is arranged on the shell 100, corresponds to the card-inserting bottom plate 103, and detects the insertion of the detection card into the upper limit position of the card-inserting bottom plate 103 in the instrument through the card slot 108; the silica gel pad 109 can play a role in shock resistance of the instrument so as to protect the counter; the printer 110 is arranged in the shell 100, can be electrically connected with the man-machine interaction unit through the main control panel 101, and can be used for printing and outputting a detection result; the heat dissipation port 111 is correspondingly provided with a heat dissipation fan 104 for reducing the ambient temperature of the counter during working; the USB interface 112 can be used for data transmission, and the power interface 113 is correspondingly connected to the battery compartment 105 to provide a power source in the battery compartment 105.

As shown in fig. 4, the detection card 4 of the raw milk somatic cell counter provided by the present invention is shown, which comprises a card holder 4c and two parallel detection sheets installed on the card holder 4 c: a test piece 4a and a test piece 4b, wherein the front ends of the two test pieces protrude out of the card holder 4c and comprise a breast somatic cell analysis area. In order to facilitate the pretreatment of the biological milk somatic cell sample, a holding cavity 4d is arranged on the tray 4c, the holding cavity 4d is cylindrical, inverted truncated cone-shaped or hemispherical, the capacity is 30-100 muL, the depth is 5-14 mm, a reagent which can react with the biological milk somatic cell sample can be fixed in advance in the holding cavity 4d, and the reagent can be fixed in the holding cavity 4d in a freeze-drying or drying mode, wherein the reagent comprises a cytofluorescent dye which can be one or more of SYTO9 dye, propidium iodide, ethidium bromide, acridine orange, Hoechst dye, DAPI dye, Cy3, Cy5 and other fluorescent dyes. The raw milk sample to be entered into the analysis zone of the test strip can be previously reacted with the reagent therein in the housing chamber 4d to stain the somatic cells of the raw milk. Still be provided with two at least joint portions 4e on the card holds in the palm 4c for the joint detects

piece

4a and 4b, the utility model provides a be provided with two the

same detection pieces

4a and 4b on the detection card 4, can carry out the accuracy recheck to the testing result of each detection piece on the one hand (detect two detection pieces promptly in proper order, the difference of the two testing result is no longer than preset threshold value promptly effectively, get the two testing result sum or mean value as final testing result), on the other hand, because the fluorescence of living milk somatic cell is more weak, thereby can guarantee the accuracy that detects to the testing result stack of a plurality of detection pieces. In order to facilitate assembling and clamping with the clamping part 4e of the clamping support 4c, the

detection sheets

4a and 4b are further provided with inner positioning grooves 4f, and correspondingly, the clamping support 4c is provided with bulges matched with the inner positioning grooves 4f so as to realize accurate assembling and clamping of the detection sheets on the clamping support 4 c. In order to facilitate the use of the detection card 4 in a lactogenic somatic cell counter, the detection card 4 is further provided with an outer positioning groove 4g, and the outer positioning groove 4g is arranged on the bottom surface of the tail end of the card support 4c and used for detecting the positioning of the detection card 4 on the card insertion bottom plate 103.

As shown in fig. 5A-5D, the schematic structural diagrams of the detection sheets 4a and 4B are shown, wherein fig. 5A is a schematic structural diagram of the front side of the detection sheet, fig. 5B is a schematic structural diagram of the side of the detection sheet, fig. 5C is a schematic structural diagram of the three-dimensional structure of the detection sheet, and fig. 5D is a schematic structural diagram of the cross-section taken along line AA in fig. 5A. The utility model provides a detection sheet which is a slit micro-fluidic chip structure and comprises a base 21 and a sample injection detection cavity 22, wherein the front end of the base 21 extends to form an arc-shaped edge; the half open cavity that advances a kind detection chamber 22 and be two chip lateral walls 23 formation that have certain clearance and be parallel, including introduction port 25, advance a kind opening 28, analysis area 24, exhaust hole 26 and the drainage channel district 27 that communicates introduction port 25 and analysis area 24, wherein:

as shown in fig. 5A-5C, the sample inlet 25 is located at the opening of the upper edge of the two chip side walls 23 of the sample detection chamber 22, and the upper edge of the two chip side walls 23 located at the sample inlet 25 is provided with a sample notch 28 for facilitating positioning of the raw milk sample pipette, so that the raw milk cell sample can be injected from the sample notch 28 by injection, and the raw milk cell sample can smoothly enter the sample detection chamber 22 to the analysis region 24 through a flow path formed by the drainage groove region 27 (detailed structure is described below) under the combined action of the injection power and capillary force of the pipette. In order to avoid the relatively large air bubbles in the raw milk somatic cell sample from entering the sample detection cavity 22, a notch is formed in one side (non-opened side) of the sample inlet 25 close to the base 21 at the closed end of the sample detection cavity 22 to serve as a vent groove 29, the thickness of the vent groove 29 is consistent with that of the drainage groove region 27, and the bottom of the vent groove 29 does not cross the innermost end of the notch of the sample inlet notch 28. When the raw milk sample in the pipette is injected from the injection notch 28, the larger air bubbles in the raw milk sample escape from the vent groove 29 on the side of the injection port 25, thereby preventing the air bubbles from entering the injection detection cavity 22.

As shown in FIGS. 5A and 5C, the sample inlet 25 is in the shape of a concave arc, and the included angle α between the tangent of the downward sliding arc (left arc in FIG. 5A) and the horizontal reference plane of the sample inlet 25 can determine the flow direction of the raw milk sample to be tested entering the drainage channel region 27, so as to ensure that the raw milk sample to be tested which has been injected into the notch 28 spontaneously flows into the analysis region 24 and fills the analysis region in a predetermined manner, the angle α is in the range of 15-85 degrees, preferably 45-85 degrees, more preferably 80 degrees, in view of the high fat and protein content and high viscosity of the raw milk sample, the angle α is preferably 45-85 degrees, wherein the larger the angle α is the higher the flow rate of the injected sample, but the higher the speed is the higher the probability that the front surface of the sample deforms and generates.

As shown in fig. 5A and 5C, the analysis region 24 is located in the sample detection chamber 22, the shape of the analysis region 24 may be a combination of rectangle, square, trapezoid, circle or arc and other shapes, and each shape may have a fillet, a right angle or a combination of fillet and right angle, the present invention is not limited to the specific shape of the analysis region 24; the analysis zone 24 may have a single thickness H_{Is divided into}The raw milk sample enters the analysis zone 24 to form a detection surface, the thickness of which is generally 50 μm-400 μm; the analysis zone 24 may also be divided into a plurality of interconnected sections of the same or different thickness, for example two interconnected sections of the same or different thickness, which may be used for comparative analysis of a sample of lactogenic somatic cells on the same test strip. The total area of one side of the analysis area 24 accounts for 50-90% of the total area of one side of the sample detection cavity 22, the analysis area 24 has a larger area proportion, the loading capacity and the spreading area of the lactogenic cells can be simultaneously improved, and the total accurate cell counting and the accurate single cell analysis are considered; meanwhile, the chip side wall 23 at the analysis region 24 is thicker, so that the chip side wall 23 with a larger area is not easy to deform, and the analysis region 24 is ensured to have uniform thickness.

As shown in FIGS. 5A and 5D, the flow guide channel region 27 is located in the sample detection chamber 22 and is communicated with the sample inlet 25 and the analysis region 24, and the thickness H of the flow guide channel region 27_{Guiding device}The range is generally 120 μm to 500. mu.m. Thickness H of analysis zone 24_{Is divided into}Is less than the thickness H of the drainage groove area 7_{Guiding device}The raw milk somatic cell sample enters a flow path formed by the drainage groove area 27 from the sample inlet 25 and can be uniformly and quickly guided into the analysis area 24 and filled. Wherein the thickness H of the analysis zone 24_{Is divided into}Less than the thickness H of the gutter area 27_{Guiding device}And can facilitate the discharge of air bubbles. To ensure that the sample of lactogenic somatic cells can flow continuously from the drain channel region 27 into the analysis region 24 and fill it up under capillary forces, capillary pressures above zero are required. Capillary force and thickness H of analysis zone 24_{Is divided into}Thickness H of the gutter area 27_{Guiding device}Liquid surface tension to be detected, and liquid to be detectedThe contact angle of the material surface in the drainage channel region 27 has the following relationship:

as can be seen from the above equation, by designing the thickness H of the gutter area 27_{Guiding device}And the thickness H of the analysis zone 24_{Is divided into}The adjustment of different liquid flow rates and liquid laminar flow characteristics can be realized, so that the formation of bubbles is avoided.

As shown in fig. 5A and 5D, in order to further avoid the generation of bubbles, at least one vent hole 26 is disposed on the sample detection chamber 22, and the vent hole 26 is a through hole communicating the interior of the sample detection chamber 22 with the outside atmosphere, and may be located on one side or both sides of the analysis region 24 or the drainage groove region 27, and may be a symmetrical or asymmetrical through hole, that is, the vent hole 26 penetrates through a side wall of one side of the analysis region 24 or the drainage groove region 27 or symmetrically penetrates through two side walls of the analysis region 24 or the drainage groove region 27. Preferably, the exhaust hole 26 is a symmetrical inverted cone-shaped through hole, i.e. the conical small end opens towards the inside of the drainage channel region 27, and the conical large end opens towards the external atmosphere. The inverted cone-shaped exhaust hole has the advantages that firstly, the surface tension of the raw milk sample to be measured and the gas is utilized to enable the bubbles to be discharged more easily, and the exhaust mode based on the inverted cone-shaped exhaust hole is not influenced by the sampling angle and distance and can effectively discharge the bubbles; under the condition that the reagent needs to be packaged in the sample injection detection cavity 22 in advance, because the contact surface between the reagent and the external environment is very small, after the reagent is added into the sample injection detection cavity 22, the drying process is long, the contact surface between the reagent and the external environment can be increased by utilizing the design of the exhaust holes 26, the drying and uniform distribution of the reagent are accelerated, and therefore the generation of bubbles under various conditions is avoided; the sample amount can be accurately controlled, and the raw milk sample to be detected is difficult to overflow under the action of surface tension after entering the vent hole 26; the exhaust holes 26 are arranged on one side or two sides of the sample detection cavity 22, and only the side surface of the chip needs to be wiped after sample introduction is finished, so that the loss of a liquid sample caused by wiping a sample inlet is avoided; and meanwhile, the inverted taper hole structure further reduces the possibility of wiping loss of the sample.

As shown in fig. 5D, in order to prevent the to-be-measured raw milk sample from flowing out under the action of gravity when the microfluidic chip is moved, the inner edge of the end edge of the side wall 23 of the chip is provided with a transition fillet 210, and the radius R of the transition fillet is in the range of 0.2mm to 1.5 mm. After the micro-fluidic chip is subjected to sample injection, a stable liquid bridge surface 211 is formed on the transitional fillet 210 at the end edge of the side wall 23 of the two chips by the raw milk sample, so that the gravity of the liquid to be detected can be effectively balanced and the liquid to be detected cannot flow out.

As shown in fig. 6, the schematic structural diagram of the photoelectric detection unit of the raw milk somatic cell counter provided by the present invention is shown, and 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 photoelectric detection modules which are the same in structure and are symmetrically arranged, imaging detection can be synchronously performed on the analysis areas of the detection sheet 4a and the detection sheet 4b on the detection card 4, and a moving mechanism is avoided. The photoelectric detection unit mainly comprises a light source plate 1, an optical filter 2, an optical filter 3, a reflector 5, a reflector 9, an imaging lens 6, an imaging lens 10, an optical filter 7, an optical filter 11, an image sensor 8 and an image sensor 12, wherein 2 independent light sources, a light source 1a and a light source 1b are arranged on the light source plate 1. The light source 1a, the optical filter 2, the reflector 5, the imaging lens 6, the optical filter 7 and the image sensor 8 form a first photoelectric detection module, the light source 1b, the optical filter 3, the reflector 9, the imaging lens 10, the optical filter 11 and the image sensor 12 form a second photoelectric detection module, and the two groups of photoelectric detection modules form the same structure and respectively detect analysis areas of 2 detection pieces on the detection card 4. Taking the first photoelectric detection module as an example, the working principle is as follows: the light source 1a is an LED light source and is positioned under one of the analysis areas of the detection card 4 at a distance of 10-30 mm; the main control board 101 drives the lighting light source 1a to emit light, firstly, the light passes through the optical filter 2, and the central wavelength of the transmission spectrum of the optical filter 2 is consistent with the central wavelength of the light emitted by the light source 1 a; the optical filter 2 is positioned between the detection card 4 and the light source 1 a; the signal light emitted by somatic cells in a raw milk sample under the illumination of a light source 1a is reflected by a reflector 5 and then polarized by 90 degrees, and then is focused and imaged into an image sensor 8 (the image sensor 8 is an area array image sensor, the resolution is more than 300 ten thousand pixels) through an imaging lens 6 and an optical filter 7, so that the image is detected and imaged, and finally, the image acquired by the image sensor 8 is processed by a data processing unit to obtain a detection result. The working principle of the second photoelectric detection module is the same as that of the first photoelectric detection module, and the description is omitted.

The data processing unit processes the fluorescence images acquired by the image sensor 8 and the image sensor 12, respectively counts the number of fluorescence bright spots in the corresponding fluorescence images, each fluorescence bright spot represents one raw milk somatic cell, obtains the total concentration of the raw milk somatic cells in each analysis area, and obtains the final total number of the raw milk somatic cells through the integration of the statistical results of the two analysis areas. Meanwhile, the morphology of the lactogenic somatic cells can be directly observed according to the fluorescence image.

Based on the above raw milk somatic cell counting instrument, the utility model also provides a raw milk somatic cell counting method, including following steps:

1) sample preparation

Freshly sampled raw milk can be tested directly, or in a buffer (e.g., containing PO)₄ ^3-、Cl^-、CO₃ ^2-Aqueous solutions of Na +, K +, etc.) was diluted 2 to 20 times and then subjected to detection. Adding 50-100 mul of raw milk sample into the accommodating cavity of the detection card, blowing and uniformly mixing, obtaining reaction liquid after the reaction with a reaction reagent (including cytofluorescent dye) in the accommodating cavity is finished, sucking 40-90 mul of reaction liquid, and respectively adding the reaction liquid into the analysis areas of the two detection sheets of the detection card until the analysis areas are filled;

2) imaging detection

When the sample imaging detection is carried out, a user clicks a detection function key to prompt the user to put a detection sample. Placing the detection card loaded with the reaction liquid obtained in the step 1) into a limit position of a counter, and then clicking a confirmation key to start a detection program.

The main control board drives the light sources corresponding to the

detection sheets

4a and 4b to be lightened, and then controls the image sensors of the two groups of photoelectric detection modules to acquire the fluorescence images of the samples in the corresponding analysis areas. The first photoelectric detection module and the second photoelectric detection module synchronously or sequentially acquire fluorescence images.

3) Image processing

And the data processing unit processes the fluorescence images respectively collected by the image sensors of the two groups of photoelectric detection modules. Before treatment, calibrating the magnification of an imaging light path by using a reticle in advance; then, the number of characteristic points (i.e. the number of fluorescent bright spots, which represents the number of the raw milk somatic cells) in the detection range of the sample is calculated, the area of the detection card analysis region chamber corresponding to the detection range is calculated according to the amplification rate, the volume of the solution corresponding to the detection range is calculated based on the known thickness (the thickness of the detection card analysis region chamber), and the raw milk somatic cell concentrations of the raw milk cells, namely, the MilkNum1 and the MilkNum2, based on the fluorescence images collected by the first photoelectric detection module and the second photoelectric detection module are respectively obtained through the following calculation formulas:

wherein: MilkNum is the total cell number concentration of the raw milk; n is the number of the characteristic points; a is the dilution ratio of the raw milk sample; s is the imaging analysis area (the area of the detection card analysis area cavity corresponding to the detection range) in mm²(ii) a d is the chamber thickness in μm;

the total somatic cell count concentration in the raw milk sample is (MilkNum1+ MilkNum 2)/2.

4) Result output

And displaying and outputting the detection result after the image processing on a display screen. Meanwhile, a user can click a printing button, and the main control panel controls the printer to print and output the detection result.

The present invention will be described in more detail and further illustrated with reference to specific examples, which are not intended to limit the present invention in any way.

Example 1:

the embodiment provides a lactogenesis somatic cell counter, which comprises a shell, a main control panel, a photoelectric detection unit, a data processing unit, a human-computer interaction unit and a detection card, wherein the shell, the main control panel, the data processing unit and the human-computer interaction unit are described above with reference to the attached drawings and are not repeated here.

The photoelectric detection unit comprises a first photoelectric detection module and a second photoelectric detection module, the system principle is shown in fig. 6, an illumination light source 1a and a light source 1b are LED light sources with the central wavelength of 480nm, the central wavelength of 480nm of an optical filter 2 is 480nm, and the bandwidth is 30 nm; under the excitation of a light source, the somatic cells in one analysis area in the detection card 4 emit fluorescence, a fluorescence signal is deflected by 90 degrees after being reflected by the reflector 5 and then is imaged into the image sensor 8 through the imaging lens 6 and the optical filter 7, the optical filter 7 is an optical filter with the central wavelength of 540nm, the transmittance of 520nm to 550nm is more than 80 percent, and the transmittance of the light with the wavelength less than 500nm is less than 0.01 percent, so that the optical filter 7 can filter the influence of the illumination light emitted by the light sources 1a and 1b on the fluorescence imaging of the sample. The fluorescence signal emitted by the somatic cell in the other analysis region of the detection card 4 is reflected by the reflector 9, deflected by 90 °, and imaged into the image sensor 12 through the imaging lens 10 and the optical filter 11.

Example 2:

as shown in fig. 4 and fig. 5A to 5D, this embodiment provides a detection card 4 for the raw milk body cell counter provided in embodiment 1, which includes a card holder 4c, and a housing cavity 4D,

detection pieces

4a and 4b, a card-connecting portion 4e, and an outer positioning groove 4g for positioning on a card-insertion base plate 103, which are provided on the card holder 4 c. The

detection sheets

4a and 4b are in a slit microfluidic chip structure and comprise a base 21 and a sample injection detection cavity 22, wherein the front end of the base 21 extends to form an arc-shaped edge; the sample detection cavity 22 is a semi-open cavity formed by two parallel chip side walls 23 with a certain gap, and includes a sample inlet 25, a sample opening 28, a vent groove 29, an analysis region 24, an exhaust hole 26, and a flow guide groove region 27 communicating the sample inlet 25 and the analysis region 24, wherein the structures of the sample inlet 25, the sample opening 28, and the vent groove 29 are as above, and are not described herein again.

In this embodiment, the total area of the analysis region 24 on one side accounts for 80% of the total area of the sample detection chamber 22 on one side, and has a uniform thickness of 300 μm. The drainage channel region 7 is located in the sample detection cavity 22, is communicated with the sample inlet 5 and the analysis region 4, and has a thickness H_{Guiding device}Is 450 mu m, the included angle α between the tangent line of the downward sliding arc line of the injection port 5 and the horizontal reference plane of the injection port 5 is 80 degrees, and the number of the exhaust holes 26 is two, which are respectively positioned in the drainage groove area7 and penetrates through two side walls of the drainage groove area 7. The radius R of the transition fillet 210 of the edge fillet of the chip side wall 23 is 1.0 mm. Other structures are as described above with reference to the drawings, and are not described in detail herein.

Example 3:

in this embodiment, the fresh raw milk sample with 5-4717 thousand cells/ml is tested based on the raw milk somatic cell counter provided in example 1 and the test card provided in example 2, and the total number of raw milk somatic cells is measured by fluorescent staining.

The method for counting the somatic cells of the raw milk in the embodiment comprises the following steps:

1) sample preparation

Fresh sampled raw milk can be directly detected, or diluted by 2-20 times with buffer solution (same as above) and then detected. Adding 50-100 mul of raw milk sample into a containing cavity (in which SYT09 dye and/or acridine orange are fixed in advance, excitation wavelength of somatic cells is 480nm after fluorescent staining, emission fluorescent wavelength is 530nm) of a detection card, blowing, uniformly mixing, obtaining reaction liquid after reaction is finished, sucking 40-90 mul of reaction liquid, adding into an analysis area of the detection card, and filling the analysis area;

2) imaging detection

When the sample imaging detection is carried out, a user clicks a detection function key to prompt the user to put a detection sample. Placing the detection card loaded with the reaction liquid obtained in the step 1) into a limit position of a counter, and clicking a confirmation key to start a detection program.

The main control board synchronously or sequentially drives and lights the light sources corresponding to the

detection sheets

4a and 4b, and then controls the corresponding image sensors of the two groups of photoelectric detection modules to respectively collect fluorescence images of samples in the corresponding analysis areas.

3) Image processing

And the data processing unit respectively processes the fluorescence images acquired by the image sensors of the two groups of photoelectric detection modules. Before treatment, calibrating the magnification of an imaging light path by using a reticle in advance; then, the number of characteristic points (i.e. the number of fluorescent bright spots, which represents the number of the raw milk somatic cells) in the detection range of the sample is calculated, the area of the detection card analysis region chamber corresponding to the detection range is calculated according to the amplification ratio, the volume of the solution corresponding to the detection range is calculated based on the known thickness (the thickness of the detection card analysis region chamber), and the raw milk somatic cell concentrations of the raw milk cells, namely, the MilkNum1 and the MilkNum2, based on the fluorescence images collected by the first photoelectric detection module and the second photoelectric detection module are respectively obtained through the following calculation formulas:

the final total somatic cell concentration of the raw milk sample is then: (MilkNum1+ MilkNum 2)/2.

4) Result output

Comparative example 1:

the comparative example uses a flow cytometer (model: Foss BacSomatic all-in-one machine) to detect a fresh raw milk sample with the cell density of 5 thousand cells/ml to 4717 thousand cells/ml, and the specific operation method comprises the following steps: sucking 100 mu L of raw milk sample, adding into a centrifuge tube, then adding 1mL of the matched processing reagent of the flow cytometer, fully shaking and uniformly mixing. And finally, putting the mixed sample into a sample port of a flow cytometer, and detecting the scattered light and the fluorescence of the particles in the sample one by one to realize the identification and counting of the breast somatic cells.

Based on the above detection results of example 3 and comparative example 1, as shown in fig. 7, the comparison result between the detection result obtained by using the raw milk somatic cell counter and the detection result obtained by using the flow cytometer is shown, it can be seen that the raw milk somatic cell counter provided by the present invention has the accurate quantification capability between 5 thousand cells/ml and 4717 thousand cells/ml, and has 0.9776 consistency with the flow cytometer. But the utility model provides a living milk somatic cell count appearance has a plurality of advantages for flow cytometer, and it not only can be through fluorescence image direct observation to living milk somatic cell's form, and easy operation, instrument convenient to carry, count are accurate moreover, consume the time short, have compensatied the vacancy in current living milk somatic cell detection application.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A lactogenic somatic cell counter, comprising:

a housing;

a main control board disposed within the housing;

2. Raw milk according to claim 1Somatic cell counter, its characterized in that, it still holds in the palm including the card to detect the card, first detection piece and second detect the piece setting and are in the card holds in the palm, it is slit micro-fluidic chip structure to detect the piece, including the base and locate the appearance detection chamber on the base, it has the semi-open cavity that the parallel chip lateral wall of certain clearance formed for two to advance the appearance detection chamber, has one or more analysis district, introduction port and the drainage channel district that communicates introduction port and analysis district, and the thickness H of analysis district_{Is divided into}Is less than the thickness H of the drainage groove area_{Guiding device}Further, any one or more of the following technical means are superposed:

the second means: a notch is arranged on the sample inlet at the closed end of the sample injection detection cavity and is used as a vent groove, and the bottom of the vent groove is not lower than the innermost end of the notch of the sample injection notch;

the third means: the sample injection detection cavity is provided with at least one exhaust hole which is a through hole for communicating the inside of the sample injection detection cavity with the outside atmosphere and penetrates through the side wall of one side of the analysis area or the drainage groove area or symmetrically penetrates through the two side walls of the analysis area or the drainage groove area.

3. The raw milk somatic cell counter of claim 2, wherein the vent is an inverted cone shaped through hole with the small end opening into the interior of the analysis zone or the drainage channel zone and the large end opening into the outside atmosphere.

4. The raw milk somatic cell counter of claim 2 or 3, wherein the total area of the analysis area on one side accounts for 50% -90% of the total area of the detection cavity on one side, and the thickness of the analysis area is less than the thickness H of the drainage channel area_{Guiding device}Thickness H of the analysis zone_{Is divided into}In the range of 50-400 μm, the thickness H of the drainage channel region_{Guiding device}The range is 120-500 mu m; the analysis zone is a zone of single thicknessA domain, or two spatially independent but connected partitions of equal or unequal thickness.

5. The lactosomal cytometer of claim 4, wherein the sample inlet is concave arc shaped, and an included angle α between a tangent of the downward sliding arc and a horizontal reference plane of the sample inlet ranges from 15 ° to 85 °.

6. The lactogenic somatic cell counter of claim 2 or 3, wherein the sample inlet is concave arc-shaped, and an included angle α between a tangent line of a downward sliding arc line and a horizontal reference plane of the sample inlet ranges from 15 ° to 85 °.

7. The raw milk somatic cell counter of claim 5, wherein the included angle α is between 45 ° and 85 °.

8. The raw milk somatic cell counter of claim 6, wherein the included angle α is between 45 ° and 85 °.

9. The raw milk somatic cell counter of claim 7, wherein the included angle α is 80 °.

10. The raw milk somatic cell counter of any one of claims 2-3, wherein the holder is further provided with a holding chamber for storing reagents and serving as a mixing container for reagents and raw milk samples.

11. The raw milk somatic cell counter of claim 10, wherein the containment chamber is pre-packaged with a reagent; the reagent comprises a cytofluorescent dye, wherein the cytofluorescent dye is one or more of SYTO9 dye, propidium iodide, ethidium bromide, acridine orange, Hoechst dye, DAPI dye, Cy3 and Cy 5.

12. The raw milk somatic cell counter of any one of claims 1-3, wherein the first and second photodetecting modules each further comprise:

a light source for emitting light;

the second optical filter is used for transmitting light with a fixed wavelength range in the imaging fluorescence;

the first optical filter is arranged between the detection card and the light source, the detection card is arranged between the first optical filter and the reflector, the imaging lens is arranged at a position capable of receiving fluorescence reflected by the reflector, and the second optical filter is arranged between the image sensor and the imaging lens.