CN102213715A - New animal mastitis detection method and detector - Google Patents
New animal mastitis detection method and detector Download PDFInfo
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- CN102213715A CN102213715A CN2010101619701A CN201010161970A CN102213715A CN 102213715 A CN102213715 A CN 102213715A CN 2010101619701 A CN2010101619701 A CN 2010101619701A CN 201010161970 A CN201010161970 A CN 201010161970A CN 102213715 A CN102213715 A CN 102213715A
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Abstract
The invention provides a new method for rapidly detecting lactation livestock mastitis comprising clinical and sub-clinical mastitis. In the method provided by the invention, the number of somatic cells in the milk of the ill livestock is detected by measuring the time for the mixed gel to pass through a fiber filter membrane after reaction reagents are added to the milk of the ill livestock, thereby further evaluating the degree of the livestock which are infected with the mastitis. The invention has the principle that the nucleic acid DNA (Deoxyribo Nucleic Acid) of the somatic cells in the milk and electronegative SDS (Sodium Dodecyl Sulfate) are combined to form the gel; and the more number of the somatic cells is, the more gel forms, thereby increasing the viscosity of the milk and proportionally prolonging the time for the gel to pass through the fiber membrane. The detector which is designed by the method provided by the invention can rapidly and quantitatively measure the number of the somatic cells in the milk, thereby providing the simple and effective method and tool for rapidly distinguishing the clinical and sub-clinical mastitis and effectively controlling the spread of the mastitis in livestock groups in the early days.
Description
Technical field
The invention belongs to the scorching technology that detects of lactation animal's mammary gland, the technique for detection of the subclinical mastitis of lactation animal of saying so more specifically.
Background technology
Mastitis is a modal disease in the lactation domestic animal, and all can bring the big economic loss of tool to dairy industry every year, and the milk quality and the milk yield that show as ill domestic animal significantly reduce, and treatment cost and overhead cost increase.Mastitis is divided into clinical type and subclinical type according to its clinical degree.Clinical mastitis causes the breast redness, or with whole body and breast local symptom.Output has grumeleuse, flocculus and water sample milk, can with the naked eye detect immediately; Detecting of subclinical type (or non-clinical) mastitis then need be by instrument or other chemical method.The subclinical mastitis of domestic animal accounts for 90% of the mammitis incidence of disease, is one of important diseases of harm lactation domestic animal.In time detect subclinical mastitis, just timely further developing of control disease improved result of treatment.Thereby reduce individual ill domestic animal to the chance that whole drove infects, reduce economic loss.
The diagnostic method of mastitis has the cytolgical examination of emulsion, conductance measurement, the inspection of pH value, whey electrophoresis diagnosis, enzyme detection method, PCR detection method etc.California mastitis detection method (CMT) is to use at present more general a kind of easy, method fast, but this method only can be used for the qualitative detection of clinical mastitis, because its poor sensitivity, false-positive recall rate can not be used for the detection of subclinical mastitis up to 50%.The laboratory is with a kind of somatic number that makes flow cytometer detect emulsion, its principle is that instrument can write down the somatic number that passes through automatically when samples of latex is passed through the unicellular passage of instrument, but this instrument costs an arm and a leg, volume is big, can not be used for on-the-spot sick ox and detect.Once had a kind of device (MSA-D-Tec) that detects the milk conductivity that uses to detect mastitis on the market, the method is according to the electrolyte (Na in the milk
+And Cl
-) principle of rising designs along with the increase of the somatic number in the milk, the method is easy, quick.But remolding sensitivity is relatively poor, false-positive recall rate is also high, and each mensuration all needs to carry out instrumental correction with the milk sample of tested ox, in addition, experimental results show that not to be that all conductivity that infect the mastitis milk all can raise, and the rising of milk electric conductivity is not to come from the milk cow that suffers from mastitis fully yet.In recent years, a kind of use occurs and detected myeloperoxidase (MPer) (myoloperoxidase in the milk sample, MPO) technology is diagnosed subclinical mastitis, it be according to milk in the sample somatic number and the concentration of MPO the strong correlativity (facies relationship numerical value 0.91) of group is arranged, use enzyme linked immunosorbent assay (ELISA) detection of the MPO in the milk sample to be judged the activated state of neutrophil leucocyte in the breast, can make early diagnosis to the subclinical mastitis of milk cow, but this technical operation complexity, time-consuming, only be confined to breadboard detection.Milk somatic cell electronics analyzer (PortaSCC) is to appear at U.S.'s product that clinical and subclinical mastitis are diagnosed in the home market recently, the design of this instrument derives from the vitro detection technology of human leukocytes, promptly by measuring the reaction of body cell film surface biological enzyme and specific substrate, this reaction can make the test paper color change, somatic number in the milk sample is many more, and color is dark more.The characteristics of this instrument are, field conduct is easy and simple to handle; But the instrument cost height detects and takes length, and the test condition of sample is strict.
Summary of the invention
The object of the present invention is to provide a kind of technical design novelty, easy and simple to handle, detect rapidly, the detection technique of lactation animal's mammary gland inflammation that cost is low, can overcome the defective of above-mentioned mastitis detection technique.
Technology contents of the present invention is as follows: measure the time of the mixed gel that forms behind the adding reaction reagent in ill domestic animal milk liquid by one deck fiber filter film, come the detection of cancerous patient and his family to raise the somatic number of milk in the liquid, thereby further assess the degree of animal infection mastitis.Its principle is that after the animal infection mastitis, somatic number increases thereupon in the milk.A kind of electronegative saponified (SDS) mixed with the milk sample, somatic nucleic acid DNA can discharge in the milk, and plant electronegative saponified reacting to each other therewith in conjunction with forming gel, somatic quantity gel more at most forms many more, the viscosity of liquid of causing suckling increases, and is just long by the time of fiber filter film.The detector that designs with method provided by the present invention can be measured the somatic number in the milk quantitatively, debates not clinical and subclinical mastitis fast.
The device design is made up of Fig. 1 to Fig. 5: detection method provided by the invention is finished jointly by sample container and microprocessor.The design is a kind of design of testing by the method pair cell number that direct employing electric signal interrupts clocking, also can by other signal interruption such as electric capacity, electromagnetism, light, etc. the method pair cell number that clocks of signal interruption test.
Description of drawings
Device design overall assembly drawing as depicted in figs. 1 and 2.Comprise: 1. instrument box cover; 2. power switch; 3. sample container signal input output end and Microprocessor Interface part a; 4. instrument box base; 5. sample container platform; 6. the mounting hole of mouthpiece (3,7) on the instrument box base; 7. sample container signal input output end and Microprocessor Interface part b; 8. sample container combination; 9. microprocessor control panel; 10 counting is strong again; 11. display.
The sample container combination is formed as shown in Figure 3 and Figure 4, comprising: 12. sample container loam cakes; 13. signal input output end and tool interface system spare a, 14. signal transduction pathway a, 15. sample gelatinous fibre filtering membrane .16. base, 17. the filtration membrane bracing frame on the base, 18. signal transduction pathway b, 19. signal input output ends and tool interface system spare b, 20. sample receiving portion funnel, 21. the sensor monitoring mouth, 22. sample container handles, the pilot hole of 23. sample container and signal input output end and tool interface system spare.Microprocessor control synoptic diagram as shown in Figure 5.
Fig. 6, Fig. 7 and Fig. 8 figure are embodiment 1-4 result schematic diagram, be respectively under the different body cell concentration, the mixed gel that the milk sample forms is by different apertures sample gelatinous fibre filtering membrane (sample receiving portion funnel 20, sensor monitoring mouth 21.) time and somatic number are the relation of linear growth, the horizontal ordinate of Fig. 6, Fig. 7, Fig. 8 is a somatic number, and ordinate is the time.
Embodiment
Operating process of the present invention is as follows: sample milk and reaction reagent are evenly mixed 30-60 after second in proportion, form mixed gel, the mixed gel that forms is poured in the sample container (sample container combination 8), and mixed gel is from narrow mouthful of (sample receiving portion funnel 20) process of funnel fiber filter film (sample gelatinous fibre filtering membrane 15) of sample container.At mixed gel from the lower end funnel of sample container narrow mouthful (sensor monitoring mouth 21) through out-of-date, sensor will produce reaction signal, this signal imports microprocessor (9) by signal input output end and tool interface system spare (13 and 19) into through signal transduction pathway (14 and 18), the microprocessor opening entry time, when mixed gel when the narrow mouth of the funnel of sample container all flows through, the reaction signal of sensor side will disappear, cell number is handled and calculated to microprocessor to the writing time of mixed gel process filtrator, and show on miniscope (11).Sensor can adopt various inductance, electric capacity, and electromagnetism, optical sensor is etc. switching signal.
The present invention uses the concentration of reagent SDS to be generally 1%-5%, is preferably 3%, is preferably 2%.The ratio of milk sample and reagent SDS is 1: 0.5-3 is preferably 1: 0.75-2 is preferably 1: 1.The average pore size of the fiber filter film that the present invention uses is 1-20 μ m, is preferably 1-10 μ m, is preferably 1-5 μ m.
The present invention can be used for the detection of the milk sample of on-the-spot diseased individuals livestock, also can be used for big tank stores milk liquid and contains the detection of somatic number, thereby identify the quality that stores milk liquid.The good effect that detection method of the present invention is compared with prior art had is: instruments design is simple, cost is low, detection time is short, the degree of accuracy height, be convenient to the preparation of sample and the cleaning of sample container, to milk sample to be measured source, as holding time length, the sample impurity content does not all have strict demand simultaneously.
The present invention will be further described below in conjunction with embodiment.
Prepare the simulation mastitis milk of known somatic number.Collect 500ml fresh bovine blood, change over to then in the centrifuge tube that contains the anticoagulant factor, left standstill on ice 10 minutes, centrifugal 300Xg (4 ℃) outwells the blood feelings, with PBS buffer solution for cleaning cell, use the Ficoll-Isopaque method to separate leucocyte, measure cell content with cell counter.(somatic number is lower than 50,000/ml) to select the high-quality milk that market sells, detect the somatic number of this milk with flow cytometer, leucocyte (body cell) with separator well is added in the milk according to quantity then, make the simulation mastitis sample milk that contains known somatic number, sample milk is answered 4 ℃ of preservations.
Get 1ml and contain the simulation mastitis milk of somatic number 250,000/ml in glass test tube one, get 0.5ml in the glass test tube one and contain the simulation mastitis milk of somatic number 250,000/ml in glass test tube two, add 0.5 ml somatic number and be lower than the high-quality milk of 50,000/ml in glass test tube two, mix, somatic number in this test tube two is 12.5 ten thousand/ml, getting the 0.5ml sample in the test tube two is added in the glass test tube three, add the 0.5ml somatic number and be lower than the high-quality milk of 50,000/ml in glass test tube three, mix, somatic number in this test tube three is 6.25 ten thousand/ml, in test tube three, get the 0.5ml sample, discard.Add high-quality milk that the 0.5ml somatic number is lower than 50,000/ml in glass test tube four, sample tube four is blank (zero), with adding the 2% reagent SDS of 0.5ml in above-mentioned each sample tube, mixes 1 minute.Be that the sample gelatinous fibre filtering membrane of 3 μ m is put into to take out by detection part and helped mouthful to put into sample container with the aperture, the open detection instrument is poured the sample tube potpourri in the sample container into and to be measured.Experimental result as shown in Figure 6.
2 preparations contain the simulation mastitis milk of obstructed somatic number with reference to embodiment, be respectively, 800 ten thousand/ml, 600 ten thousand/ml, 400 ten thousand/ml, 200 ten thousand/ml and 100 ten thousand/ml, add each sample of 0.5ml in a glass test tube, add high-quality milk that the 0.5ml somatic number is lower than 50,000/ml in a glass test tube, as blank (zero), with adding the 2% reagent SDS of 0.5ml in above-mentioned each sample tube, mixed 1 minute.Be that the sample gelatinous fibre filtering membrane of 8 μ m is put into to take out by detection part and helped mouthful to put into sample container with the aperture, the open detection instrument is poured the sample tube potpourri in the sample container into and to be measured.Experimental result as shown in Figure 7.
3 preparations contain the simulation mastitis milk of obstructed somatic number with reference to embodiment, be respectively, 2.4 10,000,000/ml, 1.2 10,000,000/ml and 0.6 thousand ten thousand/ml, add each sample of 0.5ml in a glass test tube, add high-quality milk that the 0.5ml somatic number is lower than 50,000/ml in a glass test tube, as blank (zero), with adding the 2% reagent SDS of 0.5ml in above-mentioned each sample tube, mixed 1 minute.Be that the sample gelatinous fibre filtering membrane of 15 μ m is put into to take out by detection part and helped mouthful to put into sample container with the aperture, the open detection instrument is poured the sample tube potpourri in the sample container into and to be measured.Experimental result as shown in Figure 8.
Claims (9)
1. fast detecting lactation mastitis in domestic animal, the new method that comprises clinical and subclinical mastitis, it is characterized in that, reaction reagent SDS is added in the ill domestic animal milk liquid, somatic DNA interacts in reaction reagent SDS and the milk liquid, changed the viscosity of milk liquid, detected mastitis by the time that the sample container tunica fibrosa adsorbs by assaying reaction milk liquid.Use microprocessor to count the record time automatically, calculate the somatic number in the milk liquid.
2. mastitis detection method as claimed in claim 1, wherein the concentration of reagent SDS is 1%-5%.
3. mastitis detection method as claimed in claim 1, wherein the aperture of sample fiber filter film is 1-20 μ m.
4. mastitis detection method as claimed in claim 1, wherein sample container is a funnel-form, can be various funnel shapeds, is not limited to Fig. 3 and circle shown in Figure 4.Wherein the sample container sample is 1-10mm by aperture (20).
5. mastitis detection method as claimed in claim 1, wherein assaying reaction is suckled the time that liquid is adsorbed by the sample container tunica fibrosa, the method that is adopted can be signals such as any electromagnetism, electric capacity interruption, light sensation interruption, is not limited to the method for direct employing current interruptions as shown in Figure 3 and Figure 4.
6. mastitis detection method as claimed in claim 1 comprises sample container.Wherein sample container has quick web member (signal input output end and tool interface system spare 13 and 19), can be connected efficiently with measurement mechanism.
7. mastitis detection method as claimed in claim 1 comprises sample container.Wherein sample container has the filtering membrane support member, and there is port support member and sample receptacle bottom, and filter liquide is passed through.
8. mastitis detection method as claimed in claim 1 comprises measurement mechanism.Wherein measurement mechanism has quick web member (sample container signal input output end and Microprocessor Interface part 3 and 7), can be connected efficiently with sample container.
9. mastitis detection method as claimed in claim 1 has sample mounting table (sample container platform 5) on the measurement mechanism, sample container is positioned on the sample mounting table, and sample container can conveniently pick and place, and is convenient to the preparation of sample and the cleaning of sample container.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101619701A CN102213715A (en) | 2010-04-08 | 2010-04-08 | New animal mastitis detection method and detector |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101619701A CN102213715A (en) | 2010-04-08 | 2010-04-08 | New animal mastitis detection method and detector |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001035728A1 (en) * | 1999-11-18 | 2001-05-25 | Sensortec Limited | Mastitis detection |
| CN1544941A (en) * | 2003-11-25 | 2004-11-10 | 浙江大学 | Method for detecting somatic cell content in milk and sample collector therefor |
| CN1587975A (en) * | 2004-08-23 | 2005-03-02 | 浙江大学 | Method for determining milk somatic cell number |
| CN1724998A (en) * | 2005-03-17 | 2006-01-25 | 浙江大学 | Diagnosis reagent for dairy cattle recessive mammitis and its preparation method |
-
2010
- 2010-04-08 CN CN2010101619701A patent/CN102213715A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001035728A1 (en) * | 1999-11-18 | 2001-05-25 | Sensortec Limited | Mastitis detection |
| CN1544941A (en) * | 2003-11-25 | 2004-11-10 | 浙江大学 | Method for detecting somatic cell content in milk and sample collector therefor |
| CN1587975A (en) * | 2004-08-23 | 2005-03-02 | 浙江大学 | Method for determining milk somatic cell number |
| CN1724998A (en) * | 2005-03-17 | 2006-01-25 | 浙江大学 | Diagnosis reagent for dairy cattle recessive mammitis and its preparation method |
Non-Patent Citations (3)
| Title |
|---|
| 潘永兰等: "关于中药水提液的粘度特征及其与膜通量的相关性初步研究", 《化工时刊》 * |
| 程文键等: "快速检测牛奶中体细胞数方法的改进", 《中国食品学报》 * |
| 郭士伦等: "核孔膜测定溶液浓度的原理, 实验及应用前景", 《原子能科学技术》 * |
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Application publication date: 20111012 |