CN111678915A - Detection method of brucellosis in sheep whole blood - Google Patents
Detection method of brucellosis in sheep whole blood Download PDFInfo
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- CN111678915A CN111678915A CN202010363489.4A CN202010363489A CN111678915A CN 111678915 A CN111678915 A CN 111678915A CN 202010363489 A CN202010363489 A CN 202010363489A CN 111678915 A CN111678915 A CN 111678915A
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- 210000004369 blood Anatomy 0.000 title claims abstract description 100
- 239000008280 blood Substances 0.000 title claims abstract description 100
- 241001494479 Pecora Species 0.000 title claims abstract description 50
- 238000001514 detection method Methods 0.000 title claims abstract description 49
- 206010006500 Brucellosis Diseases 0.000 title claims abstract description 26
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 48
- 241000282376 Panthera tigris Species 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 30
- 230000004520 agglutination Effects 0.000 claims abstract description 10
- 238000010241 blood sampling Methods 0.000 claims abstract description 6
- 239000011521 glass Substances 0.000 claims description 28
- 210000002966 serum Anatomy 0.000 claims description 25
- 230000000670 limiting effect Effects 0.000 claims description 12
- 241001148106 Brucella melitensis Species 0.000 claims description 9
- 229940038698 brucella melitensis Drugs 0.000 claims description 9
- 238000005070 sampling Methods 0.000 claims description 5
- 210000000601 blood cell Anatomy 0.000 claims description 4
- 210000005069 ears Anatomy 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000011241 protective layer Substances 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 244000144992 flock Species 0.000 abstract description 6
- 241000589562 Brucella Species 0.000 description 6
- 241000283690 Bos taurus Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000282887 Suidae Species 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 241000194103 Bacillus pumilus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000283898 Ovis Species 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L9/00—Supporting devices; Holding devices
- B01L9/06—Test-tube stands; Test-tube holders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
Abstract
The invention provides a detection method of brucellosis in sheep whole blood. The detection method of the brucellosis sheep whole blood comprises the following steps: s1, blood sampling: selecting sheep to be tested, collecting blood at the ear margin of the sheep by using a special blood collector, storing the collected blood in a test tube, and placing the test tube in a test tube holder. The invention provides a method for detecting brucellosis in sheep whole blood, which needs a small amount of blood collection, can not cause sheep flocks to generate excessive stress during actual blood collection, thereby greatly reducing the difficulty during blood collection, leading the blood collection to be simpler and more convenient, using tiger red reagent for detection, leading the effective time to be controlled at about two minutes, leading the reaction to be more sensitive, solving the problems of weak positive and difficult observation in the traditional method, being more than 2 times of the agglutination speed of the traditional method, only needing 10ul of tiger red reagent, and simultaneously detecting five blood samples, and greatly reducing the cost required by detection.
Description
Technical Field
The invention relates to the field of blood sample detection, in particular to a method for detecting brucellosis ovis whole blood.
Background
The Brucella is a gram-negative bacillus pumilus, and animals such as cattle, sheep, pigs and the like are most susceptible to infectious abortion of dams; human beings can be infected by contacting with bacteria-carrying animals or edible diseased animals and dairy products thereof, and the human beings have already prevailed in partial areas of China and are basically controlled at present. Brucella is also one of the incapacitating biological warfare agents listed by empire; the Brucella is divided into 6 Brucella strains of sheep, cattle, pigs, mice, sheep and dogs, 20 biotypes, and the Brucella strains of sheep, cattle and pigs are popular in China, wherein Brucella disease of sheep is the most common.
In order to detect whether brucellosis exists in sheep flocks, blood samples of the sheep flocks need to be detected, in the prior art, the brucellosis detection method is a tiger red flat plate detection method and a test paper card detection method, and the two methods have certain defects, for example, the required blood sampling amount is large, the steps in the detection process are complicated, weak positive results are difficult to judge, the time spent is long, in addition, a certain amount of detection reagent needs to be used, and the detection cost is high.
Therefore, it is necessary to provide a method for detecting brucellosis in sheep whole blood to solve the above technical problems.
Disclosure of Invention
The invention provides a method for detecting brucellosis in sheep whole blood, and solves the problems that the conventional brucellosis detection method needs a large amount of blood collection, the steps are complicated in the detection process, and the weak positive result is difficult to judge.
In order to solve the technical problem, the detection method of the brucellosis in sheep whole blood provided by the invention comprises the following steps:
s1, blood sampling: selecting a sheep to be detected, collecting blood at the ear margin of the sheep by using a special blood collector, placing the collected blood sample in a test tube for storage, and placing the test tube in a test tube holder;
s2, blood sample pretreatment: placing the test tube with the blood sample in S1 into a special centrifugal device, and centrifuging the blood sample in the test tube through the centrifugal device to separate serum from the blood;
s3, preparation of detection vessels: sucking the tiger red reagent by using a sampler, and dripping the tiger red reagent on the glass slide so that the glass slide is fully paved with the tiger red reagent;
s4, serum detection: sucking the serum separated in the S2 by using a sampler, then dripping the serum into the tiger red reagent, and then slightly shaking the glass slide to fully mix the tiger red reagent and the serum;
s5, microscopic observation: placing the processed fragment loading piece in the S4 on a microscope, and observing a detection result;
s6, judging the result: and (4) judging according to the conditions observed in the step (S5), wherein the judgment standard is that a positive result shows that laces appear on the edge, a light purple worm-shaped stripe is agglutinated in the middle of the sample, a negative result shows that blood cell particles are uniformly distributed, agglutination does not occur, an invalid result does not occur, and the former two conditions do not occur.
The detection method needs less blood collection amount, can not cause the sheep flock to generate excessive stress during actual blood collection, thereby greatly reducing the difficulty during blood collection, and making the blood collection simpler and more convenient, the tiger red reagent is used for detection, so that the effective time of the tiger red reagent is controlled to be about two minutes, the reaction is more sensitive, the problems of weak positive and difficult observation in the traditional method can be solved, the detection speed is greatly improved, the agglutination speed is more than 2 times of the traditional method, only 10ul of tiger red reagent is needed, five blood samples can be simultaneously detected, the cost required by detection is greatly reduced, and the agglutination speed is less than one third of the traditional method.
Preferably, in the step S1, when the blood sample is taken, the sample can be taken at any position of the edge of the sheep ear, and the two ears of the sheep are separately sampled.
Preferably, in S2, when the centrifugal device is used, the rotation speed of the centrifugal device needs to be adjusted to 1500 rpm, and the centrifugal time is 15-20 minutes.
Preferably, the tiger red reagent in S3 is absorbed by 10ul, and when the tiger red reagent is dropped onto the slide glass, the slide glass needs to be cleaned first, so as to ensure that the slide glass is clean and tidy.
Preferably, the serum in S4 is sucked by 10ul, dropped into the middle of the tiger red reagent, and shaken to ensure that the reagent cannot overflow and that the two reagents are uniformly mixed, so that one slide glass can simultaneously detect five different blood samples.
Preferably, in S5, the observation is performed immediately after completion of S4 in microscopic observation, and the effective time is two minutes, which requires observation using a microscope.
Preferably, the positive result in S6 generally appears at about 20S, and the observation needs to be ensured within a valid time to ensure the accuracy of the detection result.
A test tube placer for detecting the Brucella melitensis whole blood, which comprises a placer for placing a blood sample test tube in the detection method of the Brucella melitensis whole blood in claim 1, and the test tube placer for detecting the Brucella melitensis whole blood comprises:
a main body case;
the test tube placing groove is formed in the top of the main body box, and a protective layer is arranged inside the test tube placing groove;
the two movable grooves are respectively arranged on the left side and the right side of the top of the main body box, the bottom of the inner surface of each movable groove is fixedly connected with two limiting rods, the outer surfaces of the limiting rods are sleeved with movable blocks, and the outer surfaces of the limiting rods and the bottoms of the movable blocks are sleeved with elastic pieces;
the two handles are respectively arranged on the front side and the back side of the main body box.
Compared with the related technology, the detection method of the brucellosis in sheep whole blood provided by the invention has the following beneficial effects:
the invention provides a method for detecting brucellosis in sheep whole blood, which needs a small amount of blood collection, can not cause sheep flocks to generate excessive stress during actual blood collection, thereby greatly reducing the difficulty during blood collection, leading the blood collection to be simpler and more convenient, using a tiger red reagent for detection, leading the effective time to be controlled at about two minutes, leading the reaction to be more sensitive, solving the problems of weak positive and difficult observation in the traditional method, further greatly improving the detection speed which is more than 2 times of the agglutination speed of the traditional method, only needing 10ul of the tiger red reagent, and simultaneously detecting five blood samples, greatly reducing the cost required by detection and being less than one third of the cost of the traditional method.
Drawings
FIG. 1 is a schematic structural diagram of a test tube placer for detecting Brucella melitensis whole blood provided by the invention;
FIG. 2 is a schematic view of the interior of the main body case shown in FIG. 1;
fig. 3 is a schematic structural view of the top of the main body case shown in fig. 1.
Reference numbers in the figures: 1. main part box, 2, test tube standing groove, 3, inoxidizing coating, 4, activity groove, 5, gag lever post, 6, movable block, 7, elastic component, 8, handle.
Detailed Description
The invention is further described with reference to the following figures and embodiments.
Please refer to fig. 1, fig. 2 and fig. 3 in combination, wherein fig. 1 is a schematic structural diagram of a test tube holder for detecting brucellosis whole blood according to the present invention; FIG. 2 is a schematic view of the interior of the main body case shown in FIG. 1; fig. 3 is a schematic structural view of the top of the main body case shown in fig. 1. The detection method of the brucellosis in sheep whole blood comprises the following steps:
s1, blood sampling: selecting a sheep to be detected, collecting blood at the ear margin of the sheep by using a special blood collector, placing the collected blood sample in a test tube for storage, and placing the test tube in a test tube holder;
s2, blood sample pretreatment: placing the test tube with the blood sample in S1 into a special centrifugal device, and centrifuging the blood sample in the test tube through the centrifugal device to separate serum from the blood;
s3, preparation of detection vessels: sucking the tiger red reagent by using a sampler, and dripping the tiger red reagent on the glass slide so that the glass slide is fully paved with the tiger red reagent;
s4, serum detection: sucking the serum separated in the S2 by using a sampler, then dripping the serum into the tiger red reagent, and then slightly shaking the glass slide to fully mix the tiger red reagent and the serum;
s5, microscopic observation: placing the processed fragment loading piece in the S4 on a microscope, and observing a detection result;
s6, judging the result: and (4) judging according to the conditions observed in the step (S5), wherein the judgment standard is that a positive result shows that laces appear on the edge, a light purple worm-shaped stripe is agglutinated in the middle of the sample, a negative result shows that blood cell particles are uniformly distributed, agglutination does not occur, an invalid result does not occur, and the former two conditions do not occur.
In the step S1, when the blood sample is taken, the sample can be taken at any position of the edge of the sheep ear, and the two ears of the sheep are separately sampled.
When the centrifugal device is used in S2, the rotation speed of the centrifugal device needs to be adjusted to 1500 rpm, and the centrifugation time is 15-20 minutes.
The S3 Tiger red reagent absorbs 10ul, and when the reagent drops on the glass slide, the glass slide needs to be cleaned first, so that the glass slide is clean and tidy.
10ul of serum in the S4 is absorbed and dropped into the middle of the tiger red reagent, the reagent is required to be prevented from overflowing during shaking, the two reagents are uniformly mixed during shaking, and one glass slide can be used for detecting five different blood samples simultaneously.
In S5, in the microscopic observation, observation needs to be performed immediately after completion of S4, and the effective time is two minutes, and observation needs to be performed using a microscope.
The positive result in S6 generally appears in about 20S, and it is necessary to ensure observation within a valid time during observation to ensure the accuracy of the detection result.
A test tube placer for detecting the Brucella melitensis whole blood, which comprises a placer for placing a blood sample test tube in the detection method of the Brucella melitensis whole blood in claim 1, and the test tube placer for detecting the Brucella melitensis whole blood comprises:
a main body case 1;
the test tube placing groove 2 is formed in the top of the main body box 1, and a protective layer 3 is arranged inside the test tube placing groove 2;
the two movable grooves 4 are respectively arranged on the left side and the right side of the top of the main body box 1, two limiting rods 5 are fixedly connected to the bottoms of the inner surfaces of the movable grooves 4, movable blocks 6 are sleeved on the outer surfaces of the limiting rods 5, and elastic pieces 7 are sleeved on the outer surfaces of the limiting rods 5 and the bottoms of the movable blocks 6;
and the two handles 8 are respectively arranged on the front surface and the back surface of the main body box 1.
The main box 1 is mainly made of plastic materials with good elasticity and is a rectangular block, ten test tube placing grooves 2 are arranged for placing test tubes containing blood samples, the number of the placing grooves 2 can be adjusted according to specific requirements, the pore size of the placing grooves 2 is matched with that of most existing test tubes, and the protective layer is made of 3-bit sponge materials, so that the test tubes in the placing grooves 2 can be effectively protected, and the test tubes are prevented from being damaged due to collision in the using process;
the movable groove 4 is matched with the movable block 6, the movable block 6 can move in the movable groove 4, the two limiting rods 5 are respectively positioned at the front side and the rear side of the bottom of the movable groove 4, the surfaces of the limiting rods 5 are in sliding connection with the inside of the movable block 6, a limiting effect is achieved on the movable block 6, the elastic part 7 can be pressed when the movable block 6 contracts in the movable groove 4, the elastic part 7 can contract, the top of the main body box 1 has a buffering effect through the matching arrangement of the movable block 6 and the elastic part 7, and when the top of the main body box 1 is collided, a certain buffering effect can be achieved on the top of the main body box 1 through the movement of the movable block 6, so that the top of a test tube on the main body box 1 is protected, and the test tube is prevented from being damaged;
the two handles 8 are used for taking up the main body box 1, when the main body box 1 is taken up, the two handles 8 are rotated to the top of the main body box 1, then the main body box 1 can be taken up by holding with one hand, and the use is very convenient.
The working principle of the detection method of the brucellosis in sheep whole blood provided by the invention is as follows:
s1, blood sampling: selecting a sheep to be detected, collecting blood at the ear margin of the sheep by using a special blood collector, storing the collected blood sample in a test tube, placing the test tube in a test tube holder, and sampling at any position of the ear margin of the sheep and separately sampling two ears of the sheep when the blood sample is collected;
s2, blood sample pretreatment: putting the test tube with the blood sample in S1 into a special centrifugal device, centrifuging the blood sample in the test tube by the centrifugal device, separating serum in the blood, and when the centrifugal device is used, regulating the rotating speed of the centrifugal device to 1500 revolutions per minute for 15-20 minutes;
s3, preparation of detection vessels: sucking the tiger red reagent by using a sampler, dripping the tiger red reagent on a glass slide to ensure that the glass slide is fully paved with the tiger red reagent, sucking 10ul of tiger red reagent, and cleaning the glass slide when the tiger red reagent is dripped on the glass slide to ensure that the glass slide is clean and tidy;
s4, serum detection: sucking the serum separated in the S2 by using a sampler, then dripping the serum into the tiger red reagent, and then slightly shaking the glass slide to fully mix the tiger red reagent and the serum, sucking 10ul of the serum, dripping the serum into the tiger red reagent, wherein the reagent cannot overflow when shaking, and the two reagents are uniformly mixed when shaking, and one glass slide can simultaneously detect five different blood samples;
s5, microscopic observation: placing the processed ground piece in the S4 on a microscope, observing the detection result, and observing immediately after S4 is finished during microscopic observation, wherein the effective time is two minutes, and the observation needs to be carried out by using microscope equipment;
s6, judging the result: and judging according to the conditions observed in the step S5, wherein the judgment standard is positive results, laces appear on the edges, light purple worm-shaped stripe agglutination appears in the middle of the sample, negative results, blood cell particles are uniformly distributed, agglutination does not occur, invalid results do not appear in the former two conditions, the positive results generally appear in about 20S, and observation within effective time needs to be ensured during observation so as to ensure the accuracy of the detection results.
Compared with the related technology, the detection method of the brucellosis in sheep whole blood provided by the invention has the following beneficial effects:
the invention provides a method for detecting brucellosis in sheep whole blood, which needs a small amount of blood collection, can not cause sheep flocks to generate excessive stress during actual blood collection, thereby greatly reducing the difficulty during blood collection, leading the blood collection to be simpler and more convenient, using a tiger red reagent for detection, leading the effective time to be controlled at about two minutes, leading the reaction to be more sensitive, solving the problems of weak positive and difficult observation in the traditional method, further greatly improving the detection speed which is more than 2 times of the agglutination speed of the traditional method, only needing 10ul of the tiger red reagent, and simultaneously detecting five blood samples, greatly reducing the cost required by detection and being less than one third of the cost of the traditional method.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (8)
1. The detection method of the brucellosis in sheep whole blood is characterized by comprising the following steps:
s1, blood sampling: selecting a sheep to be detected, collecting blood at the ear margin of the sheep by using a special blood collector, placing the collected blood sample in a test tube for storage, and placing the test tube in a test tube holder;
s2, blood sample pretreatment: placing the test tube with the blood sample in S1 into a special centrifugal device, and centrifuging the blood sample in the test tube through the centrifugal device to separate serum from the blood;
s3, preparation of detection vessels: sucking the tiger red reagent by using a sampler, and dripping the tiger red reagent on the glass slide so that the glass slide is fully paved with the tiger red reagent;
s4, serum detection: sucking the serum separated in the S2 by using a sampler, then dripping the serum into the tiger red reagent, and then slightly shaking the glass slide to fully mix the tiger red reagent and the serum;
s5, microscopic observation: placing the processed fragment loading piece in the S4 on a microscope, and observing a detection result;
s6, judging the result: and (4) judging according to the conditions observed in the step (S5), wherein the judgment standard is that a positive result shows that laces appear on the edge, a light purple worm-shaped stripe is agglutinated in the middle of the sample, a negative result shows that blood cell particles are uniformly distributed, agglutination does not occur, an invalid result does not occur, and the former two conditions do not occur.
2. The method for detecting brucellosis in sheep whole blood as claimed in claim 1, wherein the step of sampling blood in S1 is carried out by sampling blood at any position of edge of sheep ear and separately sampling two ears of sheep.
3. The method for detecting brucellosis in sheep whole blood as claimed in claim 1, wherein in step S2, when the centrifugal device is used, the rotation speed of the centrifugal device needs to be adjusted to 1500 rpm, and the centrifugation time is 15-20 minutes.
4. The method for detecting brucellosis in sheep whole blood as claimed in claim 1, wherein the sucking of tiger red reagent in S3 is 10ul, and when dropping on the slide glass, the slide glass needs to be cleaned first to ensure that the slide glass is clean and tidy.
5. The method for detecting brucellosis in sheep whole blood as claimed in claim 1, wherein the serum in S4 is sucked by 10ul, dropped into the middle of the tiger red reagent, shaken to ensure that the reagent cannot overflow, and shaken to ensure that the two are mixed uniformly, so that one slide glass can detect five different blood samples simultaneously.
6. The method for detecting brucellosis in sheep whole blood as claimed in claim 1, wherein the observation in S5 is performed immediately after completion of S4 in microscopic observation, and the effective time is two minutes, which is required to be performed by using a microscope.
7. The method for detecting brucellosis in sheep whole blood as claimed in claim 1, wherein the positive result in S6 appears in about 20S, and the observation needs to be carried out within a valid time to ensure the accuracy of the detection result.
8. The method for detecting Brucella melitensis whole blood according to claim 1, comprising a container for containing a blood sample tube in the method for detecting Brucella melitensis whole blood according to claim 1, wherein the container for containing the blood sample tube comprises:
a main body case;
the test tube placing groove is formed in the top of the main body box, and a protective layer is arranged inside the test tube placing groove;
the two movable grooves are respectively arranged on the left side and the right side of the top of the main body box, the bottom of the inner surface of each movable groove is fixedly connected with two limiting rods, the outer surfaces of the limiting rods are sleeved with movable blocks, and the outer surfaces of the limiting rods and the bottoms of the movable blocks are sleeved with elastic pieces;
the two handles are respectively arranged on the front side and the back side of the main body box.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010363489.4A CN111678915A (en) | 2020-04-30 | 2020-04-30 | Detection method of brucellosis in sheep whole blood |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010363489.4A CN111678915A (en) | 2020-04-30 | 2020-04-30 | Detection method of brucellosis in sheep whole blood |
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| CN111678915A true CN111678915A (en) | 2020-09-18 |
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|---|---|---|---|
| CN202010363489.4A Pending CN111678915A (en) | 2020-04-30 | 2020-04-30 | Detection method of brucellosis in sheep whole blood |
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| CN (1) | CN111678915A (en) |
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- 2020-04-30 CN CN202010363489.4A patent/CN111678915A/en active Pending
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Application publication date: 20200918 |