CN107589117A - A kind of quick microscopy diagnostic kit of swine eperythrozoonosis and preparation method thereof - Google Patents
A kind of quick microscopy diagnostic kit of swine eperythrozoonosis and preparation method thereof Download PDFInfo
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- CN107589117A CN107589117A CN201710726649.5A CN201710726649A CN107589117A CN 107589117 A CN107589117 A CN 107589117A CN 201710726649 A CN201710726649 A CN 201710726649A CN 107589117 A CN107589117 A CN 107589117A
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Abstract
The invention provides quick microscopy diagnostic kit of a kind of swine eperythrozoonosis and preparation method thereof, the kit is made up of detection reagent, vessel apparatus, reagent is made up of Rui Shi dyestuffs, Giemsa, glycerine, polysorbas20, methanol, vessel apparatus is made up of reagent bottle, rubber head dropper, its is supporting scientific and reasonable, detection method is simple, and Detection accuracy is up to 98%, suitable with swine eperythrozoonosis PCR diagnostic kit accuracys rate.
Description
Technical field：
The invention belongs to veterinary inspection field, and in particular to a kind of quick microscopy diagnostic kit of swine eperythrozoonosis
And preparation method thereof.
Background technology：
Eperythrozoonosis is that eperythrozoon colonizes in erythrocyte surface, blood plasma and the marrow of people or many animals and drawn
A kind of zoonosis risen.Eperythrozoon is classified as rickettsia by the past in the world, but in recent years according to 16SrRNA
Gene sequencing, eperythrozoon is in classification closer to Mycoplasma (Mycoplasma).Eperythrozoon suis
(Eperythrozoonsuis, E.suis) is a kind of pathogen for parasitizing swine erythrocyte surface or being free on blood plasma, can be caused
Newborn piglet and weanling pig weakness and anaemia, Growth of Pigs is slow, and sow breeding difficulty, conception rate are low, out of heat, miscarriage
Development and the human health of animal husbandry with summary symptom, serious threats such as stillborn foetuses.Huge economy is had resulted at present
Loss, and expand trend in scope, thus cause the extensive concern of domestic and foreign scholars.
Clinically the sick diagnostic method is mainly based on direct microscopy, including blood tabletting and smear, but it is false
Positive rate is high.Animal experiment method is the effective means made a definite diagnosis, but time-consuming oversize, costly.Serological method includes indirect
Hemagglutination test (IHA), complement fixation test (CFT) (CFT) or EUSA (ELISA) etc., due to eperythrozoonosis
Antibody changes big rise and fall, and these methods are not suitable for disease diagnosis yet.PCR diagnostic methods have very strong specificity, but technology
It is required that higher, detection operation influence factor is more.
The content of the invention：
The present invention is in order to solve the above problems, there is provided a kind of quick microscopy diagnostic kit of swine eperythrozoonosis and its
Preparation method, the kit is supporting scientific and reasonable, and detection method is simple, and Detection accuracy is up to 98%, with eperythrozoon suis
Sick PCR diagnostic kits accuracy rate is suitable.
Specifically, the invention provides a kind of quick microscopy diagnostic kit of swine eperythrozoonosis, by detection reagent, device
Ware apparatus is formed, and described reagent is made up of Rui Shi dyestuffs, Giemsa, glycerine, Tween-20, methanol, described vessel device
Tool is made up of reagent bottle, rubber head dropper.
Reagent purity described above is required to as top pure grade.
Present invention also offers a kind of preparation method of the quick microscopy diagnostic kit of swine eperythrozoonosis, including it is following
Step：
1st, the washing of quick mirror kit vessel apparatus：
Required glassware is put into soaked overnight in the glassware cylinder that cleaning agent is aoxidized equipped with strong acid by 1.1；
1.2 take out the vierics that cleaning agent soaked overnight is aoxidized through strong acid, are cleaned with hot liquid soap, repeatedly with originally
Water rinses 5 times, and take out within 1 hour glassware with distilled water immersion is filtered dry naturally.
Glassware is put into 138 DEG C of dry disinfection case dry heat sterilization 1 hour by 1.3, is taken out after cooling and is put into 35 DEG C of constant temperature
Drying box is standby.
The washing of 1.4 dropper heads, plastics reagent bottle bottle：Dropper head and plastic bottle are put into clear water and soaked 30 minutes；Will
The dropper head and plastic bottle soaked in clear water is cleaned with hot liquid soap, is rinsed 5 times with running water repeatedly, with distillation water logging
Bubble 1 hour, takes out dropper head and plastic bottle is filtered dry naturally.
Dropper head and plastic bottle are put into 15 pounds of autoclave sterilizer by 1.5 to be sterilized 30 minutes, is taken out after cooling and is put into 35 DEG C of perseverances
Warm drying box drying for standby.
2nd, the preparation of quick mirror kit reagent：
2.1 Rui Shi dyestuffs and Giemsa are put into 30-40 DEG C of incubator drying box before weighing and stayed overnight in advance；
2.2 weigh respectively：Rui Shi dyestuff powder 1-1.8g, Giemsa 1-1.8g.
2.3 measure respectively：Glycerine 20-36ml, Tween-20 4-8ml, methanol 1000ml.
2.4 place the Rui Shi dyestuffs and Giemsa that weigh in mortar, and dyestuff is gently broken into pieces into powder with newborn rod, then
Row is ground to fine powder；
2.5 add glycerine dissolving dye in mortar, dyestuff is shown bright luster in newborn Bowls, and attached without dyestuff powder
, add proper amount of methanol to be ground, stand a moment, supernatant liquid is poured into the 2000ml volumetric flasks of a cleaning；
2.6 add methanol trituration, are repeated several times, untill in mortar without dyestuff；
2.7 add Tween-20 in volumetric flask, with methanol constant volume in 2000ml；Shake up and seal bottleneck, deposit room temperature dark place
Filter once, refiltered after 2 weeks once after 24 hours, you can used；Dyeing liquor storage is more long, then dyestuff dissolving, decomposition is more filled
Point, this process is the maturing process of dyeing liquor, and more than 3 months Colors of storage more preferably, notice that sealing is kept in dark place, had for a long time
Effect.
3rd, kit package processing technology：
Packing box specification is 120mm × 70mm × 70mm, and two 20mL reagent bottles and two rubber head droppers can be deposited in box,
The label of kit, specification, sealing label are tested.
4th, the packing of kit：
4.1 taking methanol 15ml to load sterilized dried white reagent bottle, label is labeled as I liquid；
4.2 take the dye liquor 15ml after curing to load sterilized dried brown reagent bottle, and label is labeled as II liquid；
4.3 are put into I liquid, II liquid in box, and are put into two rubber head droppers and kit operation instructions, packaging seal,
The as quick microscopy diagnostic kit of swine eperythrozoonosis；
Step 2.1 described above is to be put into 38 DEG C of incubator drying box mistakes in advance before weighing Rui Shi dyestuffs and Giemsa
Night；
Step 2.2 described above weighs respectively：Rui Shi dyestuff powder 1.4g, Giemsa 1.4g.
Step 2.3 described above measures respectively：Glycerine 28ml, Tween-20 6ml, methanol 1000ml.
Strong acid oxidation cleaning agent washing lotion described above is formulated as：300g potassium bichromates are weighed, are placed in 2500mL distillations
In water, dissolve by heating, cooling, addition concentrated sulfuric acid 1700mL, stirring while adding slowly, is bottled after cold standby.
Beneficial effects of the present invention are：
The kit is solved in prior art microscopy, including blood tabletting and smear staining, and its false positive rate is high, operation
Easily pollution, the low problem of Detection accuracy, not only preparation method is simple for the kit, and cost is low, and detection method is simple, detection
Accuracy rate is up to 98%, suitable with swine eperythrozoonosis PCR diagnostic kit accuracys rate.
Brief description of the drawings：
Accompanying drawing 1：The making schematic diagram of standard Blood piece：
Accompanying drawing 2：Microscopy judges compares figure：
Embodiment：
With reference to preferred embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate
The present invention rather than limit the scope of the invention.In addition, it is to be understood that after present disclosure has been read, this area
Technical staff can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims
Limited range.
Embodiment 1：The preparation of the quick microscopy diagnostic kit of swine eperythrozoonosis
1st, the washing of quick mirror kit vessel apparatus：
Required glassware is put into soaked overnight in the glassware cylinder that cleaning agent is aoxidized equipped with strong acid by 1.1；
1.2 take out the vierics that cleaning agent soaked overnight is aoxidized through strong acid, are cleaned with hot liquid soap, repeatedly with originally
Water rinses 5 times, and take out within 1 hour glassware with distilled water immersion is filtered dry naturally.
Glassware is put into 138 DEG C of dry disinfection case dry heat sterilization 1 hour by 1.3, is taken out after cooling and is put into 35 DEG C of constant temperature
Drying box is standby.
The washing of 1.4 dropper heads, plastics reagent bottle bottle：Dropper head and plastic bottle are put into clear water and soaked 30 minutes；Will
The dropper head and plastic bottle soaked in clear water is cleaned with hot liquid soap, is rinsed 5 times with running water repeatedly, with distillation water logging
Bubble 1 hour, takes out dropper head and plastic bottle is filtered dry naturally.
Dropper head and plastic bottle are put into 15 pounds of autoclave sterilizer by 1.5 to be sterilized 30 minutes, is taken out after cooling and is put into 35 DEG C of perseverances
Warm drying box drying for standby.
2nd, the preparation of quick mirror kit reagent：
2.1 Rui Shi dyestuffs and Giemsa are put into 35 DEG C of incubator drying boxes before weighing and stayed overnight in advance；
2.2 weigh respectively：Rui Shi dyestuff powder 1.4g, Giemsa 1.4g.
2.3 measure respectively：Glycerine 28ml, Tween-20 6ml, methanol 1000ml.
2.4 place the Rui Shi dyestuffs and Giemsa that weigh in mortar, and dyestuff is gently broken into pieces into powder with newborn rod, then
Row is ground to fine powder；
2.5 add glycerine dissolving dye in mortar, dyestuff is shown bright luster in newborn Bowls, and attached without dyestuff powder
, add proper amount of methanol to be ground, stand a moment, supernatant liquid is poured into the 2000ml volumetric flasks of a cleaning；
2.6 add methanol trituration, are repeated several times, untill in mortar without dyestuff；
2.7 add Tween-20 in volumetric flask, with methanol constant volume in 2000ml；Shake up and seal bottleneck, deposit room temperature dark place
Filter once, refiltered after 2 weeks once after 24 hours, you can used；Dyeing liquor storage is more long, then dyestuff dissolving, decomposition is more filled
Point, this process is the maturing process of dyeing liquor, and more than 3 months Colors of storage more preferably, notice that sealing is kept in dark place, had for a long time
Effect.
3rd, kit package processing technology：
Packing box specification is 120mm × 70mm × 70mm, and two 20mL reagent bottles and two rubber head droppers can be deposited in box,
The label of kit, specification, sealing label are tested.
4th, the packing of kit：
4.1 take methanol 15ml to load sterilized dried white reagent bottle, and label is labeled as I liquid；
4.2 take the dye liquor 15ml after curing to load sterilized dried brown reagent bottle, and label is labeled as II liquid；
4.3 are put into I liquid, II liquid in box, and are put into two rubber head droppers and kit operation instructions, packaging seal,
The as quick microscopy diagnostic kit of swine eperythrozoonosis.
Embodiment 2：The quick microscopy diagnostic kit operation of swine eperythrozoonosis：
1st, the collection of blood sample
1.1 ear veins are taken a blood sample：From middle and lower part on the outside of the ear of pig left or right side, the venous blood of median size and protrusion is selected
Pipe, is first carried out disinfection with the tincture of iodine, then carries out de- iodine and sterilization with 75% alcohol, and rear No. 8 intravenous needles collection blood 1 drip.
1.2 vena cava anteriors are taken a blood sample：Front depressed part between pig right side clavicle and right fore is found, is first disappeared with the tincture of iodine
Poison, then de- iodine and sterilization are carried out with 75% alcohol, it is rear to carry out vena cava anterior blood with 20ml disposable syringes installation blood taking needle
Collection.
2nd, prepared by Blood piece sample
2.1 taking slide 4 to open, 3 are cooked slide glass, and 1 is made push jack (having smooth edge).Left hand thumb and forefinger hold load
Slide both ends, the μ l of new blood 1~1.5 are added dropwise on slide (side leaves enough marks, sees accompanying drawing 1 (1))；
2.2, with the middle part of right hand thumb and forefinger clamping push jack lateral margin, push jack lower edge are flatted against the center line of slide；
2.3 use push jack end thereof contacts drop of blood, blood is deployed between push jack and slide, allow blood in slide with pushing away
(see accompanying drawing 1 (2)) when extending to that about 2cm is wide to both sides between piece；
2.4 make two slides keep 30~45 degree of angles, are at the uniform velocity pushed into forward from right to left on the left of slide (see accompanying drawing 1 (3))；
Push jack is pushed into left side by 2.5 mores than at 1/5 i.e. pushing into Blood piece to be checked (see accompanying drawing 1 (4))；
Speed is uniform during 2.6 push jack, and the speed of corner dimension and expansion blood film is fast between drop of blood size, push jack and slide glass
It is slow to wait the thickness for often influenceing blood film.Manufactured Blood piece should form the haemocyte of tiling on slide, contacted with each other between cell and
Do not overlap.
3rd, the processing and dyeing of Blood piece
Processing before the dyeing of 3.1 Blood pieces
3.1.1 after Blood piece natural drying (winter can appropriate heat drying), be adopted with mark mark of the pencil in Blood piece
The relevant informations such as sample place, time, animal species, numbering；
3.1.2 I drops are added in dried blood film and (cover whole blood films), be fixed 5 minutes, unnecessary is returned
Receive or move and abandon.
3.1.3 untreated Blood piece need to be handled as early as possible, and summer standing time, winter was no more than 72 no more than 48 hours
Hour.
The dyeing of 3.2 Blood pieces
3.2.1 in Blood piece be added dropwise dye liquor make covering whole blood film, after 2 minutes to 5 minutes (regard dyeing environment temperature and
Fixed, then dyeing time is short for environment temperature height, on the contrary then grow)；
3.2.2 slowly rinsed and (paid attention to from slide one end with distilled water or running water：Dye liquor is not removed first or directly to blood
Film rinses), dry rear microscopy.
4th, microexamination
Slide sample after dyeing is placed under light microscope, first with 400-600 times of sem observation, finds the red thin of deformation
Reconvert is observed to (1000-1500 times) under oil mirror after born of the same parents and its surface attachment have eperythrozoon.
5th, inspection result judges
Under 5.1 400-600 times of light microscopic dark fields, find oval a large amount of RBC deformations (30-95%), triangle,
The polymorphy such as polygon change, and some even ruptures.Erythrocyte surface has little particle attachment, and visible yellowish folding is finely tuned in regulation
Light dot；
For 5.2 reconverts to observing under oil mirror (1000-1500 times), eperythrozoon is in polymorphy, such as oval, spherical,
Disc, comma shape, crescent etc., into it is single be dispersed in, cluster or chain are attached to erythrocyte surface, make red blood cell shaped like gear
Shape, pineapple shape, asterism shape etc.；
5.3 infection intensities are 1-12, and eperythrozoon size is 0.73-1.82 μm.Every tested Blood piece observes 20
The visual field, it is found that have an eperythrozoon is judged to the positive, otherwise be negative (see accompanying drawing 2).
Experimental example：
1st, by " the quick microscopy diagnostic kit of swine eperythrozoonosis " of development and " swine eperythrozoonosis PCR diagnosis
Kit " carries out clinical diagnosis contrast test, is compared by the laboratory diagnosis to 850 blood samples." swine eperythrozoonosis is fast
Fast microscopy diagnostic kit " accuracy rate 98%, it is 98% with " swine eperythrozoonosis PCR diagnostic kits " coincidence rate.
2nd, completed using " the quick microscopy diagnostic kit of swine eperythrozoonosis " in 2006 to 2011 to Kweiyang,
The cities and counties 40 of the south of Guizhou Province, the southeast of Guizhou Province, the southwest of Guizhou Province, Liu Panshui, Zun Yi, Tongren, Anshun, Bijie etc. nine pig Base Counties and 24 weights
The epidemiology of the swine eperythrozoonosis of point pig 1142 plants (family) in county (city, area) 133 443, small towns village is adjusted
Look into；Find swine eperythrozoonosis original and Eperythrozoon wenyonii cause of disease.For the pig incidence of disease from 23.3% to 100%, morbidity is dead
Rate is died between 33.33% to 80.14%.Blood sample checks positive rate average 70.33%.RBC deformation rate 30%~80%,
Infected Erythrocytes rate 50% to 80%, infection intensity are 1~12.The preliminary infection danger for having found out Guizhou Province's eperythrozoon suis
Evil situation.
3rd, 2013 to 2016 " the quick microscopy diagnostic kits of swine eperythrozoonosis " by development in Guiyang, abide by
296,41 counties (area, city) scale field popularization and application eperythrozoon suis of 4 cities and counties such as adopted city, In Qiannan and Affected It In Qianxinan
Sick quick mirror 7.65 ten thousand parts of kit.Because application quick diagnosis reagent kit carries out early stage diagnosis in time to 7.65 ten thousand pigs,
Clinical case 2236 is diagnosed to be altogether, and examination goes out subclinical infection case 27735.The timely correctly taking drugs treatment of plant is instructed,
Prevented in time, timely processing, reduce medication, reduce dead, plant's head is averaged 121.50 yuan of new output value, newly
Increase 928.75 ten thousand yuan of the gross output value, 108.10 ten thousand yuan of new returns (profits tax), obtain preferable economic results in society.
Claims (7)
- A kind of 1. quick microscopy diagnostic kit of swine eperythrozoonosis, it is characterised in that：By detection reagent, vessel apparatus group Into described reagent is made up of Rui Shi dyestuffs, Giemsa, glycerine, Tween-20, methanol, and described vessel apparatus is by reagent Bottle, rubber head dropper composition.
- 2. the quick microscopy diagnostic kit of swine eperythrozoonosis according to claim 1, it is characterised in that：Described examination Agent purity is required to as top pure grade.
- A kind of 3. preparation method of the quick microscopy diagnostic kit of swine eperythrozoonosis, it is characterised in that：Comprise the following steps：1st, the washing of quick mirror kit vessel apparatus：Required glassware is put into soaked overnight in the glassware cylinder that cleaning agent is aoxidized equipped with strong acid by 1.1；1.2 take out the vierics that cleaning agent soaked overnight is aoxidized through strong acid, are cleaned with hot liquid soap, are rushed repeatedly with running water Wash 5 times, take out within 1 hour glassware with distilled water immersion is filtered dry naturally.Glassware is put into 138 DEG C of dry disinfection case dry heat sterilization 1 hour by 1.3, is taken out after cooling and is put into 35 DEG C of freeze-day with constant temperature Case is standby.The washing of 1.4 dropper heads, plastics reagent bottle bottle：Dropper head and plastic bottle are put into clear water and soaked 30 minutes；By clear water Middle the dropper head soaked and plastic bottle are cleaned with hot liquid soap, are rinsed 5 times with running water repeatedly, with distilled water immersion 1 Hour, take out dropper head and plastic bottle is filtered dry naturally.Dropper head and plastic bottle are put into 15 pounds of autoclave sterilizer by 1.5 to be sterilized 30 minutes, is taken out after cooling and is put into 35 DEG C of constant temperature and does Dry case drying for standby.2nd, the preparation of quick mirror kit reagent：2.1 Rui Shi dyestuffs and Giemsa are put into 30-40 DEG C of incubator drying box before weighing and stayed overnight in advance；2.2 weigh respectively：Rui Shi dyestuff powder 1-1.8g, Giemsa 1-1.8g.2.3 measure respectively：Glycerine 20-36ml, Tween-20 4-8ml, methanol 1000ml.2.4 place the Rui Shi dyestuffs and Giemsa that weigh in mortar, gently break dyestuff into pieces with newborn rod and are ground into powder, then row It is milled to fine powder；2.5 add glycerine dissolving dye in mortar, dyestuff is shown bright luster in newborn Bowls, and adhere to without dyestuff powder, Add proper amount of methanol to be ground, stand a moment, supernatant liquid is poured into the 2000ml volumetric flasks of a cleaning；2.6 add methanol trituration, are repeated several times, untill in mortar without dyestuff；2.7 add Tween-20 in volumetric flask, with methanol constant volume in 2000ml；Shake up and seal bottleneck, it is small to deposit room temperature dark place 24 When after filter once, refiltered after 2 weeks once, you can use；Dyeing liquor storage is more long, then dyestuff dissolving, decomposition are more abundant, this Process is the maturing process of dyeing liquor, and more than 3 months Colors of storage more preferably, notice that sealing is kept in dark place, permanently effective.3rd, kit package processing technology：Packing box specification is 120mm × 70mm × 70mm, and two 20mL reagent bottles and two rubber head droppers can be deposited in box, to examination Label, specification, the sealing label of agent box are tested；4th, the packing of kit：4.1 take methanol 15ml to load sterilized dried white reagent bottle, and label is labeled as I liquid；4.2 take the dye liquor 15ml after curing to load sterilized dried brown reagent bottle, and label is labeled as II liquid；4.3 are put into I liquid, II liquid in box, and are put into two rubber head droppers and kit operation instructions, packaging seal, are The quick microscopy diagnostic kit of swine eperythrozoonosis.
- 4. the preparation method of the quick microscopy diagnostic kit of swine eperythrozoonosis according to right 3, it is characterised in that：Institute It is to be put into 35 DEG C of incubator drying boxes in advance before weighing Rui Shi dyestuffs and Giemsa to stay overnight to state step 2.1.
- 5. the preparation method of the quick microscopy diagnostic kit of swine eperythrozoonosis according to right 3, it is characterised in that：Institute Step 2.2 is stated to weigh respectively：Rui Shi dyestuff powder 1.4g, Giemsa 1.4g.
- 6. the preparation method of the quick microscopy diagnostic kit of swine eperythrozoonosis according to right 3, it is characterised in that：On The step 2.3 is stated to measure respectively：Glycerine 28ml, Tween-20 6ml, methanol 1000ml.
- 7. the preparation method of the quick microscopy diagnostic kit of swine eperythrozoonosis according to right 3, it is characterised in that：Institute The strong acid oxidation cleaning agent washing lotion stated is formulated as：300g potassium bichromates are weighed, are placed in 2500mL distilled water, are dissolved by heating, Cooling, addition concentrated sulfuric acid 1700mL, stirring while adding slowly, is bottled after cold standby.
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| CN201710726649.5A CN107589117A (en) | 2017-08-22 | 2017-08-22 | A kind of quick microscopy diagnostic kit of swine eperythrozoonosis and preparation method thereof |
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Application publication date: 20180116 |