CN206512204U - A kind of PCR detection kit for detecting this burnt worm of dog BABEI - Google Patents
A kind of PCR detection kit for detecting this burnt worm of dog BABEI Download PDFInfo
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- CN206512204U CN206512204U CN201621477107.6U CN201621477107U CN206512204U CN 206512204 U CN206512204 U CN 206512204U CN 201621477107 U CN201621477107 U CN 201621477107U CN 206512204 U CN206512204 U CN 206512204U
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Abstract
The utility model discloses a kind of PCR detection kit for detecting this burnt worm of dog BABEI, include packing box 1, liner 2 is provided with several positioning holes on liner 2, Reagent Tube is installed by positioning hole and positioned inside it, and the Reagent Tube includes what is installed side by side：5xbuffer pipes 3, Tag enzymes pipe 4, dNTPmix pipes 5, primer mix pipes 6, positive control pipe 7, negative control pipe 8, deionization water pipe 9.The utility model be have developed for detecting dog Babesiosis PCR quick diagnosis reagent kits, and dog Babesiosis etiology nucleic acid, the features such as kit has high quick, sensitivity, high specificity can be directly detected from dog whole blood clinical sample.Suitable for the detection, diagnosis and epidemiology survey of dog Babesiosis.
Description
Technical field
The utility model is a kind of PCR detection kit for detecting this burnt worm of dog BABEI, belongs to the detection neck of animal pathogenic
Domain, the single-minded PCR detection kit for this burnt worm of dog BABEI, it is adaptable to the quick detection kit of this burnt worm of dog BABEI.
Background technology
The Babesia Gibsoni of dog is mainly propagated through hard tick and parasitizes caused entomophila property bloodprotozoonoses in dog red blood cell.
Babesia is subordinate to Subkingdom Protozoa, Sporozoa, Eucoccida, pyriform worm suborder, Babesia section, the one of Babesia category
Class protist parasite (equal 2001) of Gu Jun.Babesia main parasitic is in the red blood cell of vertebrate, area prevailing
Including Asia, Africa, Europe, the Middle East and North America.The category includes more than 90 kinds of Babesia, and except polypide out of the ordinary such as vole
Babesia (Babesiamicroti) not only parasitize vole and can also encroach on human body sometimes, remaining most of Babesia is all
There is strict host specificity (Zhang Xichen and Zhao Quan 2005).
Classification on infecting dog Babesia cause of disease, was broadly divided into big young waiter in a wineshop or an inn's type according to the size of Babesia in the past,
(2.5~5.0 μm) of volume the greater is dog Babesia (Babesiacanis), and polypide resembles a pear in shape；(the 1.0~2.5 of small volume
μm) it is Ji Shi Babesias (Babesiagibsoni), polypide is in ring sample.It is quick due to Protocols in Molecular Biology in recent years
Development, experiment is using to rRNA small ylidene gene (18SrDNA), the first and second the Internal Transcribed Spacers (ITS1, ITS2)
Further investigate, table site, intervention 5.8SrRNA gene coding regions, heat shock protein 70 (HSP70) and cytochrome b gene
The bright Babesia for infecting dog has two kinds incessantly, and the infection of each Babesia Gibsoni can show different clinical conditions
Shape.The Babesia having now found that has dog Babesia, Webster Babesia, Roche Babesia, Ji Shi Babesias, Kang La
Moral Babesia, microti sample pyriforms worm (also known as Taylor protozoon or " Spain's separation strains "), its also a kind of unnamed form
Similar to this large-scale Babesia of double bud BABEIs, the dog of main infection immunologic hypofunction.
Or according to clinical symptom characteristic (hyperpyrexia, anaemia, blood red egg blood urine and splenomegaly) and with the presence of tick bite
Medical history can make tentative diagnosis.Adopt disease dog peripheral blood (have sharp ears blood) and make smear, microscopy after the dyeing of Rui Shi liquid finds have in red blood cell
Babesia can be made a definite diagnosis.At present, the microscopic examination of perpheral blood smear technology of blood sample is still the diagnosis most suitable method of dog Babesia Gibsoni,
But this method has a very big shortcoming-hyposensitivity, therefore it is very low dynamic to may not apply to periphery hematozoic parasite content
The detection with echiuran after thing and infection are resistance to.Serological testing is also that dog Babesia epidemiological study is effective
Method, but such method can not distinguish animal be in infection in or infected resistance to mistake, it is impossible to identification band worm move
Thing and reservoir host.And found during the Babesia Gibsoni of detection dog, dog Babesia and two-pressure humidity generator it
Between often there is cross reaction.The higher method of sensitiveness, such as Standard PCR detection technique, the molecule diagnosis based on DNA level,
The number that its sensitiveness is expanded with target gene is closely bound up.Therefore the gene of one high copy number in genome of selection is made
The sensitiveness of existing PCR method will be greatly improved for target gene.Set up a kind of easy, efficiently, practical DNA detection methods.
This method can be used for dog Babesia Gibsoni quick detection, have a extensive future.
Utility model content
The purpose of this utility model is to avoid the deficiencies in the prior art from providing a kind of PCR inspections for detecting this burnt worm of dog BABEI
Test agent box.
To achieve the above object, the technical scheme that the utility model is taken is：A kind of PCR inspections for detecting this burnt worm of dog BABEI
Test agent box, it is characterised in that the PCR detection kit of this burnt worm of the detection dog BABEI, includes packing box 1, liner 2,
Several square positioning holes are provided with liner 2, Reagent Tube is installed by positioning hole and positioned inside it, the Reagent Tube bag
Include what is installed side by side：5xbuffer pipes 3, Tag enzymes pipe 4, dNTPmix pipes 5, primer mix pipes 6, positive control pipe 7, negative control
Pipe 8, deionization water pipe 9；
It is preferred that, positive control pipe 7 is brown pipe, and negative control pipe 8 is managed for blueness；
It is preferred that, 5xbuffer pipes 3 are managed for translucent PE；
It is preferred that, Tag enzymes pipe 4 is completely black opaque PE pipes.
Above-mentioned various liquid are attached separately in container, in insertion container hole.
Described nucleotides sequence is classified as：
Sense primer is 5 '-TAGCGATGGACCATTCAAGT-3 '；
Anti-sense primer is 5 '-CGTTTGTCTGGACCTGGTGAG-3 '；
Wherein, described positive control is：The dog BABEI separated from one dog Babesiosis positive sample of hospital
This burnt worm builds plasmid.
Described negative control is distilled water.
PCR response procedures are：95℃5min；95℃30s,56℃40s,72℃1min,35Cycle；72℃10min；4℃
∞。
Result of determination：If expanded from sample to be detected to 892bp fragments, the detection sample contain BABEI this
Babesiasis is former.
The beneficial effects of the utility model are：By adopting the above-described technical solution, the utility model have developed for examining
The PCR kit of this burnt worm of dog BABEI is surveyed, dog Babesiosis protokaryon acid can be directly detected from dog whole blood clinical sample,
The sensitivity of kit is 34.2fg, and the whole detection process of result is only needed 5 hours from starting to going out.As can be seen here, the kit
With quick, sensitivity is high, high specificity the features such as.Suitable for the detection, diagnosis and epidemiology survey of this burnt worm of dog BABEI.
Brief description of the drawings
Fig. 1 is schematic perspective view of the present utility model；Wherein, packing box 1, liner 2,5xbuffer pipes 3, Tag enzymes pipe 4,
DNTPmix pipes 5, primer mix pipes 6, positive control pipe 7, negative control pipe 8, deionization water pipe 9；
Testing result in Fig. 2 utility models embodiment 2, wherein M representation DNAs 2000Marker, 1 represents positive sample, 2
Represent negative control.
The testing result of Fig. 3 utility model sensitivity Detections, M representation DNAs 2000Marker, 1, which represents 00,2, represents 10-1、3
Represent 10-2, 4 represent 10-3, 5 represent 10-4, 6 represent 10-5, 7 represent 10-6, 8 represent 10-7, 9 represent negative control.
The testing result of Fig. 4 utility model specific detections, M representation DNA 2000Marker are 1 respectively, 2,3,4,5,6,
7th, 8,9,10 sample, 11 represent negative control.
Embodiment
Principle of the present utility model and feature are described with reference to embodiments, example is served only for explaining this reality
With new, scope of the present utility model is not intended to limit.
Embodiment 1：A kind of PCR detection kit for detecting this burnt worm of dog BABEI, the PCR of this burnt worm of the detection dog BABEI
Detection kit includes packing box 1, and liner 2 is provided with several square positioning holes on liner 2, will be tried by positioning hole
Agent pipe, which is installed, to be positioned inside it, and the Reagent Tube includes what is installed side by side：5xbuffer pipes 3, Tag enzymes pipe 4, dNTPmix pipes 5,
Primer mix pipes 6, positive control pipe 7, negative control pipe 8, deionization water pipe 9；
It is preferred that, positive control pipe 7 is brown pipe, and negative control pipe 8 is managed for blueness；
It is preferred that, 5xbuffer pipes 3 are managed for translucent PE；
It is preferred that, Tag enzymes pipe 4 is completely black opaque PE pipes.
Above-mentioned various liquid are attached separately in container, in insertion container hole.
Described nucleotides sequence is classified as：
Sense primer is 5 '-TAGCGATGGACCATTCAAGT-3 '；
Anti-sense primer is 5 '-CGTTTGTCTGGACCTGGTGAG-3 '；
Wherein, described positive control is：The dog BABEI separated from one dog Babesiosis positive sample of hospital
This burnt worm builds plasmid.
Described negative control is distilled water.
PCR response procedures are：95℃5min；95℃30s,56℃40s,72℃1min,35Cycle；72℃10min；4℃
∞.Embodiment 2：A kind of application method for the PCR detection kit for detecting this burnt worm of dog BABEI, comprises the following steps：
It is research object from a positive sample, usesDNA Mini kit kits extract DNA.Extract
Flow is as follows：20ul Proteinase Ks are added in 1.5ml centrifuge tubes, 200ul whole bloods are added；200ul AL liquid is added, 56 are mixed
DEG C, 10min；200ul absolute ethyl alcohol is added, is mixed；700ul liquid is transferred in 2ml centrifugal column, centrifuges, outwells waste liquid；
750ulAW1 is added, waste liquid is outwelled in centrifugation, add 750ulAW2, centrifugation outwells waste liquid, repeats this step once, and sky is from 2 points
Clock, opens pillar cool a few minutes；50TEbuffer is added, -80 preserve
2nd, PCR is detected
PCR reaction systems 25ul, 5Xbuffer5ul, dNTP2ul, primer mix2ul, Tag enzyme 0.2ul extract DNA2ul,
ddH2O 13.8ul
95℃5min；95℃30s,56℃40s,72℃1min,35Cycle；72℃10min；4℃∞.
PCR primer carries out 1.5% agarose gel electrophoresis, using DNA2000Marker, after Ethidium Bromide color upper gel into
As system, Figure of description Fig. 2, M representation DNA 2000Marker is specifically shown in, 1 represents positive sample, and 2 represent negative control.
Embodiment 3：Sensitivity Detection
The plasmid built is chosen, the mass concentration extracted with spectrophotometric determination is 342ng/ul, take out 1ul and make ladder
Degree dilution, is followed successively by 00,10-1、10-2、10-3、10-4、10-5, 10-6、10-7、10-8, on institute's method carry out method enter performing PCR,
PCR primer is shown in Figure of description Fig. 3, M representation DNA 2000Marker, and 1, which represents 00,2, represents 10-1, 3 represent 10-2, 4 represent 10-3, 5 represent 10-4, 6 represent 10-5, 7 represent 10-6, 8 represent 10-7, 9 represent 10-8, 10 represent negative control.
Embodiment 4：Specific detection
10 parts of Babesiosis positive sample whole bloods are have collected from each of Beijing pets hospital, through PCR testing results such as
Electrophoretogram 4, M representation DNA 2000Marker are 1,2,3,4,5,6,7,8,9,10 samples respectively, 11 represent negative control.
Preferred embodiment of the present utility model is the foregoing is only, it is all in this practicality not to limit the utility model
Within new spirit and principle, any modification, equivalent substitution and improvements made etc. should be included in guarantor of the present utility model
Within the scope of shield.
Claims (1)
1. a kind of PCR detection kit for detecting this burnt worm of dog BABEI, it is characterised in that this burnt worm of the detection dog BABEI
PCR detection kit includes packing box (1), and liner (2) is provided with several square positioning holes on liner (2), by fixed
Reagent Tube is installed and positioned inside it by position hole, and the Reagent Tube includes what is installed side by side：5xbuffer manages (3), Tag enzymes pipe (4),
DNTPmix manages (5), primer mix pipes (6), positive control pipe (7), negative control pipe (8), deionization water pipe (9)；Wherein, it is positive
Control tube (7) is brown pipe, and negative control pipe (8) is managed for blueness；5xbuffer manages (3) and managed for translucent PE；Tag enzyme pipes
(4) it is completely black opaque PE pipes.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201621477107.6U CN206512204U (en) | 2016-12-30 | 2016-12-30 | A kind of PCR detection kit for detecting this burnt worm of dog BABEI |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111534625A (en) * | 2020-06-29 | 2020-08-14 | 天津拓瑞医药科技有限公司 | PCR detection method for Babesia gibsoni |
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2016
- 2016-12-30 CN CN201621477107.6U patent/CN206512204U/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111534625A (en) * | 2020-06-29 | 2020-08-14 | 天津拓瑞医药科技有限公司 | PCR detection method for Babesia gibsoni |
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