CN102154483A - Detection kit for diagnosing babesia caballi and preparation and use methods - Google Patents
Detection kit for diagnosing babesia caballi and preparation and use methods Download PDFInfo
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Abstract
The invention discloses a detection kit for detecting whether an equus animal is infected with a babesia caballi disease, and a preparation method and a use method of the kit. The detection kit for diagnosing babesia caballi comprises at least four babesia caballi specific primers. The use method of the detection kit comprises the following steps of: with blood genome DNA (Deoxyribonucleic Acid) extracted from equus animal blood to be detected as a template, uniformly mixing the template with a mixture of DNA polymerase, a buffer solution, sterilized ultrapure water and the babesia caballi specific primers in the kit according to a proportion; performing loop-mediated isothermal amplification; filling an amplification product in ethidium-bromide-containing agarose gel; performing electrophoresis; observing under an ultraviolet lamp; and determining whether the detected animal suffers from the babesia caballi disease according to the fact whether a specific lane has a specific electrophoretic band pattern.
Description
Technical field
The present invention relates to the detection authentication technique of a kind of zooparasite, the invention provides the detection kit whether tested equus of a kind of rapid differential diagnosis infects dull this parasitosis of BABEI exactly, and the preparation method of this test kit and using method.
Background technology
This worm of an inferior horse BABEI (Babesia caballi Nutall et Strickland, 1910) (old name Ma Jiao worm, proplasma caballi) is a member of this section of BABEI (Babesiidae) this genus of BABEI (Babesia) in Protozoa (Protozoa), top double action thing subphylum (Apicomplexa), sporozoa (Sporozoea), pyriform worm subclass (Piroplasmia), the pyriform worm order (Piroplasmida).This worm of an inferior horse BABEI is one of cause of disease of horse piroplasmosis, cause that the disease of equus is called this parasitosis of dull BABEI, be through arthropods---tick as vector-borne, parasitize in the equus red corpuscle of (comprising horse, donkey, mule and zebra), cause the high heat of animal, anaemia, jaundice, hemorrhage, have difficulty in breathing, become thin and the body surface swollen lymph node is a class blood protozoon property parasitosis of feature.(Office International Des Epizooties OIE) classifies it as category-B eqpidemic disease (OIE.Equine piroplasmosis.2008), and China classifies them as two class eqpidemic diseases in International Office of Epizootics.The cause of disease of horse piroplasmosis comprises this worm of dull BABEI and horse Taylor worm (Theileria equi Mehlhorn, Schein1998, the burnt worm of old name Na Shi).In China, an inferior horse BABEI this parasitosis mainly is popular in ground such as northeast, east Inner Mongolia, Qinghai and Xinjiang, grassland that certified main communication media is Dermacentor leather tick, dermacentor silvarum, silver-colored shield leather tick, China's leather tick (the Wang Ming chief editor. veterinary parasitology [M]. Beijing: Chinese agriculture press, 2008:366-369).This sick popularity in season is very strong, it is main being acute process more, M ﹠ M is all very high, and especially the harm to the equus of external introduction, thoroughbred and hybridization horse is bigger, is one of principal disease of restriction developing country equus aquaculture industry development.At present, the diagnosis of dull this parasitosis of BABEI mainly contains following several means: (1) blood smear dyeing microscopic examination, lymph node puncture test, vitro culture, clinical diagnosis and animal inoculation pvaccination test etc.(2) serological method, as: complement fixation test (CFT) (CFT), indirect fluorescent antibody test (IFAT), enzyme linked immunosorbent assay (ELISA) and competition enzyme-linked immunosorbent adsorption test (C-ELISA) etc., but false positive or omission appear in these method regular meetings, referring to Donnelly, J., Phipps, L.P., Watkins, K.L., 1982.Evidence of maternal antibodies to Babesia equi and B.caballi in foals of seropositive mares.Equine Vet.J.14,126-128.; Weiland, G., Aicher, B.M., Boch, J., 1984.Serodiagnosis and therapy control of equine piroplasmosis by CFT and IFAT.Berl.Munch.Tierarztl.Wochenschr.97,34134-34139.; Knowles Jr., D.P., Perryman, L.E., Goff, W.L., Miller, C.D., Harrington, R.D., Gorham, J.R., 1991.A monoclonal antibody defines a geographically conserved surface protein epitope of Babesia equi merozoites.Infect.Immun.59,2412-2417.; R., Jorgensen, W.K., Dalgliesh, R.J., Friedhoff, K.T., de Vos, A.J., 1995.Current state and future trends in the diagnosis of babesiosis.Vet.Parasitol.57,61-74.; Br ü ning, A., Phipps, P., Posnett, E., Canning, E.U., 1997.Monoclonal antibodies against B abesia caballi and B abesia equi and their application in serodiagnosis.Vet.Parasitol.68,11-26.; Kappmeyer, L.S., Perryman, L.E., Hines, S.A., Baszler, T.V., Katz, J.B., Hennager, S.G., Knowles, D.P., 1999.Detection of equine antibodies to Babesia caballi by recombinant B.caballi rhoptry-associated protein 1in a competitive-inhibition enzyme-linked immunosorbent assay.J.Clin.Microbiol.37,2285-2290..(3) molecular detection technology, as PCR, Chao Shi round pcr, multiplex PCR and reverse wire blot hybridization technique (RLB) etc., compare with the method for routine have the susceptibility height, high specificity, recall rate advantages of higher, specifically referring to BASHIRUDDIN J.B., CAMMA C.﹠amp; REBELOE. (1999) .Molecular detection of Babesia equi and Babesia caballi in horse blood bv PCR amplification of part of the 16S rRNA gene.Vet.Parasitol., 84,75-83; SAHAGUN-RUIZ A., WAGNELA S.D., HOLMAN P.J., CHIEVES L.P.﹠amp; WAGNER G.G. (1997) .Biotin-labeled DNA probe in a PCR-based assay increases detection sensitivity for the equine hemoparasite Babesia caballi.Vet.Parasitol., 73,53-63.But above two class methods all have an inevitable shortcoming---need advanced person, the instrument of costliness and the technician of professional training, and testing process is consuming time more, come this parasitosis of differential diagnosis an inferior horse BABEI so need work out a kind of simple, convenient, practical method, this has important Special Significance in clinical diagnosis, treatment and research.
Summary of the invention
But the invention provides the detection kit of a kind of this worm of rapid differential diagnosis an inferior horse BABEI, the using method of this test kit is provided simultaneously.Test kit of the present invention and using method can overcome the deficiencies in the prior art, need not expensive instrument, and detection sensitivity are higher, and operative technique is simple relatively, quick simultaneously, can implement whole testing processes under the common lab condition.
Have at least this worm Auele Specific Primer of four dull BABEIs to form in the detection kit of dull this worm of BABEI of diagnosis of the present invention, these four primers are: the outside, upstream primer is the nucleotide sequence Bc F3 of SEQ ID NO:1 in the sequence table; The outside, downstream primer is the nucleotide sequence Bc B3 of SEQ ID NO:2 in the sequence table; Upstream inner primer Bc FIP is formed by connecting through connexon L1 by primer Bc F2 in the middle of the upstream of the nucleotide sequence of the nucleotide sequence Bc F1c of SEQ ID NO:3 in the sequence table and SEQ ID NO:4; Downstream inner primer Bc BIP is formed by connecting through connexon L2 by primer Bc B2 in the middle of the downstream of the nucleotide sequence of SEQ ID NO:6 in the nucleotide sequence Bc B1c of SEQ ID NO:5 in the sequence table and the sequence table, and connexon L1 and L2 are made up of 4～6 nucleic acid bases.Connexon L1 and L2 can be identical, also can be different, as long as meet some requirements, that is: behind the adding connexon, do not influence primer specificity, the various secondary structures of the amplification efficiency that yet can not exert an influence.In goal gene, the concrete sequence of each primer correspondence sees Table 1, and each primer mutual alignment concerns that synoptic diagram and primer structure form synoptic diagram and ask for an interview accompanying drawing 1.
An optimum combination of having verified among the present invention is: the outside, upstream primer Bc F3; The outside, downstream primer Bc B3; Upstream inner primer Bc FIP is the nucleotide sequence Bc FIP-I of SEQ ID NO:7 in the sequence table; Downstream inner primer Bc BIP is the nucleotide sequence BcBIP-I of SEQ ID NO:8 in the sequence table
The concrete sequence of the specific primer of each this worm of dull BABEI sees Table 1 among the present invention.
The concrete sequence of each this worm Auele Specific Primer of dull BABEI among table 1 the present invention
| Bc?F3： | 5’-GTCGGGGATTGATTTTTGCA-3’ |
| Bc?B3： | 5’-TTCACAAAACTTCCCAAGGC-3’ |
| Bc F2 | 5 '-CGAGGAATGCCTAGTATGCG-3 ' |
| Bc F1c | 5 '-GTGTACAAAGGGCAGGGACGTA-3 ' |
| Bc B2 | 5 '-GACGAATCGGAAAAGCCAC-3 ' |
| Bc B1c | 5 '-CGTCGCTCCTACCGATCGAG-3 ' |
| BcFIP-I: | 5 '-GTGTACAAAGGGCAGGGACGTA GAATTCGAGGAATGCCTAGTATGCG-3 ' |
| Bc BIP-I: | 5 '-CGTCGCTCCTACCGATCGAG AATTCGACGAATCGGAAAAGCCAC-3 ' |
| Remarks: the sequence of drawing the horizontal line part in the primer sequence is a connexon. |
Use for convenient, can also be placed with the pure super water of LAMP reaction buffer, archaeal dna polymerase and sterilization more respectively in the detection kit of dull this worm of BABEI of diagnosis of the present invention.These reagent of placing respectively can be by the service requirements proportioning when concrete use.
Be to have placed following content in specific embodiment:
A) genomic dna of this worm of standard an inferior horse BABEI;
B) standard-bred Taylor molitor genomic dna;
C) the horse genomic dna of standard-bred pyriform worm feminine gender (that is, this worm of dull BABEI and horse Taylor worm all are negative);
D) the donkey genomic dna of standard-bred pyriform worm feminine gender;
E) the mule genomic dna of standard-bred pyriform worm feminine gender;
F) sterilization ultrapure water;
G) LAMP reaction buffer;
H) archaeal dna polymerase;
And by Bc FIP, the Bc BIP of 40p mol and the Bc F3 of 5p mol, this worm special primer mixture of dull BABEI that Bc B3 forms.
The using method of the detection kit of dull this worm of BABEI of diagnosis of the present invention is: extracting poba gene group DNA with equus blood to be measured is template, with itself and archaeal dna polymerase, damping fluid, after mixing in proportion, this worm special primer mixture of dull BABEI in sterilization ultrapure water and the test kit encircles the constant-temperature amplification of mediation, deactivation archaeal dna polymerase at once after the amplification, getting amplified production is damping fluid with TAE, place the sepharose that contains ethidium bromide again, carry out electrophoresis, under ultraviolet lamp, observe, specificity electrophoresis banding pattern whether occurs according to specific swimming lane and determine whether tested animal suffers from this parasitosis of dull BABEI.
The present invention is actually isothermal amplification technology (the Loop-mediated isotheral amplification method that adopts the ring mediation, LAMP) a kind of detection method, this method is susceptibility very high DNA cloning technology (the Notomi T. of Japanese scientist such as Notomi T. in invention in 2000, Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., Hase T., 2000.Loop-mediated isothermal amplification of DNA.Nucleic Acids Research 28, E63.).The present invention differentiates in the detection kit of dull this worm of BABEI and the detection method that being is target gene with 18S rRNA gene, design the specific LAMP primer of this worm of dull BABEI, the poba gene group DNA that extracts with animal via blood sampling to be checked back is the constant-temperature amplification that template is encircled mediation.Do not need expensive plant and instrument when adopting this worm of method differential diagnosis of the present invention an inferior horse BABEI, only need conventional whizzer, water-bath and simple electrophoresis equipment just can finish detection, can implement in ordinary laboratory, and can obtain having the susceptibility height, detected result that specificity is good.
Description of drawings
Fig. 1 for each primer among the present invention in goal gene the mutual alignment relation and the composition synoptic diagram of each amplimer.
Fig. 2 is for adopting among the present invention with the result of this worm specificity optimum combination primer of dull BABEI through the constant-temperature amplification rear electrophoresis detection of ring mediation, wherein: M is DNA standard molecular weight (Wide Range DNA Marker (100～6000)), the tested DNA sample that the 1-6 swimming lane is corresponding respectively is: 1, and this worm male horse genomic dna (standard positive control) of standard an inferior horse BABEI; 2, blank (sterilization ultrapure water); 3, the horse genomic dna of standard-bred pyriform worm feminine gender; 4, the donkey genomic dna of standard-bred pyriform worm feminine gender; 5, the genomic dna of the negative mule of standard-bred pyriform worm; 6, standard-bred Taylor molitor genomic dna.
Embodiment
Preferred forms of the present invention below is provided.
The detection method of present embodiment can be carried out in the centrifuge tube of 200 μ L or 50 μ L, and employed concrete primer and reagent are as follows:
1) this worm special primer of dull BABEI:
Bc?F3：5’-GTCGGGGATTGATTTTTGCA-3’；
Bc?B3：5’-TTCACAAAACTTCCCAAGGC-3’；
Bc?FIP-I：5’-GTGTACAAAGGGCAGGGACGTA
GAATTCGAGGAATGCCTAGTATGCG-3’；
Bc?BIP-I：5’-CGTCGCTCCTACCGATCGAG
AATTCGACGAATCGGAAAAGCCAC-3’；
2) this worm Auele Specific Primer mixture of dull BABEI (that is, Bc LAMP primer premixed liquid contains Bc F3, the Bc B3 of 5p mol and Bc FIP-I, the Bc BIP-I of 40p mol).
3) 2 * LAMP reaction buffer (40mM Tris-HCl (pH 8.8), 20mM KCl, 16mMMgSO
4, 20mM (NH
4)
2SO
4, 0.2%Tween 20,1.6M betaine (trimethyl-glycine) and 2.5mM dNTP).
4) the Bst archaeal dna polymerase (M0275L, BioLabs).
5) this molitor genomic dna of standard an inferior horse BABEI;
6) standard-bred Taylor molitor genomic dna;
7) the horse genomic dna of standard-bred pyriform worm feminine gender;
8) the donkey genomic dna of standard-bred pyriform worm feminine gender;
9) the mule genomic dna of standard-bred pyriform worm feminine gender;
10) sterilization ultrapure water
11) 6 * sample-loading buffer (30mM EDTA, 36% (V/V) glycerine (Glycerol), 0.05% (W/V) tetrabromophenol sulfonphthalein (Bromophenol Blue), the aqueous solution of 0.05% (W/V) dimethylbenzene nitrile indigo plant (Xylene CyanolFF)).
12) 1 * TAE damping fluid (0.04M Tris-acetate, 0.002M EDTA).
13) 2% agarose (with the preparation of 1 * TAE damping fluid)
14) 10mg/mL ethidium bromide
Its concrete working method is as follows:
(1) acquisition of this molitor genomic dna of standard an inferior horse BABEI
Containing the live blood inoculation experiments equus respectively of worm kind of this worm of dull BABEI with what preserve in the liquid nitrogen, reach 1% when above when dying the worm rate, is antithrombotics with 20% Sodium Citrate, venous blood collection, with centrifugal 10 minutes of 1000 * g under 4 ℃ of conditions of institute's blood sampling, supernatant discarded was inhaled white corpuscle as far as possible and is abandoned.With three times (same pre-treatment, centrifugal 10 minutes of 1000 * g) of 2% Sodium Citrate washing, at last supernatant is removed, the red corpuscle branch is packed in the 1.5mL centrifuge tube, and-20 ℃ of preservations are standby.Blood promptly obtains this molitor genomic dna of standard an inferior horse BABEI after extracting genomic dna thus.
(2) acquisition of standard-bred Taylor molitor genomic dna
Contain the live blood inoculation experiments equus respectively of worm kind of horse Taylor worm with what preserve in the liquid nitrogen, reach 1% when above, the same processing when dying the worm rate.Blood promptly obtains standard-bred Taylor molitor genomic dna after extracting genomic dna thus.
(3) acquisition of the animal blood genomic dna of standard-bred pyriform worm feminine gender
Menses plate coating checking and PCR detect this worm of dull BABEI and horse Taylor worm, (continuous more than 40 days) all is the equus of jack to jack adapter for a long time, gather anticoagulation through vein, obtain the animal blood genomic dna of standard-bred pyriform worm feminine gender behind the extraction animal gene group DNA ,-20 ℃ of preservations are standby.Promptly obtain the animal blood genomic dna of standard-bred pyriform worm feminine gender thus behind the blood extraction genomic dna.
(4) method of extraction genomic dna from blood
A) adding of 300 μ L blood is filled in the 1.5mL centrifuge tube of 900 μ L BRC Lysis Solution, put upside down 10 times, in incubated at room 1 to 3 minute (that is, red corpuscle is wanted complete cracking, liquid clear).
B) 2000 * g is centrifugal 2 minutes, careful supernatant discarded, visible white precipitate and about 10～20 μ L liquid below keeping, 20 seconds mixings of vortex or blow and beat mixing with suction nozzle.
C) add 300 μ L Cell Lysis Solution in every pipe, the piping and druming mixing.
D) add 100 μ L Protein Preciptation Solution, 20 seconds mixings of spiral or blow and beat mixing with suction nozzle are until the brown precipitation particle occurring.
E) 13000 * g moves to supernatant liquor in the new excessively centrifuge tube of mark after centrifugal 3 minutes, adds 300 μ L Virahols, puts upside down 50 times.
F) 13000 * g is centrifugal 5 minutes.Abandon supernatant, on the filter paper of cleaning, carefully be inverted, residual liquid is blotted.
G) add 300 μ L, 70% ethanol.Centrifugal 3 minutes of 13000 * g carefully removes supernatant liquor, blots residual liquid with thieving paper.
H) room temperature or 37 ℃ are dry air 3-5 minute.
I) add 100 μ L DNA Hydration Solution, 5 seconds mixings of spiral or repeatedly with suction nozzle piping and druming.
J) 65 ℃ of incubations 5 minutes are with dissolving DNA.
K) room temperature shaken over night or a few hours then ,-20 ℃ of preservations are standby.
The preparation of (five) 2 * LAMP reaction buffers
After at first using 2 * LAMP reaction buffer storage liquid of ultrapure water according to the no dNTP of ratio preparation in the table 2 of sterilization, with the filter filtration of 0.45 μ m.The storage liquid of being prepared can be preserved-20 ℃ of prolonged preservation 3 months at 4 ℃.
Preparation and required each ratio of reagents of 2 * LAMP reaction buffer storage liquid of the no dNTP of table 2
| Reagent | Cumulative volume 900mL | Cumulative volume 90mL |
| 1M?Tris-Cl(pH?8.8) | 40mL | 4mL |
| KCl | 1.49g | 0.15g |
| MgSO 4(MgSO 4·7H 2O) | 1.93g(3.94g) | 0.19g(0.39g) |
| (NH 4) 2SO 4 | 2.64g | 0.26g |
| Tween?20 | 2mL | 0.2mL |
| Betaine (trimethyl-glycine) | 187.4g | 18.7g |
Add 100 μ L 25mM dNTP before using in 2 * LAMP reaction buffer storage liquid of per 900 μ L, mixing promptly is prepared into 2 * LAMP reaction buffer, and-20 ℃ of preservations are standby, can preserve more than three months.
(6) testing process
In the centrifuge tube of 50 μ L or 200 μ L, add each reagent and sample DNA to be checked, set up standard positive control, standard negative control, blank and the contrast of standard-bred Taylor molitor genomic dna simultaneously in the described ratio of table 3.
Table 3Bc LAMP reacts the adding proportion and the standard testing result thereof of required all ingredients
Mixing gently, moment is centrifugal.(experimental screening obtains under the specified temp in water-bath, this worm of the present invention an inferior horse BABEI is 65 ℃) (experimental screening obtains the amplification appropriate time, the best proliferation time of this worm Auele Specific Primer of dull BABEI is 40 minutes among the present invention, can extend to 60 minutes), deactivation 2 minutes in 80 ℃ of water-baths at once then.Getting amplified production 5 μ L, is damping fluid with TAE, in the sepharose 2% (containing 0.5 μ g/mL ethidium bromide), carries out nucleic acid electrophoresis and detect under 75 volts of voltages.Observation and result of determination under ultraviolet lamp.
(7) detecting example and result judges
1. the detection of dull this worm of BABEI:
In the centrifuge tube of 6 200 μ L, add all ingredients respectively in ratio in the table 3, set up standard positive control, standard negative control, blank and the contrast of standard-bred Taylor molitor genomic dna simultaneously, wherein: Bc LAMP primer premixed liquid is the mixture of this worm special primer of dull BABEI; Genome DNA sample in different reaction tubess is respectively the horse genomic dna of this molitor genomic dna of dull BABEI, standard-bred pyriform worm feminine gender, the donkey genomic dna of standard-bred pyriform worm feminine gender, genomic dna, the horse Taylor molitor genomic dna of the negative mule of standard-bred pyriform worm.Mixing gently after all application of samples finish, moment is centrifugal; Increased 40 minutes under 65 ℃ of conditions in thermostat water bath or in the PCR instrument; Inactivator 2 minutes under 80 ℃ condition at once then; Getting amplified production 5 μ L, is damping fluid with TAE, in the sepharose 2% (containing the 0.5ug/mL ethidium bromide), carries out electrophoresis detection under 75 volts of voltage conditions, and detected result is seen accompanying drawing 1.The result shows can be by specific amplification in the reaction tubes (standard positive) that has only this molitor genomic dna correspondence of dull BABEI, the specific DNA band occurs, and specific band all do not occur in the genomic dna of horse Taylor molitor genomic dna, the negative equus of horse pyriform worm worm and the corresponding reaction tubes of sterilization ultrapure water.
When judging whether tested equus has infected this worm of dull BABEI, with the poba gene group DNA of tested equus after above detection, if occur in the electrophoresis result consistent with result's (being designated as 1 electrophoresis road in the accompanying drawing) of the corresponding reaction tubes of this molitor genomic dna of dull BABEI in the accompanying drawing 1, simultaneously set various contrasts are also consistent, and in the time of getting rid of tested animal DNA sample by this molitor genomic dna of dull BABEI and reaction product pollution thereof, can determine that tested equus has infected this worm of dull BABEI.
By above example as seen, it is good that detection kit of the present invention has specificity, and testing process is simple relatively, can finish all testing processes in 2 hours the soonest, need not expensive instrument and equipment.
Claims (4)
1. a detection kit of diagnosing dull this worm of BABEI is characterized in that having at least in the detection kit this worm Auele Specific Primer of four dull BABEIs to form, and these four primers are: the outside, upstream primer is the nucleotide sequence Bc F3 of SEQ IDNO:1 in the sequence table; The outside, downstream primer is the nucleotide sequence Bc B3 of SEQ ID NO:2 in the sequence table; Upstream inner primer Bc FIP is formed by connecting through connexon L1 by primer Bc F2 in the middle of the upstream of the nucleotide sequence of SEQ ID NO:4 in the nucleotide sequence Bc F1c of SEQ ID NO:3 in the sequence table and the sequence table; Downstream inner primer Bc BIP is formed by connecting through connexon L2 by primer Bc B2 in the middle of the downstream of the nucleotide sequence of SEQ ID NO:6 in the nucleotide sequence Bc B1c of SEQ ID NO:5 in the sequence table and the sequence table, and connexon L1 and L2 are made up of 4～6 nucleic acid bases.
2. the detection kit of dull this worm of BABEI of diagnosis according to claim 1 is characterized in that upstream inner primer Bc FIP is the nucleotide sequence Bc FIP-I of SEQ ID NO:7 in the sequence table; Downstream inner primer Bc BIP is the nucleotide sequence BcBIP-I of SEQ ID NO:8 in the sequence table.
3. the detection kit of dull this worm of BABEI of diagnosis according to claim 1 and 2 is characterized in that also being placed with respectively in the detection kit LAMP reaction buffer, archaeal dna polymerase, sterilization ultrapure water.
4. the using method of the detection kit of dull this worm of BABEI of the described arbitrary diagnosis of claim 1 to 3, it is characterized in that extracting poba gene group DNA with equus blood to be measured is template, with itself and archaeal dna polymerase, damping fluid, after this worm Auele Specific Primer mixture of dull BABEI in sterilization ultrapure water and the test kit mixes in proportion, encircle the constant-temperature amplification of mediation, deactivation archaeal dna polymerase at once after the amplification, getting amplified production is damping fluid with TAE, place the sepharose that contains ethidium bromide again, carry out electrophoresis, under ultraviolet lamp, observe, specificity electrophoresis banding pattern whether occurs according to specific swimming lane and determine whether tested animal suffers from this parasitosis of dull BABEI.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710433A (en) * | 2013-11-14 | 2014-04-09 | 中国检验检疫科学研究院 | Babesiidae protozoon detection primer and real-time fluorescent quantitative PCR (polymerase chain reaction) kit |
| CN104593493A (en) * | 2015-01-09 | 2015-05-06 | 中国农业科学院兰州兽医研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) kit for detecting ovine babesia |
| CN109633160A (en) * | 2018-11-13 | 2019-04-16 | 新疆农业大学 | Horse Babesia caballi disease colloidal gold colloidal gold detection test paper strip and its preparation and application method |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009049630A1 (en) * | 2007-10-16 | 2009-04-23 | Aarhus Universitet | Isothermal amplification method for detection of nucleic acid mutation |
| CN101838690A (en) * | 2010-01-28 | 2010-09-22 | 中国水产科学研究院东海水产研究所 | Method for fast detecting Alexandrium mimutum Halim by using loop-mediated isothermal amplification technology |
-
2011
- 2011-02-15 CN CN 201110041865 patent/CN102154483B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009049630A1 (en) * | 2007-10-16 | 2009-04-23 | Aarhus Universitet | Isothermal amplification method for detection of nucleic acid mutation |
| CN101838690A (en) * | 2010-01-28 | 2010-09-22 | 中国水产科学研究院东海水产研究所 | Method for fast detecting Alexandrium mimutum Halim by using loop-mediated isothermal amplification technology |
Non-Patent Citations (2)
| Title |
|---|
| 《Experimental Parasitology》 20080418 Guiquan Guan等 The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China 第120卷, * |
| 《Veterinary Parasitology》 20071231 Andy Alhassan等 Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis 第143卷, * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710433A (en) * | 2013-11-14 | 2014-04-09 | 中国检验检疫科学研究院 | Babesiidae protozoon detection primer and real-time fluorescent quantitative PCR (polymerase chain reaction) kit |
| CN104593493A (en) * | 2015-01-09 | 2015-05-06 | 中国农业科学院兰州兽医研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) kit for detecting ovine babesia |
| CN109633160A (en) * | 2018-11-13 | 2019-04-16 | 新疆农业大学 | Horse Babesia caballi disease colloidal gold colloidal gold detection test paper strip and its preparation and application method |
| CN110016514A (en) * | 2019-04-12 | 2019-07-16 | 中国农业科学院兰州兽医研究所 | A kind of primer sets and kit can be used for the detection of horse pyriform worm |
| CN110551836A (en) * | 2019-07-09 | 2019-12-10 | 沈阳农业大学 | Dual-fluorescence RPA detection method for equine piroplasmosis |
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