CN101597650A - Detect detection film and the method for cattle and sheep pyriform worm in the blood tick body of Qinghai - Google Patents
Detect detection film and the method for cattle and sheep pyriform worm in the blood tick body of Qinghai Download PDFInfo
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Abstract
The present invention relates to detect in the blood tick body of Qinghai and have or not cattle and sheep pyriform worm, and can to distinguish be the detection film of which kind of pyriform worm.The detection film that the present invention is used to detect Taylor Lv Shi worm, Taylor You Shi worm and pyriform worm in the cattle and sheep body is the specific oligonucleotide that is fixed with Taylor Lv Shi worm and Taylor You Shi worm on Biodyne C film, perhaps also is fixed with this worm specific oligonucleotide of BABEI and the general specific oligonucleotide of Taylor worm on it.Detection method of the present invention is that the interior DNA that extracts of blood tick body to be checked is as template, carry out pcr amplification with specific primer, the Taylor worm of its product and detection and the oligonucleotide of this worm of BABEI are hybridized, film after will hybridizing after enhanced chemiluminescence is hatched is again placed in the exposure cassette, on film, put processing such as X-ray film exposes, development, determine according to the strength signal that produces on the film whether tested animal has and suffer from corresponding disease.
Description
Technical field
The present invention relates to whether have cattle and sheep pyriform worm in the blood tick body of a kind of detection Qinghai, and can distinguish the infected animal parasitism detection film of which kind of pyriform worm, the preparation method and the using method of this detection film are arranged.
Background technology
Piroplasmosis be by in the multiple door in top, the pyriform Piroplasmea, the general name of the blood protozoal disease that causes of the protozoon of pyriform worm purpose this section of BABEI and Taylor section.Piroplasmosis has another name called " babesiasis " or " haemosoridiasis ".Be that the multiple pyriform worm that this belongs to and the Taylor of Taylor section belongs to by the BABEI of this section of the tick-borne BABEI of media parasitizes caused blood protozoal disease in the red corpuscle, clinical is principal character with high heat, anaemia, jaundice and hemoglobinuria, can cause death in the time of seriously.Because all parts of the world are distributed with various tick classes, so distribution that should disease is also very wide, be found in Egypt in 1914 at first, subsequently in Africa, many countries in Europe, Asia all find this cause of disease.Such as the former Soviet Union, India, Sri Lanka, Japan, South America, Africa, the Asia, and all there is this pathogenetic report on ground such as southern Europe.The cowboying of China, Yang Qu mainly are distributed in the northern territory.Correlation study shows, and is very popular at these regional piroplasmosis.Qinghai blood tick is under the jurisdiction of hard tick section, is a novel species of Deng state fence name in 1978.It is distributed in the Qinghai of China, Gansu, and Ningxia, Sichuan, sea level elevations such as Yunnan are in the zone of 1600-4200 rice, no record still abroad, this tick life and meadow, mountain area or thicket belong to three host tick, finish 1 generation in 3 years at nature
[2], in recent years studies show that Qinghai blood tick is a sheep Taylor worm, comprise Taylor You Shi worm and Taylor Lv Shi worm, the communication media of Chinese Taylor worm and four kinds of piroplasmosis of this worm of sheep Mohs BABEI.
Diagnostic method to piroplasmosis in the prior art mainly comprises three kinds of methods: (1) is by the method and the clinical symptom of blood smear, referring to Leemans I, Hooshmand-Rad P, Uggla A.The indirectfluorescent antibody test based on schizont antigen for study of the sheep parasiteTheileria lestoquardi[J] .Veterinary Parasitology, 1997,69 (1-2): 9～18..(2) use some serological methods; as: complement fixation test (CFT) (CFT) and indirect fluorescent antibody test (IFAT) detect; but the cross reaction between the very difficult eliminating of these methods is not of the same race; and false positive and omission appear in regular meeting; referring to Bruning A.Equine piroplasmosis an update on diagnosis[J] .Br Vet J; 1996; 152:139～151 and Papadopoulos B; Brossard M, Perie NM (1996) Piroplasms of domesticanimals in the Macedonia region of Greece.Piroplasms of small ruminants.VetParasitol 63:67-74..(3) round pcr is to detect the most frequently used molecular diagnosis method of Taylor worm at present, compare with the method for routine have the susceptibility height, high specificity, recall rate advantages of higher.But PCR method can not detect the generation of polyinfection usually simultaneously, referring to Alhassan A, Pumidonming W, Okamura M, Hirita H, Battsetseg B, Fujisaki F, Yokoyama N, Igarashi I (2005) Development of asingle-round and multiplex PCR method for the simultaneous detection of Babesia caballiand Babesia equi in horse blood.Vet Parasitol 129:43-49..
Provide a kind of and can detect tested cattle and sheep simultaneously and whether suffer from piroplasmosis, particularly can detect polyinfection, and distinguish infected animal trouble and be which kind of piroplasmosis, raising, the diseases prevention and treatment of cattle and sheep are had positive meaning.
Summary of the invention
The invention provides and a kind ofly can detect tested cattle and sheep simultaneously and whether suffer from piroplasmosis, particularly can detect polyinfection, and can distinguish the detection film of infected animal trouble for which kind of piroplasmosis, and the preparation method of this detection film and using method.
The detection film that the present invention is used to detect Taylor Lv Shi worm, Taylor You Shi worm and pyriform worm in the cattle and sheep body is the specific oligonucleotide that is fixed with Taylor Lv Shi worm and Taylor You Shi worm on Biodyne C film, perhaps also is fixed with this worm specific oligonucleotide of BABEI and the general specific oligonucleotide of Taylor worm on it.
The present invention is used to detect Taylor Lv Shi worm in the cattle and sheep body, in the detection film of Taylor You Shi worm and pyriform worm, fixed Taylor Lv Shi worm specific oligonucleotide sequences is atcttctttttgatgagttg on Biodyne C film, fixed Taylor You Shi worm specific oligonucleotide sequences is tgcattttccgagtgttact, fixed pyriform worm general oligonucleotide sequence is ctgtcagaggtgaaattct, this worm general oligonucleotide sequence of fixed BABEI is ccttggtaatggttaataggaa, fixed China Taylor worm specific oligonucleotide probe sequence is tcgcatctcttgctgagtg, and this worm specific oligonucleotide sequences of fixed Mohs BABEI is gaatgatgccgacttaaaccct.
The preparation method that the present invention is used to detect the detection film of cattle and sheep pyriform worm in the blood tick body of Qinghai is that synthetic goes out pyriform worm universal oligonucleotide probe respectively, Taylor worm universal oligonucleotide probe, this worm universal oligonucleotide probe of BABEI, China Taylor worm specific oligonucleotide, this worm specific oligonucleotide of Mohs BABEI, the specific oligonucleotide of Taylor Lv Shi worm and Taylor You Shi worm, resulting each oligonucleotide 5 ' end is modified with amine groups, it is covalently bound to respectively on the Biodyne C film that has negative charge again.
The method that adopts detection film of the present invention to detect cattle and sheep pyriform worm in the blood tick body of Qinghai is as template with the DNA that extracts in the blood tick body of Qinghai to be checked, the primer that a pair of clip size that amplifies of conservative region design at Taylor worm and these two ends, worm 18S rRNA gene order V4 hypervariable region of BABEI is 480bp-491bp, primer biotin labeling wherein, carry out pcr amplification then, obtain biotin labeled PCR product, biotin labeled PCR product and be fixed on Taylor worm on the Biodyne C film and the oligonucleotide of this worm of BABEI is hybridized, film after will hybridizing after enhanced chemiluminescence is hatched is again placed in the exposure cassette, putting X-ray film on film exposes, again film is developed and photographic fixing is handled, determine according to the strength signal that produces on the film whether tested animal has and suffer from corresponding disease.
In sum, the present invention designs the versatility primer of pyriform worm to being fixed on the Biodyne C film with pyriform worm specific specificity oligonucleotide probe according to the pyriform worm 18S rRNA gene order of having reported, with PCR method and RLB method combine set up a kind of special, responsive diagnostic method can be discerned corresponding pyriform worm kind or genus; Its susceptibility is apparently higher than the slot type PCR method.
The present invention has following advantage:
Adopt detection film of the present invention can detect in the blood tick body of tested Qinghai whether have the pyriform worm easily, and can distinguish in the blood tick body of Qinghai be which kind of pyriform worm, what for example carry is Chinese Taylor worm, and this worm of Mohs BABEI still is Taylor Lv Shi worm or Taylor You Shi worm.Owing to also be fixed with the universal oligonucleotide probe of pyriform worm on the detection film of the present invention, so the present invention can detect the existence of a plurality of worm kinds simultaneously, and this control and prophylactic treatment for cattle and sheep group disease has positive meaning.
Method great advantage of the present invention is to detect the situation that carries Taylor worm and this worm of BABEI in the blood tick body of Qinghai simultaneously simultaneously, and only need make PCR one time, if amplified production is arranged, illustrate that this tick is to carry a certain of this worm of BABEI or Taylor worm or several kinds certainly.In addition, behind PCR product and the probe hybridization, the kind of the polypide that tested tick is entrained can also be described, this control and prevention for disease has more positive meaning.The 3rd, susceptibility of the present invention will be higher than PCR, and can distinguish the genus and the kind of cause of disease simultaneously, and this characteristic is significant in epidemiology survey.Owing to can remove with the PCR product of probe hybridization, so Hybond membrane can use repeatedly, it is reported that this film can use more than 20 times at least, in addition, on a film, can solidify 45 probes, detects 45 PCR products simultaneously, and its cost is cheap relatively.
Adopt very simple of method that detection film of the present invention detects, and it is less to detect the time spent, it is relative also lower that it detects cost, and loss will be far smaller than prior art.
Description of drawings
Fig. 1 is four kinds of cattle and sheep pyriform worm RLB standard positive specificity test charts in the blood tick body of Qinghai.
Embodiment
One, the design of primer and probe is with synthetic
The specific oligonucleotide sequences of Taylor worm and this worm of BABEI is according to the 18S rRNA gene order design of each worm kind.The primer that a pair of clip size that amplifies of conservative region design at Taylor worm and these two ends, worm 18S rRNA gene order V4 hypervariable region of BABEI is 480bp-491bp, wherein: upstream primer: RLB-F (5-gaggtagtgacaagaaataacaata-3), downstream primer: RLB-R (biotin-5-tcttcgatcccctaactttc-3), according to the V4 hypermutation zone design of fixed 18S rRNA gene order at this worm of Mohs BABEI, China Taylor worm, (the problem here is only with present record to four specific oligonucleotide probes of the different worm kinds with Taylor You Shi worm of Taylor Lv Shi worm, whether those skilled in the art also can design this four specific oligonucleotide probes, as not all right technology and the related request that then needs to provide designing probe), that is: B.m, T.s, T.l and T.u, in addition, general probe catch-all and Taylor worm general probe at all pyriform worms, this worm general probe of BABEI Tall, Ball also is designed respectively to synthesize.All oligonucleotide probe N-(trifluoroacetamidohexyl-cyanoethyl, N, N-diisopropyl phosphoramidite[TFA])-C6 amine groups connection modification, with synthetic probe 0-800pmol/150ul 500mM NaHCO
3(pH 8.4) are sought optimum concn and are used for hybridization.
Nucleic acid probe sequence and its optimum concn that the present invention is used see Table .1, and related pyriform worm and the oligonucleotide probe recognition mode of their correspondences see Table 2.
Two, contain the preparation of the special oligonucleotide Biodyne of this worm of Taylor worm and BABEI C film:
It is synthetic that all oligonucleotide 5 ' end is modified the back by amine groups.They can be covalently bound on the Biodyne C film that has negative charge.According to 1999 laggard line operates of describing of method improvement such as Gubbels, primary process is as follows:
(1) specific oligonucleotide with 149ul 500mM NaHCO3.pH 8,4 (all will accurately proofread the pH value) dilution Taylor worm and this worm of BABEI at every turn arrives the optimum concn of having determined.
(2) Biodyne C film is cut into the 14.5cmX14.5cm size, with the marking pen Marking film so that can take one's bearings in the operation afterwards.With fresh 16% (w/v) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) of deionized water preparation, add 10ml and hatch 10 minutes activated b iodyne C films, rotate culturing bottle under the room temperature.
(3) film is placed in the plastic containers to shake and washes two minutes, put into clean trace instrument then with deionized water.Screw handle.
(4) remove liquid residual in the hole by suction.The oligonucleotide solution of 150ul dilution is added the hole slot of trace instrument, and first does not add oligonucleotide with last hole.
(5) dilution proportion of using the 2xSSPE damping fluid to press 1: 100 ink adds first hole and last hole.
(6) after all sample all adds, hatched under the room temperature 2 minutes.
(7) remove oligonucleotide solution and first and the ink diluent of last hole slot in each hole slot by suction.
(8) from the trace instrument, remove striping with tweezers, use the freshly prepared 100mM NaOH of 250ml (maximum) in plastic containers, to hatch then 10 minutes, constantly shake container and make the film passivation.
(9) use the rinsed with deionized water film.
(10) in plastic containers, use 60 ℃ of rinsings of 250ml 2x SSPE/0.1% SDS 5 minutes, slight agitation of solutions bottle during the rinsing.
(11) with 100ml 20mM EDTA pH 8,0 rinsing film in plastic containers, shook gently under the room temperature 15 minutes.
Preserve film up to using (pack with the plastics sealing or with packing film and avoid the film dehydration) for (12) 4 ℃.
Three, the purification of polypide genomic dna in the tick body
1, catches tick from zone to be detected.
2, tick is cleaned, and wash tick three times, place on the clean paper handkerchief, determine the male and female of tick with 70% alcohol.
3, take out tick, place in the lid of Ependorf pipe, to cut in the middle of the tick with two 12 syringe needles, be cut into 4 again, put into the Ependorf pipe that is added with 100ul cell lysis solution then, put upside down for several times, make all tissues enter cell lysis solution, cover pipe, the sex and the source of the good sample of mark on pipe.
4, heating 2 hours under 65 ℃, 800rpm condition on the heating oscillator.
5, add RNAse0.5ul, put upside down 25 times, 65 ℃, 15 minutes.
6, add 40ul Protein Preciptation solution
7, Vertex 20second mixes solution fully.
8,13000g is centrifugal 3 minutes
9, in the Ependorf pipe that supernatant dislocation one mark is good, add the 100ul Virahol, put upside down 50 times.
10,13000g is centrifugal 10 minutes
11, the supernatant that inclines places on the clean thieving paper, blots remaining liq.
12, the ethanol that adds 100ul70%
13,13000g is centrifugal 3 minutes, carefully removes ethanol, blots remaining liq on thieving paper.
14, dry on the DNA drying instrument.
15, add 20ulDNA Hydration solution, be heated to 65 ℃ after insulation 1 hour, be cooled to again 4 ℃ or-20 ℃ of preservations.
Adopt the DNA of the DNA extraction test kit extraction tick of GENTRA in the embodiments of the invention.
Four, pcr amplification
The reaction system of gene fragment pcr amplification and program:
H
2O 56.0μl
10x?buffer
* 7.2μl
10mM?dNTP 1.6μl
100uM upstream primer (RLB F) 3.6 μ l
100uM downstream primer (RLB R) 3.6 μ l
Genomic?DNA 1ul
Taq enzyme 0.36 μ l
*(200mM Tris-HCl (pH 8.55), 160mM (NH4) 2SO4 and 20mM MgCl2)
12000rpm is centrifugal 10 seconds after all sample mix, places the pcr amplification instrument to increase according to following circulation:
Get PCR product 1.5ul at 1.0% sepharose (containing the 0.5%ug/ml ethidium bromide), under 75 volts voltage, carry out electrophoretic analysis, observe amplification.
Five, detect the foundation of cattle and sheep pyriform worm RLB method in the blood tick body of Qinghai:
Biotin labeled PCR product and be fixed on Taylor worm on the Biodyne C film and the oligonucleotide of this worm of BABEI is hybridized.If there is this worm of Taylor worm or BABEI, then with behind the oligonucleotide specific effect hatch the back with streptavidin-peroxidase and ECL-detection (enhanced chemiluminescence) can see development on the film of the grid of black the inside, operation steps is as follows:
(1) prepare following damping fluid, with the deionized water dilution, all damping fluids are wanted preheating before using.(by the amount of a film):
250ml?2xSSPE/0.1％SDS，60℃，
500ml?2xSSPE/0.5％SDS，60℃，
500ml?2xSSPE/0.5％SDS，42℃.
500ml 2xSSPE, room temperature.
(2) add the PCR product of 20 μ l in 130 μ l 2xSSPE/0.1%SDS.
(3) the 99 ℃ of heat shocks of PCR product after the dilution were cooled off in ice in 10 minutes then immediately.
(4) use 60 ℃ of rinsing films of 250ml2xSSPE/0.1%SDS 5 minutes.
(5) in the trace instrument according to becoming vertical direction to put into film with oligonucleotide and supporting air cushion.
(6) remove liquid residual in the trace instrument by the method for suction.
(7) fill hole slot with the PCR product after the dilution, on horizontal plane, hybridized 10 minutes for 60 ℃.
(8) remove sample in the trace instrument by suction, with tweezers film is taken out then.
(9) with 60 ℃ of 250ml 2xSSPE/0.5%SDS, 10 minutes, twice of rinsing film.
(10) place film and in revolving bottle, make its cooling, in order to avoid next step is because of peroxidase effect inactivation.
(11) add 2.5ul streptomycete avidin one peroxidase (500U/ml) and be attached among the 10ml2xSSPE/0.5%SDS, this solution was added in revolving bottle 42 ℃ of hatching films 45-60 minute.
(12) 42 ℃ of rinsings in 10 minutes of 2xSSPE/0.5%SDS solution of usefulness 250ml are twice.
(13) with twice of at room temperature 5 minutes rinsing film of the 2xSSPE of 250ml.
(14) add 1ml immunoblotting luminol reagent A in a 5ml test tube, add the reagent B of 1ml subsequently, mix.
(15) film is placed in plastic cloth or the packing film, dropping A, B mixed solution contain the zone of oligonucleotide and the hybridization of PCR product on film, use the plastic layer mulch film, plastic membrane sealing device closed film.
(16) place the good film of sealing in exposure cassette, put an X-ray film (left hand corner at film gives a discount for later identification direction) on film, exposed 30 minutes.
(17) take out X-ray film and put into developing solution and developed 5 minutes, used the deionized water rinsing then 2 minutes, changed in the stop bath photographic fixing subsequently over to 5 minutes, use the deionized water rinsing again, fully observations behind the flush away stop bath.
(18), can prolong or shorten the time shutter film directly is exposed to another film if the signal on the film is too weak or too strong.The PCR product of hybridizing can be removed from film.A film can reuse about 15 times.
The result judges: by the 18S rRNA gene V4 hypermutation zone of all worm strains of listing in the pcr amplification table 1, dna fragmentation that produces and the oligonucleotide probe that is fixed on the film carry out nucleic acid hybridization, oligonucleotide probe is through the luminous effect of optics, on film, produce the signal of certain intensity, all probes only combine with their target sequences separately, do not have cross reaction between different kinds, genus and the strain, very clearly produce hybridization signal with corresponding worm kind or strain.Each Taylor worm kind can be discerned by four nucleotide probes: pyriform worm general probe (ca841-859), Taylor worm general probe (T-all 811-832) and Taylor Lv Shi worm probe (T-l 628-647) or Taylor You Shi worm probe (T-u 678-697) or Chinese Taylor worm (T.s 627-645).This worm of BABEI can be discerned by three probes: pyriform worm general probe (ca841-859), BABEI stone probe (B-all745-766) and this worm probe (B.m466-487) of Mohs BABEI.Attachedly the results are shown in Figure 1.
Carry out actual detected by 99 parts of Qinghai blood tick samples to Gannan, Gansu Province autonomous prefecture Lintan County, and compare with the slot type PCR method of primer amplified, the recall rate of RLB is apparently higher than the slot type PCR method, this method can detect the kind of pyriform worm and the situation of polyinfection, for the detection of piroplasmosis, worm kind are identified and epidemiology survey provides instrument.
Subordinate list: table .1, Taylor worm and BABEI this worm sequence oligonucleotide probe and optimum concn
Pyriform worm that table 2 is involved in the present invention and the oligonucleotide probe recognition mode of their correspondences
Claims (7)
1, a kind of detection film that is used to detect cattle and sheep pyriform worm in the blood tick body of Qinghai is characterized in that being fixed with the specific oligonucleotide probe of cattle and sheep pyriform worm on Biodyne C film.
2, the detection film that is used to detect cattle and sheep pyriform worm in the blood tick body of Qinghai according to claim 1 is characterized in that the general specific oligonucleotide probe sequence of cattle and sheep pyriform worm is ctgtcagaggtgaaattct.
3, the detection film that is used to detect cattle and sheep pyriform worm in the blood tick body of Qinghai according to claim 2 is characterized in that also being fixed with this worm universal oligonucleotide probe of BABEI and Taylor worm universal oligonucleotide probe on Biodyne C film.
4, the detection film that is used to detect cattle and sheep pyriform worm in the blood tick body of Qinghai according to claim 3, it is characterized in that the general specific oligonucleotide probe sequence of this worm of fixed BABEI is ccttggtaatggttaataggaa on Biodyne C film, fixed Taylor worm universal oligonucleotide probe sequence is taccaaagtaatggttaatagg, fixed Taylor Lv Shi worm specific oligonucleotide sequences is atcttctttttgatgagttg, fixed Taylor You Shi worm specific oligonucleotide sequences is tgcattttccgagtgttact, fixed pyriform worm specific oligonucleotide sequences is ctgtcagaggtgaaattct, fixed China Taylor worm specific oligonucleotide probe sequence is tcgcatctcttgctgagtg, and this worm specific oligonucleotide sequences of fixed Mohs BABEI is gaatgatgccgacttaaaccct.
5, claim 1 or the 2 described preparation methods that are used to detect the detection film of pyriform worm in the blood tick body of Qinghai, it is characterized in that synthetic goes out the specific oligonucleotide probe of cattle and sheep pyriform worm, resulting oligonucleotide 5 ' end is modified with amine groups, again it is covalently bound on the Biodyne C film that has negative charge.
6, claim 3 or the 4 described preparation methods that are used to detect the detection film of pyriform worm in the blood tick body of Qinghai, it is characterized in that manually synthesizing respectively the specific oligonucleotide probe of specific oligonucleotide probe, this worm universal oligonucleotide probe of BABEI, Taylor worm universal oligonucleotide probe and Chinese Taylor worm, this worm specific oligonucleotide probe of Mohs BABEI, Taylor Lv Shi worm and the Taylor You Shi worm of cattle and sheep pyriform worm, resulting each oligonucleotide 5 ' end is modified with amine groups, again it is covalently bound to respectively on the BiodyneC film that has negative charge.
7, adopt the described arbitrary detection film of claim 1 to 4 to detect the method for pyriform worm in the blood tick body of Qinghai, it is characterized in that with the DNA that in the blood tick body of Qinghai, extracts as template, the primer that a pair of clip size that amplifies of conservative region design at two ends, cattle and sheep pyriform worm 18S rRNA gene order V4 hypervariable region is 480bp-491bp, primer biotin labeling wherein, carry out pcr amplification then, obtain biotin labeled PCR product, biotin labeled PCR product and be fixed on Taylor worm on the Biodyne C film and the oligonucleotide of this worm of BABEI is hybridized, film after will hybridizing after enhanced chemiluminescence is hatched is again placed in the exposure cassette, putting X-ray film on film exposes, again film is developed and the photographic fixing processing, determine according to the strength signal that produces on the film whether tested tick has corresponding pyriform worm.
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| CN105018599A (en) * | 2015-06-11 | 2015-11-04 | 中国农业科学院兰州兽医研究所 | Detection film for detecting T. luwenshuni, T. uilenbergi and sheep Piroplasmea |
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