CN104372069A - Specific probe capable of rapidly detecting pseudocohnilembus persalinus, detection method and applications thereof - Google Patents
Specific probe capable of rapidly detecting pseudocohnilembus persalinus, detection method and applications thereof Download PDFInfo
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- CN104372069A CN104372069A CN201310350157.2A CN201310350157A CN104372069A CN 104372069 A CN104372069 A CN 104372069A CN 201310350157 A CN201310350157 A CN 201310350157A CN 104372069 A CN104372069 A CN 104372069A
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Abstract
The invention belongs to the field of diagnosis of aquatic disease caused by ciliates. A specific probe is designed to detect pseudocohnilembus persalinus, and the pseudocohnilembus persalinus can be rapidly detected by combining the specific probe with a fluorescence in-situ hybridization technology. The method has the characteristics of simple operation, high specificity, agility, and rapidness.
Description
Technical field
The invention belongs to aquatic animal disease field, be specifically related to method for quick and the application of the fine worm of the pseudo-health of a kind of water droplet.
Background technology
Along with the fast development of World Aquaculture, the various disease being difficult to cure has continued to bring huge challenge to the prevention, treatment and management of disease, the disease such as caused by the fine class ciliate of shield.The fine class ciliate of shield, be distributed widely in natural water and aquaculture water, it is the important component part of the micro-food cycle of water body, wherein with the fine Eimeria Pseudocolnilembus of pseudo-health, tailfiber Eimeria Uronema, intend boat Eimeria Paralembus, Miami Eimeria Miamiensis and belong to the polypide that Philasterides is representative addicted to tainture, become the facultative cause of disease that aquatic animal is serious.Although have ecological and economic dual importance, people are still very difficult to this kind of ciliophoran diagnosis.
Although morphological method is the ciliophoran traditional means of the fine class of qualification shield, but in culture fishery, its application receives the restriction of following 4 aspects: 1) most of shield fine class live body form is quite similar, and even concerning non-categorical expert, the form after silver dye is still difficult to distinguish; 2) the fine class of the shield of spore shape is difficult to identify with opticmicroscope; 3) AgNOR technique for Morphological Identification is very complicated and consuming time; 4), in world wide, the protobiont taxonomy expert that can be culture fishery service is very rare.Therefore, identify fast, simply and exactly the novel method of the fine class of shield be still people in the urgent need to.In recent years, emerging fluorescence in situ hybridization technique is considered to a kind of effective detection technique, finds broad application in environmental microorganism detects.Fluorescence in situ hybridization technique is the DNA sequence dna special according to population in the different category level of known organism, to utilize fluorescently-labeled specific oligos fragment as probe, with the biological genome DNA molecule hybridize of targeted environment, detect existence and the abundance of this specific biological population.But this technology is not applied in the context of detection of disease sex pili worm.
The pseudo-health of water droplet fine worm Pseudocohnilembus persalinus Evans and Thompson, 1964, the fine class ciliate of shield as a kind of high hazardness, main parasitic is in sea water fish---and the body surface of lefteye flounder Paralichthys olivaceus, the gill filament, abdominal cavity, even in brain, can cause cultured fishes massive mortality.
Summary of the invention
The object of the present invention is to provide method for quick and the application of the fine worm of the pseudo-health of a kind of water droplet.
For achieving the above object, the present invention adopts following technical scheme to be:
A method for the specific probe of the fine worm of the pseudo-health of rapid detection water droplet, specificity DNA probing needle Pp210 sequence is 5 '-ATGATTTATCCCAGAGGG-3 '.
Described probe Pp210 fluorescent mark cyanine class dyestuff Cy-3.
A detection method for the fine worm of the pseudo-health of rapid detection water droplet, detects through fluorescence in situ hybridization with the fine worm specificity DNA probing needle of the pseudo-health of the water droplet shown in fluorescently-labeled claim 1.
Specifically, in darkroom, incubation is fixed in testing sample at 46 DEG C 3 hours through stationary liquid by being added to containing the formamide hybridization drop of target-probe, after incubation at 48 DEG C through elutriant water-bath wash-out 15 minutes, after wash-out room temperature sky dry again with 4', 6-Diamidino-2-Phenylindole(DAPI) solution carries out counterstain, and after dyeing, washing sky observes fluorescence intensity after doing under fluorescent microscope.
Described formamide hybridization liquid is methane amide (formamide), the probe Pp210 of concentration 30ng/ μ l, the SDS(sodium dodecyl sulfate of massfraction 0.01% of massfraction 20%), the NaCl of volumetric molar concentration 0.9M and the TRIS-HCl of volumetric molar concentration 20mM;
Elutriant is the SDS(sodium dodecyl sulfate of massfraction 0.01%), the EDTA of the NaCl of volumetric molar concentration 0.9M, the TRIS-HCl of volumetric molar concentration 20mM and volumetric molar concentration 5mM.
Described DAPI strength of solution is 1.5ng/ml.
An application for the fine worm of the pseudo-health of rapid detection water droplet, described probe Pp210 detects the application in the fine worm test kit of the pseudo-health of water droplet in preparation.
The advantage that the present invention has: the present invention is with the fine worm of the pseudo-health of water droplet for tested object, and by developing specific oligonucleotide probe to it, and combined with fluorescent hybridization in situ technique realizes the quick diagnosis of this monoid.The present invention is simple to operate, high specific, can be sensitive and quick etc. feature.Only can be realized the quick diagnosis of this monoid with fluorescence microscopy by test kit provided by the invention, red fluorescence is the positive signal of target-probe, blue-fluorescence be DAPI for nuclear positive reaction, negative control should be unstressed configuration or faint autofluorescence.
Accompanying drawing explanation
The ciliophoran hybridization target probe (D, E, G) of sample that Fig. 1 provides for the embodiment of the present invention, general probe (A, C), negative probes (F) and chimeric 4', 6-Diamidino-2-Phenylindole(DAPI) the fluorescence developing photo of (B-G).Wherein, A. is the colour developing photo of general probe, and polypide takes on a red color fluorescence; B. be the colour developing photo of core device after DAPI is fitted together to of figure A individuality, core is mazarine fluorescence; C. be the overlap of Figure 1A and B, show red individuality and navy blue core device simultaneously; D. be that in sample, a kind of breach worm Condylostoma sp., through the colour developing overlapping photographs of target-probe and DAPI hybridization, shows navy blue core; E. be in sample the fine class Scuticociliatia sp. of another kind of shield through the colour developing overlapping photographs of target-probe and DAPI hybridization, the autofluorescence that display polypide is faint and navy blue core; F. be the colour developing overlapping photographs that negative probes and DAPI hybridize the fine worm of the pseudo-health of water droplet, only show navy blue core; G. be the fine worm of the pseudo-health of water droplet through the colour developing overlapping photographs of target-probe and DAPI hybridization, polypide takes on a red color fluorescence, and core is mazarine fluorescence.
Embodiment
Below in conjunction with Figure of description, the invention will be further described.
Embodiment 1
(1) preparation of specific probe
1. from host's lefteye flounder body surface ulcerated tissue, isolate the fine worm of the pseudo-health of water droplet, cultivate for food carries out expansions with ripe shrimp, utilize DNeasy Tissue kit(Qiagen afterwards) by specification requires extraction genome; Utilize ciliate universal primer (5 ' AAC CTG GTT GAT CCT GCC AGT3 ' and 5 ' TGA TCC TTCTGC AGG TTC ACC TAC3 ') to carry out pcr amplification SSU rRNA gene order, the amplification reaction system of 25 μ l comprises: the template DNA of about 50ng, 0.7U Taq archaeal dna polymerase, 5 ' and 3 ' primer each 1 μM (final concentration), dNTP0.2mM(final concentration), MgCl
22mM(final concentration), Takara PCR damping fluid 2.5 μ l, appropriate sterilizing distilled water (completion to whole reaction system is 25 μ l), response procedures is as follows: 94 DEG C of sex change 5 minutes, then 94 DEG C of sex change 1 minute, anneal 1 minute for 60 DEG C, 70 DEG C extend 2 minutes, totally 35 circulations, finally extend 10 minutes.Order-checking company is sent to check order after PCR primer purifying.
2., after obtaining the SSU rRNA gene order of the fine worm of the pseudo-health of water droplet, in conjunction with the homologous sequence of all populations of GenBank this kind, probe design is carried out with ARB software package.Primary election probe is through the comparison inspection of ARB software and GenBank:
1) whether match with other species sequence; 2) whether match with this kind of species sequence (containing other population sequence).Choose wherein only match with this kind (population) sequence and the sequence that do not match with other species sequence for alternate probe.With reference to the probe hybridization fluorescent brightness distribution plan of the eukaryote Saccharomyces Cerevisiae in S accharomyces cerevisiae reported, selecting the sequence being in the stronger region of fluorescent brightness is target-probe.Finally select the sequence corresponding with the Helix-12 region of Saccharomyces Cerevisiae in S SU rRNA to be target-probe sequence, sequence is 5 '-ATGATTTATCCCAGAGGG-3 ', and with cyanine class dyestuff Cy-3(C
31h
37kN
2o
8s
2) carry out marking that (fluorescent mark is by MWG Biotech biotech firm (Ebersberg, Germany) complete), called after Pspe210 oligonucleotide probe (the homologous sequence zero position of corresponding intestinal bacteria Escherichia coli16S rRNA gene is the 210th).ARB software and GenBank BLAST software is used to assess the specificity of target-probe.ARB software comparison result: target-probe and the fine worm different geographic populations of the pseudo-health of water droplet fit like a glove (reverse complemental), have at least the difference of two base mismatch (see table 1) GenBankBLAST software comparison result to show with the fine class ciliate of other shield: an environmental sample ciliate sequence Pseudocohnilembus sp.(GenBank FN430390) to fit like a glove with target-probe sequence, this environment sequence should be the fine worm of the pseudo-health of water droplet; Target-probe sequence and other is biological, as zebra fish Zebrafish(GenBank FP102983) and house mouse Mus musculus(GenBank AC168091) there is the difference of a base.The equal display-object probe of these two groups of assessment results has high specific feature.
The SSU rDNA sequence of the fine worm of the pseudo-health of the corresponding water droplet of table 1.ARB software comparison target-probe and fine class SSU rDNA sequence (will not list more than the sequence of 3 base mismatch) of other shield.
Identical base represents with "=".
3. select eukaryote general probe Euk1209R(5 '-GGGCATCACAGACCTG-3 ') complementary sequence be negative control probe, and mark Cy-3 fluorescent mark (fluorescent mark is completed by MWG Biotech biotech firm (Ebersberg, Germany)).As the negative control probe surveyed in the fine worm test kit of the pseudo-health of water droplet.
(2) fluorescence in situ hybridization detection
1. the hybridization solution concentration of optimum methane amide (Formamide) content is established.In hybridization solution, the fluorescence intensity of the lower probe of concentration of forma is higher, is optimal concentration by the fluorescent effect significant concn of alternative target-probe.The hybridization buffer of preparing hybrid liquid concentration preparation 0-50% concentration of forma gradient and elutriant (preparation is in table 2).2. and 3. after hybridization, mirror is picked up (concrete steps are according to preparing in specific probe step), show that the optimum formamide hybridization liquid concentration containing target-probe is 20%.
The hybridization buffer of the different methane amide of table 2. (FA) concentration and elution buffer liquid formula.
2. scrape from tissue, mucus and a small amount of aquaculture water mixing from lefteye flounder body surface ulcerated tissue, use small-sized micropore plankton net (bolting silk) the enrichment polypide in about 20 μm of apertures as testing sample 1.Containing two kinds of ciliates in sample 1, the fine worm of the pseudo-health of target polypide water droplet and breach worm (the comparatively large and macronucleus of build be beads shape easily and target polypide distinguish).In order to verify the specificity of target-probe further, utilizing to be separated from aquaculture water and to pass through and preliminary expanding fine class ciliate (Scuticociliatiasp.) of the another kind of shield cultivated as testing sample 2.Sample 1 or 2 fixes 10 minutes with Bonn liquid by 1:1 volume mixture respectively, uses the millipore filtration in 1.2 μm of apertures (Millipore, diameter 25mm) to carry out low press filtration polypide suspension respectively after fixing.After the sterile water wash filter membrane 5 times of 1ml, filter membrane is put in a clean culture dish, natural air drying, air-dry rear sample 1 filter membrane is divided into 4.Use the 20% formamide hybridization liquid containing target-probe to go to cover wherein 2 filter membranes respectively, cover wherein 1 filter membrane with the formamide hybridization liquid containing general probe containing 30%, cover 1 filter membrane with the 30% formamide hybridization liquid containing negative control probe.The filter membrane of sample 2 only goes to cover with the 20% formamide hybridization liquid containing target-probe.Above-mentioned each filter membrane is incubation 3 hours in 46 DEG C of hybridization casees, does not hybridize successful probe after each filter membrane incubation through elutriant 48 DEG C of water-baths 15 minutes wash-outs.Then again by empty dry for place dark after the rinse of each filter membrane distilled water, being then 4', the 6-Diamidino-2-Phenylindole(DAPI of 1 μ g/ml by concentration) solution 50 μ l carries out counterstain 3 minutes.After dyeing each filter membrane use successively distilled water, 80% alcohol rinse, empty dry.Finally to go out glue Vecta Shield mountant(Burlingame, CA with anti-Ying Guang temper) mounting.
3. fluorescent microscope (Axiostar Plus, Carl Zeiss GmbH) microscopy is used.In order to directly compare Color, the microscope parameter of every observation and shooting is consistent, wherein time shutter 10 milliseconds corresponding Cy-3 signal, and the corresponding DAPI signal of 0.4ms, observes each filter membrane fluorescence intensity (see Figure 1A-G).
To be taken on a red color fluorescence by the known target polypide of Figure 1A, show that general probe is hybridized successfully; Figure 1B is the colour developing photo of core device after DAPI hybridization of Figure 1A individuality, and core is mazarine fluorescence; Fig. 1 C is the overlap of Figure 1A and B, shows red individuality and navy blue core device simultaneously; Fig. 1 D is that in sample 1, a kind of breach worm Condylostoma sp., through the colour developing overlapping photographs of target-probe and DAPI hybridization, only shows the navy blue core of polypide, shows that target-probe fails to hybridize with polypide; Fig. 1 E is that in sample 2, the fine class Scuticociliatia sp. of another kind of shield is through the colour developing overlapping photographs of target-probe and DAPI hybridization, and the autofluorescence that display polypide is faint and navy blue core, show that target-probe fails to hybridize with polypide; Fig. 1 F is the colour developing overlapping photographs that negative probes and DAPI hybridize the fine worm of the pseudo-health of water droplet, only shows navy blue core, shows that negative probes fails to hybridize with polypide; Fig. 1 G is the fine worm of the pseudo-health of water droplet through the colour developing overlapping photographs of target-probe and DAPI hybridization, and polypide takes on a red color fluorescence, and core is mazarine fluorescence, shows that target-probe and polypide hybridize successfully.In the present embodiment, use target-probe to detect the successful of the fine worm of the pseudo-health of water droplet, and target-probe can not with the fine class of close shield and breach worm generation positive reaction, this demonstrates the feature that probe has high specific.
Containing random selecting 5 visuals field on the filter membrane of target-probe, observing polypide number for polypide number actual in the visual field with white light source, there is the polypide number of positive reaction in counting.Found that all polypides all can be detected obvious red fluorescence (indivedual polypide fluorescent brightness major part polypide is more weak a little), this demonstrate the technical program and there is high-sensitive feature.
Claims (7)
1. a method for the specific probe of the fine worm of the pseudo-health of rapid detection water droplet, is characterized in that: specificity DNA probing needle Pp210 sequence is 5 '-ATGATTTATCCCAGAGGG-3 '.
2., by the method for the specific probe of the fine worm of the pseudo-health of rapid detection water droplet according to claim 1, it is characterized in that: described probe Pp210 fluorescent mark cyanine class dyestuff Cy-3.
3. a detection method for the fine worm of the pseudo-health of rapid detection water droplet, is characterized in that: detect through fluorescence in situ hybridization with the fine worm specificity DNA probing needle of the pseudo-health of the water droplet shown in fluorescently-labeled claim 1.
4. by the detection method of the fine worm of the pseudo-health of rapid detection water droplet according to claim 3, it is characterized in that: in darkroom, incubation is fixed in testing sample at 46 DEG C 3 hours through stationary liquid by being added to containing the formamide hybridization drop of target-probe, after incubation at 48 DEG C through elutriant water-bath wash-out 15 minutes, after wash-out room temperature sky dry again with 4', 6-Diamidino-2-Phenylindole(DAPI) solution carries out counterstain, and after dyeing, washing sky observes fluorescence intensity after doing under fluorescent microscope.
5., by the detection method of the fine worm of the pseudo-health of rapid detection water droplet according to claim 4, it is characterized in that: described formamide hybridization liquid is methane amide (formamide), the probe Pp210 of concentration 30ng/ μ l, the SDS(sodium dodecyl sulfate of massfraction 0.01% of massfraction 20%), the NaCl of volumetric molar concentration 0.9M and the TRIS-HCl of volumetric molar concentration 20mM;
Elutriant is the SDS(sodium dodecyl sulfate of massfraction 0.01%), the EDTA of the NaCl of volumetric molar concentration 0.9M, the TRIS-HCl of volumetric molar concentration 20mM and volumetric molar concentration 5mM.
6., by the detection method of the fine worm of the pseudo-health of rapid detection water droplet according to claim 4, it is characterized in that: described DAPI strength of solution is 1.5ng/ml.
7. an application for the fine worm of the pseudo-health of rapid detection water droplet, is characterized in that: probe Pp210 described in claim 1 detects the application in the fine worm test kit of the pseudo-health of water droplet in preparation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079625A (en) * | 2019-04-10 | 2019-08-02 | 广西壮族自治区兽医研究所 | A kind of PCR primer, kit and method detecting balantidium Coli |
| CN110396552A (en) * | 2019-09-05 | 2019-11-01 | 大连海洋大学 | Molecular labeling, primer and the method for based on PCR identification water droplet puppet health fibre worm |
| CN112461733A (en) * | 2020-11-05 | 2021-03-09 | 闽江学院 | Method for observing three-dimensional structure of ciliate cell by multiple fluorescence markers |
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Non-Patent Citations (2)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079625A (en) * | 2019-04-10 | 2019-08-02 | 广西壮族自治区兽医研究所 | A kind of PCR primer, kit and method detecting balantidium Coli |
| CN110396552A (en) * | 2019-09-05 | 2019-11-01 | 大连海洋大学 | Molecular labeling, primer and the method for based on PCR identification water droplet puppet health fibre worm |
| CN110396552B (en) * | 2019-09-05 | 2023-02-10 | 大连海洋大学 | Molecular marker, primer and method for identifying pseudocercospora fusca based on PCR |
| CN112461733A (en) * | 2020-11-05 | 2021-03-09 | 闽江学院 | Method for observing three-dimensional structure of ciliate cell by multiple fluorescence markers |
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