CN100436597C - Fluorescence labeled cell agglutinin probe red tide biological detection reagent kit and detecting method thereof - Google Patents
Fluorescence labeled cell agglutinin probe red tide biological detection reagent kit and detecting method thereof Download PDFInfo
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- CN100436597C CN100436597C CNB200510067183XA CN200510067183A CN100436597C CN 100436597 C CN100436597 C CN 100436597C CN B200510067183X A CNB200510067183X A CN B200510067183XA CN 200510067183 A CN200510067183 A CN 200510067183A CN 100436597 C CN100436597 C CN 100436597C
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Abstract
The present invention relates to a red tide biological detection reagent kit of a fluorescence labeled cell agglutinin probe and a detecting method thereof, particularly to a cell agglutinin probe. The present invention is provided with components I, II, and III, wherein as a cell agglutinin probe system, the I comprises n kinds of fluorescence labeled cell agglutinin probes and is configured by using phosphoric acid buffer solution; the II is configured by sterilization and filtration artificial sea water or the phosphoric acid buffer solution and provides a Peck solution to be silica gel granule of ethene pyrrolidone used for separating cells in matching way; the III is a sample fixing system and is selected from a Ruge's iodine solution, paraformaldehyde and glutaraldehyde. The method has the following steps: culturing algae used in experiments; fixing the configuration of the cell agglutinin and algae cells; adding the Peck solution to a cell culture, mixing the fluorescence labeled cell agglutinin probe to the cell integration, adding the phosphoric acid buffer solution and the Peck solution, suspending and precipitating, redyeing by using double-stranded DNA fluorescent dye and observing; estimating under the qualitative dyeing, counting each dyeing cell of dyeing strength, and calculating percentage of fluorescence dyeing combination.
Description
Technical field
The present invention relates to a kind of cell agglutinin (Lectin) probe, especially relate to a kind of based on fluorescently-labeled cell agglutinin probe and the single application of planting in the target red tide frustule of low abundance in detection and evaluation pure culture red tide frustule and natural water.
Background technology
Red tide is because the change of marine environment condition impels the explosive breeding of some planktonic organism, causes a kind of ecological phenomenon that water colour is unusual.Since 20th century, the red tide particularly generation of poisonous red tide is more and more frequent, the zone also constantly enlarges, one of global main Oceanic disasters have been become, it not only seriously destroys the marine eco-environment and marine fishery resources, influences mariculture industry, infringement strand tourism, transmission, the gathering of red tide plankton toxin by biologic chain more arranged, final harm humans health.In recent years, China becomes the country of a red tide disaster pilosity rapidly, and from the eighties in 20th century, occurrence frequency increases year by year, and annual loss is in 1,000,000,000 yuan.
The marine phytoplankton that can form at present red tide in the world has kind more than 260, this than the eighties in 20th century find 150 surplus kind increase greatly, along with going deep into of red tide research, have more red tide plankton to be found.Yet, red tide plankton (algae) mostly is unicellular nannoplankton, because its individuality is small, morphological specificity is not obvious, even also being difficult under the opticmicroscope, well-trained taxonomy expert the red tide algae identified exactly kind or the subspecies level, and the method costliness both consuming time by electron microscope can't be promoted in the monitoring department of basic unit again.In addition based on the ordinary method of opticmicroscope also be difficult to differentiate morphology similar but the different kind of physiological function, for example, the plan rhombus algae of diatoms has two kinds of distortion, and is a kind of poisonous, another kind of nontoxic, but can't observe under opticmicroscope.Because the hazard approach of different red tide planktons, hazard rating difference (produce toxin as some, endanger bigger), the prophylactico-therapeutic measures of the red tide that different red tide planktons are produced is also different (when in the cage culture district poisonous red tide taking place, then must take to shift measures such as net cage), therefore inaccurate the or evaluation mistake that red tide plankton is identified will be brought very big difficulty to the control and the early stage assessment of red tide.Difficulty and restriction at the traditional monitoring method, external scientists has been devoted to develop various specific probes and has been detected various red tide monoids, the target red tide plankton can be detected by the means of optics or chemistry, fast and accurately identify, count target red tide kind.Main molecular probe has at present: antibody, nucleic acid probe etc.But most of work also is in breadboard probe design and the exploitation.Because the algae kind does not possess tangible morphological feature usually, but possesses genetic diversity, region genetic characteristics is clearly arranged, according to the probe of the specific kind exploitation in area and be not suitable for other geographic same kind, the therefore probe that needs exploitation to be suitable at Chinese marine site geographical strains.
Summary of the invention
The purpose of this invention is to provide a kind of red tide plankton detection and identification kit, i.e. fluorescence labeled cell agglutinin probe red tide biological detection reagent kit based on fluorescence labeled cell agglutinin probe that can be applied to the harmful algal biology is carried out rapid detection and evaluation.
Another object of the present invention is to identify and detect the method for red tide plankton, i.e. fluorescence labeled cell agglutinin probe red tide biological detecting method for phycology research provides a kind of fluorescently-labeled cell agglutinin probe.
The said fluorescence labeled cell agglutinin probe red tide biological detection reagent kit of the present invention is provided with box body and component I, II, III, component I, II, III are located in the box body, component I is the probe system of Lectin, the probe system of Lectin contains the fluorescently-labeled Lectin probe of n kind, n 〉=1, volume is 500～1000 μ L * n, (preferred 100 μ L * n), the concentration of each probe is 50～100 μ g/ml (preferred 100 μ g/ml), adopt PBS (phosphate buffer solution) configuration, contain 0.5%～1.5% sodiumazide (preferred 1% sodiumazide); Component I I is the reaction buffer system, and volume is 100～300mL, (preferred 200mL), the reaction buffer system is meant the artificial seawater configuration of sterilising filtration, salinity is 25～30, and pH is 7.5～8.5, (salinity preferred 28, pH preferred 8.0), perhaps be PBS buffered soln, concentration is 40～60mM (preferred 50Mm), and pH is 7.5～8.5 (preferred 8.0), selecting simultaneously provides Percoll (peck solution) to be used for cellular segregation, and concentration is 70%～90% (preferred 90%); Component III is the sample fixed system, it is 100～400mL (preferred 200mL) that volume is provided, the sample fixed system is meant selects to use Lu Geshi iodine liquid, a kind of in Paraformaldehyde 96 and the glutaraldehyde, the concentration of Lu Geshi iodine liquid is 10%～20% (preferred 10%), the final concentration of Lu Geshi iodine liquid is 0.5%～1.5% (preferred 1%), the concentration of Paraformaldehyde 96 is 10%～20% (preferred 10%), the final concentration of Paraformaldehyde 96 is 0.5%～1.5% (preferred 1%), the concentration of glutaraldehyde is 20%～25% (preferred 25%), and the final concentration of glutaraldehyde is 1.0%～2.5% (preferred 2.5%).Described component I can contain 14 kinds of fluorescently-labeled letin probe systems (be probe library, take into account the kind and the bonded glycosyl cover type of probe), and is specifically as shown in table 1.
Probe kind that this detection of table 1 identification kit uses and specificity are in conjunction with glycosyl
Its reaction product can directly utilize epifluorescence microscope to carry out qualitative and semiquantitative detection.In appropriate circumstances, its reaction product can utilize spectrophotofluorometer to carry out quantitative detection; Under the situation of needs, it also can utilize flow cytometer to carry out qualitative and quantitative detection.Above-mentioned detected result and binding pattern can utilize computer software that it is carried out kind and identify (qualitative) and statistical study (as cluster analysis, t check etc.).
The said fluorescence labeled cell agglutinin probe red tide biological detecting method of the present invention the steps include:
1) with the cultivation of algae: separate obtaining experiment and use algae from the red tide water sample in Chinese marine site, be made into substratum after boiling with the seawater of membrane filtration and cultivate, perhaps the water sample of collection in worksite passes through centrifugal or filtering and concentrating.
2) configuration of lectin and frustule is fixing: adopt filtering artificial seawater to dispose fluorescently-labeled lectin fresh solution, select to use Lu Geshi iodine liquid, Paraformaldehyde 96 and glutaraldehyde fixed cell according to detecting needs, carry out the combination experiment of lectin then.
3) after each strain is tried the algae cultivation, add Percoll to cell culture, cell centrifugation is collected, the centrifugal supernatant that goes, cell integral body is sneaked into fluorescence lentin, add PBS, add Percoll again, centrifugal, remove supernatant, add PBS suspension precipitation, can select to use the DAPI (double-stranded DNA fluorescence dye) of 50-80 μ g/ml to redye, (wave band is exciting light, 330～385nm to use UV-light then, emission light＞420nm) and blue light (exciting light 450-480nm launches light 515nm) observe respectively.
4) cell after the above-mentioned washing is observed under fluorescent microscope and photographed or make a video recording, the standard below qualitative painted situation adopts is estimated: +++: dyeing is bright, and 100% cell is caught look; ++ dyeing is bright inadequately; +: staining power is low, but with control group obvious difference is arranged;-: dye-free; " Blind Test method " taked in detection, promptly detects the people and do not know detected material situation; Staining cell to each staining power is counted, and count number is no less than 100 cells, the percentage of calculations incorporated then.
In step 1), after boiling, the seawater of the most handy 0.2 μ m membrane filtration is made into the f/2 substratum, in the 5000LX light intensity, L: D=12: in the algal species cultivation chamber, cultivate under the temperature of 12 photoperiod and 22 ℃.
In step 2) in, fluorescently-labeled lectin fresh solution is 100 μ g/ml, and Sigma company provides probe and fluorescent mark, adopts the configuration of 50mM phosphate buffer solution PBS (pH8.0) or filtering artificial seawater, and salinity is 28, and pH is 8.0.
In step 3), each strain is tried algae and was cultivated 7 days, adds in the cell culture of 100 μ l Percoll to 1ml, and cell is collected with the centrifugal 10min of 3000rpm, and the centrifugal supernatant that goes is with 10
3～10
5The cell integral body of/ml is sneaked into the fluorescently-labeled lentin of 50 μ l, adds 950 μ l 50mMPBS and is incubated 15min～1h at 20 ℃ in dark situation, and then add 100 μ l Percoll, and the centrifugal 10min of 3000rpm removes supernatant, adds 1mlPBS suspension precipitation.Can select to use the DAPI (double-stranded DNA fluorescence dye) of 50 μ g/ml to redye.
In step 4), said qualitative dyeing can be carried out qualitative dyeing and estimate that the staining cell counting to each staining power calculates fluorescent dye bonded percentage under epifluorescence microscope; Selection is carried out quantitatively or qualitative test with spectrophotofluorometer or flow cytometer; Measurement result adopts self-editing computer software to analyze and statistical treatment.
Advantage of the present invention is: can be easy, quickly red tide plankton is carried out qualitative detection and evaluation, under the situation of needs, also can carry out detection by quantitative.The present invention is the technical system that adopts fluorescently-labeled cell agglutinin probe that Chinese offshore sea waters typical case's harmful algal biology is detected and identifies, can not see that relevant report is arranged at present to plesiomorphic red tide plankton, to from the not homophyletic system of the red tide plankton of the same race of identical, adjacent marine site of CHINESE OFFSHORE or different waters and produce the different frustule of malicious level and discern and distinguish.The present invention compare with technology such as dna probes with the current domestic and international immune fluorescent probe of using morely (according to the literature, the dyeing of immune fluorescent probe and oligonucleotide probe is subjected to the influence of cell growth state, sees Rodas etc., 1997; Scholin etc., 1994,1997,1999 report etc.), it also has the influence that fluorescent mark is not subjected to cell growth state and growth phase and fixing means, and positive staining can also make the flagellum marker coloring of different algal species, can distinguish advantages such as target algae in biased sample.Handle in conjunction with self-editing supporting computer software analysis, can carry out form, Molecular Identification and chemotaxonomy research to various frustules, can also make up and understand the sibship (referring to Fig. 9) between the not homophyletic system of different algaes or identical algae by the different binding patterns of lectin.The detection kit that contains present technique has easy to use, good reproducibility, and accuracy and reliability height, testing cost is relatively low, and is time saving and energy saving, thereby can be widely used in all fields such as research of the evaluation of red tide plankton and detection and molecular ecology.
Description of drawings:
Fig. 1 is the representational lectin fluorescence probe Color figure that observes with epifluorescence microscope.Illustrate: scale=20 μ m.A) cellular control unit; B) the painted cell of lectin.A) the painted ATCI01 of ConA; B) the painted ATDH01 of DBA; C) the painted AspGX01 of LCA; D) the painted AspGX01 of PHA-E; E) the painted AMTW of s-WGA; F) the painted GIXM01 of PHA-L; G) the painted TPXM01 of PNA; H) the painted ATDH04 of PSA; I) the painted GIXM01 of UEA I; J) the painted GIXM01 of RCA120; K) the painted TPXM01 of WGA; L) the painted GIXM01 of WGA.From figure, Color can be seen clearly, fluorescence intensity, and some flagellum that can be colored.The abbreviation language sees table 1 and table 2 for details.
The epifluorescence microscope design sketch that Fig. 2 distinguishes biased sample for the WGA that uses FITC (fluorescein isothiocyanate) mark.Illustrate: a), b) among the figure, positive cell is unarmored dinoflagellate GspXM01; Negative cells is: Prorocentrum micans PMDH01, PMXM01, unarmored dinoflagellate GMDH01 and Heterosigma akashiwo; Figure c), d) in, positive cell is unarmored dinoflagellate TPXM01, negative cells be unarmored dinoflagellate GMDH01 and Prorocentrum donghaiense PDDH01. as seen from the figure, WGA can distinguish target cell significantly from biased sample.Scale=20 μ m, abbreviation language see Table 1 and table 2.
Fig. 3 is for counting the cell quantity of unarmored dinoflagellate GspXM01 when the different concns with different lectin probes in conjunction with epifluorescence microscope in biased sample.Illustrate: X-coordinate is X: and the cell quantity of estimating with the simple microscope counting before the sample mix (individual cell/ml); Ordinate zou is Y: (unit is cell/ml) to count the quantity of different concns cell with corresponding cell agglutinin probe in conjunction with epifluorescence microscope in biased sample, wherein ● the cell quantity of in biased sample, counting for the WGA probe, zero cell quantity of in biased sample, counting for the SBA probe
The cell quantity of in biased sample, counting for the PHA-E probe.As seen from the figure, the three has good dependency (r2=0.86, n=4, scale are mean value ± standard error).Biased sample is as follows with algae: unarmored dinoflagellate Gymnodinium mikimotoi, Akashiwo sanguinea, Gyrodinium instriatum, Prorocentrum micans Prorocentrum donghaiense Lu, P.minimum (PMDH01), and P.minimum (PMXM) and Heterosigma akashiwo.Abbreviation sees table 1,2 for details.
Fig. 4 is for counting regression relation between the different frustule quantity with the WGA probe in conjunction with epifluorescence microscope in biased sample.Illustrate: X-coordinate is: the AMTW that estimates with the simple microscope counting before the sample mix, the cell quantity of GspXM and TPXM (individual cell/ml); Ordinate zou is: (unit is: individual cell/ml) to count respective fine born of the same parents' quantity with the WGA probe in conjunction with epifluorescence microscope in biased sample, wherein ● be the cell quantity of WGA probe counting Alexandrium mimutum Halim by using AMTW in biased sample, zero is the cell quantity of WGA probe counting unarmored dinoflagellate GspXM01 in biased sample
Be the cell quantity of WGA probe counting unarmored dinoflagellate TPXM01 in biased sample.As can be seen from the figure, the three has good dependency (r2=0.92, n=4, scale are mean value ± standard error).Target cell is respectively AMTW, GspXM and TPXM, and other is used for the blended frustule and is: Prorocentrum micans PDDH01, PMDH01, PMXM01, Heterosigma akashiwo and unarmored dinoflagellate GMDH01.Initialism sees table 1,2 for details.
Fig. 5 is for to carry out painted flow cytometry analysis collection of illustrative plates (histogram) with different lectin probes to unarmored dinoflagellate GspXM01.Illustrate: the green fluorescence intensity that flow cytometer records the target frustule is respectively: a) no probe situation and probe arranged; B) SJA, c) PSA, d) SBA e) PNA, f) LCA, g) PHA-L, h) GSL I, i) S-WGA, j) RCA120, k) UEA I, l) the painted situation of ConA.As can be seen from the figure, each probe all can be given prominence to the green fluorescence signal of target algae significantly, but the fluorescent signal power of each probe is variant, and the quantity of every kind of cell is also different.Initialism sees Table 1,2, and wherein ordinate zou is that (event number, events), X-coordinate is a fluorescence intensity for the target cell number of flow cytometer counting.
Fig. 6 is flow cytometer is discerned WGA probe mark target frustule from biased sample a fluorescent signal histogram.Illustrate: A figure be flow cytometer in conjunction with the WGA probe at biased sample (unarmored dinoflagellate Gymnodinium mikimotoi, Prorocentrum micans Prorocentrum donghaiense, the design sketch of recognition objective frustule unarmored dinoflagellate (Gymnodinium sp) GspXM01 among the Prorocentrum minimum (PMDH01 is PMXM01) with Heterosigma akashiwo Heterosigma akashiw); B) be flow cytometer in conjunction with the WGA probe at biased sample (unarmored dinoflagellate Gymnodinium mikimotoi, Prorocentrum micans Prorocentrumdonghaiense, the design sketch of recognition objective frustule unarmored dinoflagellate Takayama cf.pulchellum (TPXM01) among the Prorocentrum minimum (PMDH01 is PMXM01) with Heterosigma akashiwo Heterosigma akashiw).The fluorescent signal of target cell and other frustule in the biased sample have tangible difference as seen from the figure.Initialism sees Table 1,2.Wherein ordinate zou is that (event number, events), X-coordinate is a fluorescence intensity for the cell number of flow cytometer counting.
Other cells: unarmored dinoflagellate Gymnodinium mikimotoi, Prorocentrum micans Prorocentrum donghaiense, (PMDH01 is PMXM01) with Heterosigma akashiwo Heterosigma akashiw for Prorocentrum minimum.
Target cells: target cell, A are unarmored dinoflagellate (Gymnodinium sp) GspXM01; B is unarmored dinoflagellate Takayama cf.pulchellum (TPXM01).
Fig. 7 is cells were tested by flow cytometry WGA probe mark Alexandrium AspGX01, AspGX02, the fluorescent signal histogram of AspDH01.Illustrate: AspGX01, AspGX02, AspDH01 are respectively the different cell strains of the big Trentepohlia in pressure mountain, and wherein ordinate zou is that (event number, events), X-coordinate is a fluorescence intensity for the target cell number of flow cytometer counting.No probes does not have probe.Initialism sees table 1,2 for details.
As seen from the figure, the fluorescent signal that produces after by identical WGA probe mark of different cells is variant.
Fig. 8 is the flow cytometry analysis collection of illustrative plates of different lectin probe mark list kinds and mixed algae sample.Illustrate: A): control group, no probe mark, B): unarmored dinoflagellate GspXM01 is by the s-WGA mark, C): GspXM01 is by the WGA mark, D): GspXM01 is by the LCA mark, E) GspXM01 is by UEA I mark, F) (be used for other algae of blended is the GspXM01 in the biased sample: unarmored dinoflagellate Gymnodinium mikimotoi by the situation of WGA mark, Prorocentrum micans Prorocentrum donghaiense, Prorocentrumminimum (PMDH01 is PMXM01) with red different curved algae tide Heterosigma akashiwo).X-coordinate is a side phase scattered light, and ordinate zou is a green fluorescence.Initialism sees Table 1,2.
As seen from the figure, and result detection by quantitative positive staining qualitative with flow cytometer show, the lectin probe can distinguish the red tide algae of specificity positive staining (can by the lectin mark on fluorescence) at biased sample or single plant in the sample at an easy rate from background particle, autofluorescence and negative staining algae (algae of fluorescence on can not be by the lectin mark).
The cluster analysis system tree graph that Fig. 9 makes up for the lectin probe binding pattern that uses FITC (fluorescein isothiocyanate) mark.Illustrate: this figure adopts the data of table 3, the dendrogram that the average chain rule between the use group (Average linkage (Between Groups)) makes up (making up with the SPSS statistical package).This is the tree graph of a unrooted, and A figure is Alexandria Trentepohlia Alexandrium; B is unarmored dinoflagellate Gymnodinium; C figure be Prorocentrum Prorocentrum. as can be seen from the figure, adopt the lectin binding pattern, can make up the dendrogram (Dendrogram) that can intuitively show each cell strain sibship, and help to understand the mutual relationship between the algae strain.Initialism sees table 1,2 for details.Case among the figure is legend, and Num is a quantity, and label is a label.
Embodiment
Following case study on implementation will the present invention is further illustrated in conjunction with the accompanying drawings.
Embodiment 1
Fluorescently-labeled lectin probe is identified different cell strains and plesiomorphic frustule in same genus or the kind and is detected
One, test material: the 14 kind of 23 strain cell title and the source of experiment usefulness see Table 2.
This detection kit of table 2 and detection method confirmatory experiment algae and source and disengaging time
Two. detection method:
Experiment is with the cultural method of algae: above-mentioned experiment separates from the red tide water sample in Chinese marine site with algae and obtains, be made into the f/2 substratum after boiling with the membrane filtration seawater of 0.2 μ m, in the 5000LX light intensity, L: D=12: in algae germplasm storehouse culturing room of ring coastal ocean environmental science National Key Laboratory of Xiamen University, cultivate under the temperature of 12 photoperiod and 22 ℃.
Fixing of the configuration of lectin and frustule: the lectin fresh solution of FITC-mark (100 μ g/ml Sigma) adopt the artificial seawater of 0.2 μ m membrane filtration to dispose, salinity 28, and pH 8.0.In order to check cell fixation methods to the influence of lectin bonded, adopt Lu Geshi iodine liquid, Paraformaldehyde 96 and glutaraldehyde fixed cell respectively, carry out the combination experiment of lectin then.
Each strain is tried algae and was cultivated 7 days, adds then in the cell culture of 100 μ l Percoll to 1ml.Above-mentioned then cell is collected with the centrifugal 10min of 3000rpm.The centrifugal supernatant that goes, about 10
3-10
5The cell integral body of/ml is sneaked into 50 μ lFITC-lentin. and is added 950 μ l 50mMPBS then at 20 ℃ of following dark situation insulation 1h, and then adds 100 μ lPercoll, and the centrifugal 10min of 3000rpm removes supernatant, adds 1mlPBS suspension precipitation.Can select to use the DAPI (double-stranded DNA fluorescence dye) of 50 μ g/ml to redye at last.Above-mentioned cell can directly detect in conjunction with vigor and photography falling to penetrating microscopically, and (emission light＞420nm) and blue light (exciting light 450-480nm launches light 515nm) are observed respectively for exciting light, 330～385nm to use UV-light.
Cell after the above-mentioned washing can be observed under fluorescent microscope and photographed or make a video recording.Standard below qualitative painted situation adopts is estimated: +++: dyeing is bright, and 100% cell is caught look; ++ dyeing is bright inadequately; +: staining power is low, but with control group obvious difference is arranged;-: dye-free." Blind Test method " all taked in all detections, promptly detects the people and do not know detected material situation.Staining cell to each staining power is counted, and count number is no less than 100 cells, calculates fluorescent dye bonded percentage then.
Three. the result
According to the preliminary qualitative and half-quantitative detection of epifluorescence microscope its in conjunction with situation (and calculating dyeing percentage), cell agglutinin probe SBA, WGA, GSL I, DBA and PHA-E (seeing Table 1) can distinguish unarmored dinoflagellate similar on the form effectively, as rice gold unarmored dinoflagellate Gymnodinium mikimotoi (GMDH01), unarmored dinoflagellate Takayama cf.pulchellum (TPXM01), with unarmored dinoflagellate Gymnodinium sp. (GspXM01), and can be preferably from biased sample with its identification, the result who positive cell is counted with fluorescent microscope and the result of microscopic counting have dependency preferably.Concrete outcome sees Table 3 and Fig. 1-4.
Each checking of table 3 is with algae and specificity lectin probe is qualitative and sxemiquantitative bonded pattern
(illustrate: the standard below qualitative painted situation adopts is estimated: +++: dyeing is bright, and 100% cell is caught look; ++ dyeing is bright inadequately; +: staining power is low, but with control group obvious difference is arranged;-: dye-free." Blind Test method " all taked in all detections, promptly detects the people and do not know detected material situation.Staining cell to each staining power is counted, and count number is no less than 100 cells, the percentage of calculations incorporated then.Shortenings sees table 1, table 2 for details, and is qualitative as can be seen from the table in conjunction with situation, and the binding ability of sxemiquantitative calculating)
Each checking of table 3 (continuous I) is with algae and specificity lectin probe is qualitative and sxemiquantitative bonded pattern
(illustrate: the standard below qualitative painted situation adopts is estimated: +++: dyeing is bright, and 100% cell is caught look; ++ dyeing is bright inadequately; +: staining power is low, but with control group obvious difference is arranged;-: dye-free." Blind Test method " all taked in all detections, promptly detects the people and do not know detected material situation.Staining cell to each staining power is counted, and count number is no less than 100 cells, the percentage of calculations incorporated then.Shortenings sees table 1, table 2 for details, and is qualitative as can be seen from the table in conjunction with situation, and the binding ability of sxemiquantitative calculating)
Each checking of table 3 (continuous H) is with algae and specificity lectin probe is qualitative and sxemiquantitative bonded pattern
(illustrate: the standard below qualitative painted situation adopts is estimated: +++: dyeing is bright, and 100% cell is caught look; ++ dyeing is bright inadequately; +: staining power is low, but with control group obvious difference is arranged;-: dye-free." Blind Test method " all taked in all detections, promptly detects the people and do not know detected material situation.Staining cell to each staining power is counted, and count number is no less than 100 cells, the percentage of calculations incorporated then.Shortenings sees table 1, table 2 for details, and is qualitative as can be seen from the table in conjunction with situation, and the binding ability of sxemiquantitative calculating)
Four. conclusion
Cell agglutinin probe can be distinguished the plesiomorphic red tide plankton unarmored dinoflagellate from East China Sea preferably from single kind or biased sample.
Embodiment 2
Fluorescently-labeled lectin probe combined with fluorescent spectrophotometer carries out detection by quantitative
One, material: with embodiment 1.
Two, method:
1. method such as the fixing and lectin probe combination of cell dyeing is seen embodiment 1.
2.lectin probe and the quantitative assay of sample bonded adopt spectrophotofluorometer to detect (exciting light of 490nm and the scattered light of 515nm), every duplicate samples reading secondary is averaged.Under every kind of situation, the cell density behind the centrifuge washing 3 times is controlled at 10
5± 10
3Need not to redye with DAPI.Quantitatively and in the qualitative test, all with cell density in the f/2 medium 10
5± 10
3The cell that does not add lectin in contrast.Statistical study is carried out in each sample replicate(determination) 4～6 times then.
Three. the result
Adopt that spectrophotofluorometer can more accurate ground quantitative assay lectin in conjunction with vigor.In this checking example, probe PHA-E can distinguish Alexandrium tamarense Alexandrium tamarense (ATDH04) and other Alexandrium tamarense from waters, the East Sea and contiguous waters effectively, also can distinguish Alexandrium tamarense A.tamarense (ATMJ02) from A.tamarense (ATMJ01) and other Alexandrium tamarense.The PNA probe can be discerned A.tamarense (ATDH01 from each Alexandrium tamarense strain system, 02,03), probe UEA I can be from detecting A.tamarense (ATCI01-JN from each Alexandrium tamarense strain system, ATCI01), RCA120 can distinguish in the Alexandrium strain system and produce inconsistent A.sp of malicious level (AspGX01) and A.sp (AspGX02), and wherein A.sp (AspGX02) produces the paralytic shellfish poison hardly.Concrete outcome sees table 4 for details.
Table 4 various red tide algaes of fluorescent spectrophotometer assay and specificity lectin probe quantitative bonded situation
(illustrate: the data in the form are the fluorescent value (mean value ± standard error) of quantitative assay, and fluorescence numerical value unit wherein is: ng FITC (fluorescein isothiocyanate)/ml.10000 cell.Initialism in the table sees Table 1,2,
*P＜0.05 shows between the algae strain of corresponding kind of plesiomorphic dinoflagellate and its or genus to be compared, and its fluorescent value has significant difference.)
Table 4 (continuing) various red tide algaes of fluorescent spectrophotometer assay and specificity lectin probe quantitative bonded situation
(illustrate: the data in the form are the fluorescent value (mean value ± standard error) of quantitative assay, and fluorescence numerical value unit wherein is: ng FITC (fluorescein isothiocyanate)/ml.10000 cell.Initialism in the table sees table 1,2 for details,
*P＜0.05 shows between the algae strain of corresponding kind of plesiomorphic dinoflagellate and its or genus to be compared, and its fluorescent value has significant difference.)
Four. conclusion
With spectrophotofluorometer accurately quantitative assay lectin probe in conjunction with situation, the result shows that the lectin probe can discern effectively and distinguish from identical and the adjacent waters and the different cell strains in the red tide algae of the same race of different geographic origin, also can distinguish producing the different algae strain of malicious level.
Fluorescently-labeled lectin probe is in conjunction with the quantitative and qualitative detection of flow cytometer
One, material: with embodiment 1.
Two, method:
Get 7 days algae liquid of growth, concentration is 100000 cell/L, and the Paraformaldehyde 96 with 1% is at 4 ℃ of following fixedly 1h.
Get the centrifugal 3000g/10min of algae liquid about 10ml, sedimentary frustule is transferred in the centrifuge tube of 1.5ml.
Decolouring: with salt-ethanolic soln (70: 30, v/v) washing is centrifugal 2 times, 7500g/2min, the silk cover filtering of wherein using 30 μ m once.
Precipitation is handled 1h with the lectin of 100 μ g/ml in 20 ℃ of dark reactions.After reaction finishes, to filter aseptic artificial seawater (salinity 28, pH8.0) centrifuge washing secondary, 7500g/2min.
Cell is suspended in the artificial seawater again, analyzes with flow cytometer (Beckman Coulter Epics Altra2 type).
Three. the result
Unarmored dinoflagellate (GspXM01) with 14 kinds of different probe marks shows different fluorescence in conjunction with behavior under the detection of flow cytometer, flow cytometer passes through green fluorescence, can be the unarmored dinoflagellate Gymnodiniumsp. (GspXM01 of WGA probe positive staining, Fig.6A) and unarmored dinoflagellate Takayama cf.pulchellum (TPXM01, Fig.6B) from biased sample, (contain the golden unarmored dinoflagellate Gymnodinium mikimotoi of rice, Prorocentrum donghaiense Prorocentrum donghaiense, prorocentrum minimum Prorocentrum minimum (PMDH01, PMXM01) and Heterosigma akashiwo Heterosigma akashiwo etc.) distinguishes.In biased sample, (AspDH01) to combine the fluorescence intensity that is showed also variant for cell and WGA probe, thereby can be discerned and sorting by flow cytometer for AspGX01, AspGX02 for Alexandrium.Flow cytometer can also be (containing the golden unarmored dinoflagellate Gymnodinium mikimotoi of rice in the mark fluorescent (GspXM01) of the positive staining unarmored dinoflagellate of WGA and the biased sample, Prorocentrum donghaiense Prorocentrum donghaiense, prorocentrum minimum Prorocentrum minimum (PMDH01 is PMXM01) with the Heterosigma akashiwo Heterosigma akashiwo) autofluorescence of frustule and other pars granulosa of non-specific binding be (seeing Fig. 5-8 for details) separately.
Four. conclusion
Show that by the qualitative and quantitative detection of flow cytometer the lectin probe can distinguish (can by the lectin mark on fluorescence) red tide algae of positive staining at biased sample or single plant in the sample at an easy rate from background particle and negative staining algae (algae of fluorescence on can not be by the lectin mark).
Cell agglutinin (lectin) is a kind of glycoprotein of non-immunogenicity, and it can be special and narrow spectrum non-covalent combination (Goldstein etc., 1980 take place miniature frustule surface specific glycosyl; Hori etc., 1996), the specificity glycosyl that utilizes little algae surface is as identification marking, and the present invention can discern it and identify easily, and this identification is not subjected to the restriction of cell growth cycle and fixing means.The present invention utilizes 14 kinds of fluorescently-labeled cell agglutinins (lectin) probe, to having carried out qualitative and quantitative detection from the typical harmful algal algae in the Chinese red tide water sample, show cell agglutinin (lectin) probe can be effectively to plesiomorphism, poisonous or nontoxic even to discerning and distinguish from the algae strain in close or adjacent marine site or the geographical strains system of different waters.In conjunction with relevant qualitative and quantitative means and self-editing software processing, the present invention has set up based on fluorescently-labeled cell agglutinin (Lectin) probe the rapid detection box of red tide plankton and supporting technical system, has realized that typical red tide plankton to China coastal waters simply and accurately detects fast and identifies.
Claims (9)
1, fluorescence labeled cell agglutinin probe red tide biological detection reagent kit is characterized in that being provided with box body and component I, component II, component III, and component I, component II and component III are located in the box body;
Component I is the probe system of cell agglutinin, and the probe system of cell agglutinin contains the fluorescently-labeled cell agglutinin probe of n kind, n 〉=1, volume is 500～1000 μ L * n, the concentration of each probe is 50～100 μ g/ml, adopts the phosphate buffer solution configuration, contains 0.5%～1.5% sodiumazide, component I contains the cell agglutinin probe system of 14 kinds of marked by fluorescein isothiocyanate: ConA, DBA, PNA, RCA120, SBA, UEA I, WGA, GSL I, LCA, PHA-E, PHA-L, PSA, SJA, the succinylation wheat germ agglutinin;
Component I I is the reaction buffer system, it is 100～300mL that volume is provided, the reaction buffer system is the artificial seawater configuration of sterilising filtration, and salinity is 25～30, and pH is 7.5～8.5, perhaps be phosphate buffer solution PBS, concentration is 40～60mM, and pH is 7.5～8.5, and the supporting peck solution that provides is the silica gel particle of V-Pyrol RC, be used for cellular segregation, concentration is 70%～90%;
Component III is the sample fixed system, it is 100～400mL that volume is provided, the sample fixed system is selected from a kind of in Lu Geshi iodine liquid, Paraformaldehyde 96 and the glutaraldehyde, the concentration of Lu Geshi iodine liquid is 10%～20%, the final concentration of Lu Geshi iodine liquid is 0.5%～1.5%, and the concentration of Paraformaldehyde 96 is 10%～20%, and the final concentration of Paraformaldehyde 96 is 0.5%～1.5%, the concentration of glutaraldehyde is 20%～25%, and the final concentration of glutaraldehyde is 1.0%～2.5%.
2, fluorescence labeled cell agglutinin probe red tide biological detection reagent kit as claimed in claim 1 is characterized in that component I contains the fluorescently-labeled cell agglutinin probe of n kind, and volume is 1000 μ L * n; The salinity of component I I is 28, and pH is 8.0; Perhaps be phosphate buffer solution, concentration is 50mM, and pH is 8.0.
3, fluorescence labeled cell agglutinin probe red tide biological detection reagent kit as claimed in claim 1, the concentration that it is characterized in that said peck solution is 90%.
4, fluorescence labeled cell agglutinin probe red tide biological detection reagent kit as claimed in claim 1, the concentration that it is characterized in that the Lu Geshi iodine liquid in the component III is 10%, the final concentration of Lu Geshi iodine liquid is 1%, the concentration of Paraformaldehyde 96 is 10%, the final concentration of Paraformaldehyde 96 is 1%, the concentration of glutaraldehyde is 25%, and the final concentration of glutaraldehyde is 2.5%.
5, fluorescence labeled cell agglutinin probe red tide biological detecting method is characterized in that the steps include:
1) with the cultivation of algae: separate obtaining experiment and use algae from the red tide water sample, the f/2 substratum that is made into after boiling with the membrane filtration seawater carries out pure culture; Or with the direct centrifugal or filtering and concentrating of the red tide water sample of collection in worksite;
2) configuration of cell agglutinin and frustule is fixing: the fresh solution that adopts filtering artificial seawater or PBS configuration fluorescence labeled cell agglutinin probe, select to use Lu Geshi iodine liquid, Paraformaldehyde 96 or glutaraldehyde fixed cell, carry out the combination experiment of cell agglutinin then;
3) after each strain is tried the algae cultivation, add peck solution to cell culture, cell centrifugation is collected, and removes supernatant, cell integral body is sneaked into fluorescently-labeled cell agglutinin probe, add phosphate buffer solution, add peck solution again, centrifugal, remove supernatant, add phosphate buffer solution suspension precipitation, redye, under fluorescent microscope, use UV-light and blue light to observe respectively then with the double-stranded DNA fluorescence dye of 50 μ g/ml; Or adopt spectrophotofluorometer and flow cytometer to carry out quantitatively and qualitative detection;
4) cell after will washing is observed under fluorescent microscope and is photographed or make a video recording, and the standard below qualitative painted situation adopts is estimated: +++: dyeing is bright, and 100% cell is caught look; ++ dyeing is bright inadequately; +: staining power is low, but with control group obvious difference is arranged;-: dye-free, sample are taked " Blind Test method ", promptly detect the people and do not know detected material situation, and the staining cell of each staining power is counted, and count number is no less than 100 cells, calculates fluorescent dye bonded percentage then.
6, fluorescence labeled cell agglutinin probe red tide biological detecting method as claimed in claim 5, it is characterized in that in step 1), be made into the f/2 substratum after boiling with the membrane filtration seawater of 0.2 μ m, in the 5000LX light intensity, L: D=12: in the algal species cultivation chamber, cultivate under the temperature of 12 photoperiod and 22 ℃.
7, fluorescence labeled cell agglutinin probe red tide biological detecting method as claimed in claim 5, it is characterized in that in step 2) in, requiring the fluorescently-labeled cell agglutinin probe fresh solution of configuration is 100 μ g/ml, adopt phosphate buffer solution 50mM, pH8.0 or the configuration of filtering artificial seawater, salinity is 28, and pH is 8.0.
8, fluorescence labeled cell agglutinin probe red tide biological detecting method as claimed in claim 5, it is characterized in that in step 3), each strain is tried algae and was cultivated 7 days, add 100 μ l peck solution to the cell culture of 1ml, cell is collected with the centrifugal 10min of 3000rpm, the centrifugal supernatant that goes is with 10
3～10
5The cell integral body of/ml is sneaked into the fluorescently-labeled cell agglutinin probe of 50 μ l, add 950 μ l 50mM phosphate buffer solutions and under 20 ℃, be incubated 15min-1h in dark situation, and then add 100 μ l peck solution, the centrifugal 10min of 3000rpm, remove supernatant, add the phosphate buffer solution suspension precipitation of trying one's best few at last, use the double-stranded DNA fluorescence dye of 50 μ g/ml to redye.
9, fluorescence labeled cell agglutinin probe red tide biological detecting method as claimed in claim 5, it is characterized in that said qualitative dyeing is for carrying out qualitative dyeing estimation under epifluorescence microscope, staining cell counting to each staining power calculates fluorescent dye bonded percentage; Selection is carried out quantitatively or qualitative test with spectrophotofluorometer or flow cytometer; Measurement result adopts self-editing computer software to analyze and statistical treatment.
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