CN101851666B - Detection method of enteromorpha micro-generation fluorescence in-situ hybridization - Google Patents
Detection method of enteromorpha micro-generation fluorescence in-situ hybridization Download PDFInfo
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Abstract
The invention relates to green-tide biological detection and monitoring research, in particular to a detection method of enteromorpha micro-generation fluorescence in-situ hybridization. An oligonucleotide probe for marking the in-situ hybridization of enteromorpha micro-generation with FITC (Fluoresceine Isothiocyanate) comprises one or more than one of the following four probes: (1) 5'-gcccgccgtttacaggat-3', (2) 5'-agccctaacccattgaaccc-3', (3) 5'-ccctgcgatagtaactgagaca-3' and (4) 5'-GGAGCCCTAACCCATTGAA-3', wherein the 5' terminals are marked by an FITC marking method. By the invention, the enteromorpha micro-generation can be rapidly and accurately evaluated and counted to confirm the biomass and the distribution situation thereof and achieve the aim of accurately detecting and forecasting.
Description
Technical field
The present invention relates to green damp biological detection and study on monitoring, specifically a kind of fluorescent in situ hybridization detecting method of enteromorpha micro-generation.
Background technology
Detection method based on molecular basis (nucleic acid, albumen, glycoprotein) has very big advantage than traditional detection method, kind is identified more accurately and reliably, and can provide quantitative information, can detect more low-abundance target microalgae kind (the miniature generation of macro), make it in routine monitoring, to use.Because the heterogeneity in rRNA gene ITS district makes it show evident difference between all kinds of biological genus, kind.Usually adopting the ITS district as the zone of classification and systematic study between various biological groups genus kinds in the world, also is the preferred region of designing probe.Comparatively speaking, transcribed spacer ITS district between the two, zone for hypermutation, for the research of all kinds of group of biology subordinate levels provides more information, it is one of each monoid subordinate interspecies level research of the biology of generally acknowledging in the world well index relatively, in vegeto-animal molecular classification, used widely, in the algae Molecular Identification, also be used.
Fluorescence in situ hybridization (fluorescence in situ hybridization, FISH) be a kind of on-radiation in-situ hybridization method, it adopts special fluorescein-labelled nucleic acid (DNA) probe, whether exists the most effective to the particular sequence that detects DNA in the cell or RNA.This technology can detect intracellular nucleotide sequence under the condition that keeps the cellular form integrity.The primary process of FISH technology be utilize fluorescently-labeled probe in cell with the nucleic acid array hybridizing of specificity complementation.Come detection signal by the fluorescence that excites hybridization probe, thereby detect corresponding nucleotide sequence.The main step of this technology comprises: 1) sample is fixing; 2) preparation of sample and pre-treatment; 3) prehybridization; 4) probe and sample sex change; 5) detect different target sequences with different probe hybridizations; 6) unconjugated probe is removed in rinsing; 7) detect hybridization signal, carry out interpretation of result.Its key link detects (Miller et al., 1998 after selection, crossover process and the hybridization of fluorescence dye; Groben et al., 2005).The fluorescence dye that the FISH technology adopts usually is fluorescein derivative (FITC, FluoX), rhodamine derivative (TRITC, TexasRed) and the indoles dicarboxyl mountain valley with clumps of trees and bamboo (Cy3, Cy5) etc., because the indoles dicarboxyl mountain valley with clumps of trees and bamboo has significant brightness and light extinguishes stability, therefore has application promise in clinical practice.
2007 and 2008, the green tide of China's Huanghai Sea middle part outburst in continuous 2 years Enteromorpha (Enteromorphaprolifera).At the beginning of 2008 6 months, the historical rare green damp phenomenon of large-scale Enteromorpha appears in marine site, the Huanghai Sea middle and south, and gradually to offshore sea waters, Qingdao drift, the green damp longer duration of Enteromorpha, scale are big, badly influence Olympic Sailing competition match marine environment quality and the coastal line view in urban district, Qingdao.This shows, the coastal severely afflicated area that not only becomes Chinese red tide generation of China, and to have become with large-scale green alga Enteromorpha (Enteromorpha prolifera) be the severely afflicated area of the green tide of representative, to red tide, green tide, particularly accurate evaluation, rapid detection and the early-warning and predicting of biology such as green little generation of tide have become the hot issue of current red tide and green damp research field, and have important theory and practical significance.
Difficulty and restriction at the traditional monitoring method, external scientists has been devoted to develop various specific molecular probes and has been detected various red tide monoids, the target red tide plankton can be detected by the means of optics or chemistry, realize fast and accurately identifying, counting the green damp kind of target, thereby determine biomass, the distribution situation of red tide, reach the purpose that green tide is forecast in accurate detection.Up to now, molecular probe technology detects with study on monitoring at red tide plankton and has obtained in the world developing rapidly, and it is few especially at the correlative study of green tide, particularly at Enteromorpha (Enteromorpha prolifera) little generation, the fluorescence in situ hybridization technique of the seed bank of namely surviving the winter (propagule bank) form yet there are no report.
Summary of the invention
The object of the present invention is to provide a kind of fluorescent in situ hybridization detecting method of enteromorpha micro-generation fast and accurately; It can realize fast and accurately identifying, counting Enteromorpha, thereby determines its biomass, distribution situation, reaches the purpose of accurate detection forecast.
For achieving the above object, the technology of the present invention's employing is:
The fluorescent in situ hybridization detecting method of enteromorpha micro-generation is used for oligonucleotide probe that the in situ hybridization of FITC mark detects enteromorpha micro-generation and is one of following 4 kinds of probes or more than one,
1)5’-GCCCGCCGTTTACAGGAT-3’
2)5’-AGCCCTAACCCATTGAACCC-3’
3)5’-CCCTGCGATAGTAACTGAGACA-3’
4)5’-GGAGCCCTAACCCATTGAA-3’
Hold the way that adopts the FITC mark in their 5 '.
Be used for oligonucleotide probe that the in situ hybridization of FITC mark detects enteromorpha micro-generation and be preferably one of following 2 kinds of probes or two kinds,
2)5’-AGCCCTAACCCATTGAACCC-3’，
3)5’-CCCTGCGATAGTAACTGAGACA-3’。
The detailed process of in situ hybridization is:
1) preparation of sample and pre-treatment
After on-the-spot seawater water body example concentrated, autofluorescence to algae is fixed, is decoloured: use centrifuge tube in the centrifugal sample 5min of 5000g/min, it is fixing to final volume concentration 1% to add ethanol, left standstill 1-2 hour, must concentrate the sample after the decolouring, on have on the strainer of polycarbonate membrane and filter, cell is stayed on the polycarbonate membrane; Adopt 1 * PBS and hybridization buffer to filter cleaning filter membranes respectively;
2) hybridization: filter membrane is placed on the slide glass, directly add hybridization buffer and fluorescent probe, make the probe final concentration reach 5-10ng/ μ L; Slide is placed warm and humid dark place 46-50 ℃ of hybridization 2-3h;
3) unconjugated probe is removed in rinsing: stop hybridization with the hybridization scavenging solution washing by soaking filter membrane that is preheated to 1 * SET of 50 ℃, filter membrane is blotted with clean filter paper in the washing back;
4) detect hybridization signal, carry out interpretation of result
Adding mountant and final concentration is the mixed solution of 1 μ g/mL dna marker thing DAPI, redyes and prevent fluorescent quenching.Covered and mounting are stored in the dark place in order to detecting.Frustule after the hybridization is with the fluorescence microscope crossbreeding effect, and the record positive mark leads; Use the excitation wavelength of 450-490nm when observing the fluorescently-labeled frustule of FITC, the spectral filter combination of 510-520nm wavelength of transmitted light is observed, observe and confirm the enteromorpha micro-generation that exists in the water sample.
Concentrate with silk cover filtering after the collection of described seawater water body example; Described hybridization buffer consist of 0.9M NaCl, 20mmol/LTris-HCl, pH7.2,0.1% (v/v) Nonidet-P40,20% (v/v) methane amide.
Whole thinking of the present invention
1, the design of probe and making
With the Enteromorpha ITS sequence that obtains (referring to the database website http://www.ncbi.nlm.nih.gov/sites/gquery of sequence, landing sequence number is FJ026732) with the ITS sequence of all strain system of the green laver that obtains from nucleic acid database kinds such as GeneBank and EMBL, using Clustal X (free software) sorts to sequence, and manually reduce the number of rank results, determine the specific sequence at target algae categorization levels; BLAST N interface by database (GeneBank and EMBL) screens these rank results then.The specific sequence that obtains after the screening is used for the suitable probe of design.
The fluorescence dye that the FISH technology adopts usually is fluorescein derivative (FITC, FluoX), rhodamine derivative (TRITC, Texas Red) and the indoles dicarboxyl mountain valley with clumps of trees and bamboo (Cy3, Cy5) etc., because the indoles dicarboxyl mountain valley with clumps of trees and bamboo has significant brightness and light extinguishes stability, therefore has application promise in clinical practice.Fluorescent marker dyes commonly used is as shown in table 1;
The FISH fluorescence dye that table 1 is commonly used
2, the acquisition of sample
1) acquisition of sample in the water body
Gather the 5-10L seawater with water sampler, concentrate with 10 μ m silk cover filterings, volume of water is reduced into 100-200mL.
2) adhere to the acquisition of sample
Adhere to the sample collecting place and be generally the tideland.The most frequently used method is that placement slide glass or other carriers are placed on the target area, regularly fetches the laboratory and detects; Another kind method is to adopt the mode of scraping target algae, and this mode causes the breakage of target algae easily, and impurity is too many, is difficult for detecting, and does not generally use this method.
3, hybridization technique flow process
The main step of this technology comprises:
1) sample is fixing;
2) preparation of sample and pre-treatment;
3) prehybridization;
4) probe and sample sex change;
5) detect different target sequences with different probe hybridizations;
6) unconjugated probe is removed in rinsing;
7) detect hybridization signal, carry out interpretation of result.
Its key link detects after selection, crossover process and the hybridization of fluorescence dye.
The fluorescent in situ hybridization detecting method of Enteromorpha (Enteromorpha prolifera) little generation that the present invention adopts has the following advantages:
1, fluorescent reagent and probe economy, safety:
The probe of in situ hybridization is divided into radio-labeling and nonradioactive labeling by the tagged molecule type.Be to strengthen strength of signal by prolonging exposure time to preparing the less demanding of sample with isotope-labeled radioactive probe advantage, so sensitive.Shortcoming is that probe instability, autography time are long, the scattering of radioactive rays make spatial resolution not high, and the isotropic substance operation more loaded down with trivial details etc.Project adopts original position fluorescent mark system then can overcome these deficiencies, and does not have radioactivity, and expense is less, therefore has the characteristics of economy, safety.
2, probe is stable, can use in two years behind the mark;
The probe of Project design is preserved under appropriate condition, can make extension of validity to two year.Therefore, probe once designs, can life-time service, reach time saving purpose.
3, experimental period short, can obtain rapidly the result,
The test operation flow process is simple, and the time is short, reaches the purpose of rapid detection.
4, amount that can the detection by quantitative enteromorpha micro-generation
The probe specificity of Project design is good, accurate positioning, under fluorescent microscope, can know and see each the spore/zygote of being hybridized/gamete, therefore, under the situation of sampling and concentrated amount record, can be by the amount of enteromorpha micro-generation seed bank in the way detection by quantitative water body of calculating.
5, polychrome FISH is by showing that in same nuclear distinct colors can detect multiple sequence simultaneously
This invention can be used multiple Enteromorpha probe simultaneously, by different fluorescence colors, and existence and the amount thereof of more correct affirmation enteromorpha micro-generation.
Description of drawings
Fig. 1 (a) and (b), (c), (d), (e), (f) are the ITS sequence alignment figure of continuous 23 samples.
Embodiment
Experimental implementation
1, design and the making of the little generation probe of Enteromorpha (Enteromorpha prolifera)
1) the green tide of whole Enteromorpha that is attained at of Enteromorpha (Enteromorpha prolifera) ITS sequence influences 23 different websites of marine site collection, the PCR fragment total length of the ITS sequence of Enteromorpha sample and 5.8SrDNA sequence is 540bp, wherein ITS1 length is 189bp, 5.8S rDNA complete sequence length is 160bp, ITS2 length is 181bp.See that from the result of sequence alignment the ITS sequence of these 23 samples and 5.8S rDNA sequence are identical, can determine to belong to a kind.Enteromorpha ITS sequence obtains for this laboratory order-checking, lands the database website http://www.ncbi.nlm.nih.gov/sites/gquery of sequence, and landing sequence number is FJ026732; Sequence alignment as shown in Figure 1;
2) screening of probe
With the Enteromorpha ITS sequence that obtains and the stone that obtains from nucleic acid database kinds such as GeneBank and the EMBL ITS sequence of all strain systems sheerly, using Clustal X (free software) sorts to sequence, and manually reduce the number of rank results, determine the specific sequence at target algae categorization levels; BLAST N interface by database (GeneBank and EMBL) screens these rank results then.The specific sequence that obtains after the screening is used for the suitable probe of design, utilizes OLIGO6.0 software to determine suitable probe length (approximately about 20bp), screens out inappropriate probe on the structure according to the characteristics such as secondary structure of Tm value and probe.
3) use of fluorescence dye
The probe that obtains is entrusted the precious biosynthesizing in commercial company Dalian and is carried out the mark of 5 ' end fluorescein isothiocyanate FITC fluorophor.
2, the acquisition of sample
Gather the 5-10L seawater with water sampler and the intensive marine site of Enteromorpha, concentrate with 10 μ m silk cover filterings, volume of water is reduced into 100-200mL, studying Lu Geshi iodine liquid, 4% formalin, 2.5% glutaraldehyde that need add 1/10 volume Paraformaldehyde 96 (10%, final concentration 1%) or 1% respectively per sample fixes.
3, hybridization technique flow process
On-the-spot sample concentrated and microscopic counting after, the autofluorescence of algae is decoloured: adopt ethanol, acetone or methyl alcohol gradient decolouring (this step decolorizing effect and time decide as the case may be), filtration.Draw the sample that concentrates about 1-2ml after decolouring with syringe, at the low press filtration of 13mm syringe filter, cell is then stayed on the filter membrane (polycarbonate membrane or nucleopore membranes).Draw 1 * PBS 1mL with syringe and clean a 5min, filter, hybridization buffer (0.9MNaCl, 20mM Tris-HCl, pH7.2,0.1% (v/v) Nonidet-P40, the 20% methane amide) 1mL that draws preheating with syringe cleans once, and 5min filters.Filter membrane can the air at room temperature drying, can at room temperature store about one month, also can be directly used in fluorescence in situ hybridization (FISH), and big filter membrane such as 25mm can be cut into some fritters, as the multiple sample analysis.Filter membrane is placed on the slide glass, directly add hybridization buffer 55 μ L and the fluorescent probe 5 μ L (66ng/ μ L) of preheating, make the probe final concentration reach 5ng/ μ L.Place warm and humid hybridizing box (self-control) in dark place 46-50 ℃ of hybridization 2-3h slide.L Water Paper in the warm and humid dark hybridizing box is wetting with hybridization buffer, and guarantees that the hybridization buffer that contains probe covers filter membrane (according to the filter membrane size, consumption can reduce) fully.With (50 ℃) 1 * SET of preheating (1mM EDTA, 20mM Tris/HCl, 0.15MNaCl, pH7.8) the hybridization scavenging solution of 100 μ L washing filter membrane stops hybridization, cleans secondary altogether, each incubation 5 minutes, filter membrane is blotted with clean filter paper in the washing back.Add 10-60 μ L mountant Citifluor/DAPI-Mix (containing 1 μ g/mL DAPI) and redye and prevent fluorescent quenching.Every slide can and discharge the filter membrane of two 25mm, and covered and mounting are stored in the dark place in order to detecting.Slide can under freezing conditions be preserved the several months and fluorescence can not decayed.
4, experimental result
Detect Enteromorpha spore/gamete/zygote that the transmitting green fluorescence that quantity do not wait is found to have in sheet in (exciting 492nm, emission 528nm) back through NIKON 80i fluorescent microscope.
Embodiment
1, the acquisition of sample
This experiment ended in July, 2008 since in June, 2008.Water body example picks up from the floating Enteromorpha close quarters in tideland, second bathing beach, Qingdao (35.35 ° of N, 119.30 ° of E, the following 10-35cm of the water surface) on June 15th, 2008.Operating process after the collected specimens is with above-mentioned experimental implementation.Gather 55 of water body examples altogether, each 5L.Test indoor 10 μ m silk cover filterings and concentrate, volume of water is reduced into 100mL.
2, the design of probe
Be used for oligonucleotide probe (according to the ITS sequences Design) design of FITC mark in situ hybridization in the Enteromorpha with above-mentioned method, 5 ' end adopts the way of FITC mark, designs 4 kinds of probes altogether:
1)5’-GCCCGCCGTTTACAGGAT-3’
2)5’-AGCCCTAACCCATTGAACCC-3’
3)5’-CCCTGCGATAGTAACTGAGACA-3’
4)5’-GGAGCCCTAACCCATTGAA-3’
3, hybridization flow process
1) preparation of sample and pre-treatment
On-the-spot sample concentrated and microscopic counting after, the autofluorescence of algae is fixed, is decoloured: in the centrifugal 15mL sample of 5000g/min 5min, abandon supernatant with the 15mL centrifuge tube, add ethanol fixing agent (volumetric concentration 1%) 3mL, left standstill 1-2 hour.
Draw the sample that concentrates about 1-2ml after decolouring with syringe, filter at the strainer that has polycarbonate membrane with the 13mm syringe, cell is stayed on the polycarbonate membrane.
2) hybridization
Filter membrane is placed on the slide glass, (50 ℃) hybridization buffer (the 0.9M NaCl that directly adds preheating, 20mmol/LTris-HCl, pH7.2,0.1% (v/v) Nonidet-P40,20% (v/v) methane amide) 55 μ L and fluorescent probe 5 μ L (50ng/ μ L) make the probe final concentration reach 5ng/ μ L.Place warm and humid hybridizing box in dark place 46-50 ℃ of hybridization 2-3h slide.L Water Paper in the warm and humid dark hybridizing box is wetting with hybridization buffer, and guarantees that the hybridization buffer that contains probe covers filter membrane fully.
3) unconjugated probe is removed in rinsing
With (50 ℃) 1 * SET of preheating (1mMEDTA, 20mMTris/HCl, 0.15MNaCl, pH7.8) the hybridization scavenging solution washing by soaking filter membrane of 100 μ L stops hybridization, cleans secondary altogether, each incubation 5 minutes, filter membrane is blotted with clean filter paper in the washing back.
4, detect hybridization signal, carry out interpretation of result
Add the mixed solution of 10-60 μ L mountant Citifluor and dna marker thing DAPI (final concentration is 1 μ g/mL), redye and prevent fluorescent quenching.Every slide can and discharge the filter membrane of two 25 * 25mm, and covered and mounting are stored in the dark place in order to detecting.Slide can under freezing conditions be preserved the several months and fluorescence can not decayed.Frustule after the hybridization is observed crossbreeding effect with fluorescent microscope (Leica, model is DMIRB), and the record positive mark leads.Use the excitation wavelength of 450-490nm when observing the fluorescently-labeled frustule of FITC, the spectral filter combination of 510-520nm wavelength of transmitted light is observed.Observations finds in 55 water samples 24 enteromorpha micro-generations of observing green-emitting fluorescence are arranged, and having rate is 43.64%; Its middle probe 2) 5 '-AGCCCTAACCCATTGAACCC-3 ' and 3) the mark effect of 5 '-CCCTGCGATAGTAACTGAGACA-3 ' is better.
Enteromorpha micro-generation
SEQUENCE LISTING
＜110〉Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science
＜120〉fluorescent in situ hybridization detecting method of enteromorpha micro-generation
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<160>4
<170>PatentIn version 3.1
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＜213〉artificial sequence
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gcccgccgtt tacaggat 18
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agccctaacc cattgaaccc 20
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Enteromorpha micro-generation
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ccctgcgata gtaactgaga ca 22
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Claims (4)
1. the fluorescent in situ hybridization detecting method of enteromorpha micro-generation is characterized in that:
Being used for the oligonucleotide probe that the in situ hybridization of FITC mark detects enteromorpha micro-generation is,
5’-AGCCCTAACCCATTGAACCC-3’
Adopt the way of FITC mark in 5 ' end.
2. according to the described detection method of claim 1, it is characterized in that: the detailed process of in situ hybridization is,
1) preparation of sample and pre-treatment
After on-the-spot seawater water body example concentrated, autofluorescence to algae is fixed, is decoloured: use centrifuge tube in the centrifugal sample 5min of 5000g/min, it is fixing to final volume concentration 1% to add ethanol, left standstill 1-2 hour, must concentrate the sample after the decolouring, on have on the strainer of polycarbonate membrane and filter, cell is stayed on the polycarbonate membrane; Adopt 1 * PBS and hybridization buffer to filter cleaning filter membranes respectively;
2) hybridization: filter membrane is placed on the slide glass, directly add hybridization buffer and fluorescent probe, make the probe final concentration reach 5-10ng/ μ L; Slide is placed warm and humid dark place 46-50 ℃ of hybridization 2-3h;
3) unconjugated probe is removed in rinsing: stop hybridization with the hybridization scavenging solution washing by soaking filter membrane that is preheated to 1 * SET of 50 ℃, filter membrane is blotted with clean filter paper in the washing back;
4) detect hybridization signal, carry out interpretation of result
Adding mountant and final concentration is the mixed solution of 1 μ g/mL dna marker thing DAPI, redyes and prevent fluorescent quenching.Covered and mounting are stored in the dark place in order to detecting.Frustule after the hybridization is with the fluorescence microscope crossbreeding effect, and the record positive mark leads; Use the excitation wavelength of 450-490nm when observing the fluorescently-labeled frustule of FITC, the spectral filter combination of 510-520nm wavelength of transmitted light is observed, observe and confirm the enteromorpha micro-generation that exists in the water sample.
3. according to the described detection method of claim 2, it is characterized in that: concentrate with silk cover filtering after the collection of described seawater water body example.
4. according to the described detection method of claim 2, it is characterized in that: described hybridization buffer consist of 0.9M NaCl, 20mmol/LTris-HCl, pH7.2,0.1% (v/v) Nonidet-P40,20% (v/v) methane amide.
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