CN101724690A - Method for detecting polymorphism of flora of prawn culture water body - Google Patents
Method for detecting polymorphism of flora of prawn culture water body Download PDFInfo
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Abstract
The invention discloses a method for detecting polymorphism of a flora of a prawn culture water body. The method comprises the following steps: (1) extracting total genomic DNA of mixed microbes in a prawn culture water body sample; (2) designing a specificity T-RFLP-PCR universal primer and performing PCR circulating reaction; (3) purifying a product and performing enzyme cutting on DNA by a specific restriction enzyme Hae III; (4) performing ionophortic separation on DNA segments by 1 percent agarose gel, performing fluorescent scanning on the DNA segments; (5) analyzing a polymorphism structure of a microbe flora; and (6) performing quantitative detection on predominant bacteria of the flora through fluorescence in situ hybridization technology. The method improves detection technology combining T-RFLP-PCR with FISH, uses fluorescence to mark a specific primer; and compared with the modern technology, the method has the characteristics of high repeatability, sensitivity, rapidness, accuracy, stability and the like, and can qualitatively and quantitatively analyze the ecological diversity of the microbes in the prawn culture water body along with time change and the dynamic change of a composite structure of the predominant flora.
Description
Technical field:
The present invention relates to a kind of molecular detecting method of prawn culturing microorganisms in water polymorphism of flora, belong to the microbial ecology field.
Background technology:
Microorganism has critical role in a lot of national economy production department, all utilize microorganism to carry out activity in production as biomedical engineering (animal, body-care), biological pesticide (endophyte of plant), light industry and fields such as environment protection, aquaculture.What in most cases, the activity in production of these departments relied on is the microorganism species allomeric function that multiple Institute of Micro-biology forms.For a long time, these utilize microbial ecosystem to carry out many industry departments of activity in production, owing to be subjected to traditional microorganism separation and Culture technology limitation, can't be comprehensively, the member of group in the recognition system objectively, more can't dynamic tracking system member population quantity change influence to system function, production efficiency is low, and unstable.
The aquaculture ecosystem also is with microorganism species close ties to be arranged, and the ecological situation of microorganism directly influences the development of culture fishery in the water, comprises the diseases prevention and treatment such as fishes and shrimps and the influence of growing thereof.In recent years, both at home and abroad in aquaculture to the microorganism situation (micro-ecological environment) of ecotope, particularly microorganism ecological environment and hydrocoles (fishes and shrimps etc.) body of water, cause the popular of disease such as fishes and shrimps because of pathogenic bacteria and virus etc., and come into one's own.But lay particular emphasis on harmful microorganism research more, the particularly research of pathogenic bacteria, and traditional separation bacterial classification method has its limitation, only some can be cultivated, its result limits to often when carrying out the microbial diversity analysis, the composition and the diversity that can not reflect mixed bacterial exactly, can not culturing micro-organisms all can't predict than harshness is perhaps many for some culture condition, therefore with the resulting investigation result of traditional cultural method can not the accurate response microorganism species the composition situation, set up and develop a kind of method that does not rely on microorganism culturing to carry out microorganism species structural research be necessity.
In recent years, along with the development of Protocols in Molecular Biology such as molecular hybridization, PCR, nucleic acid sequencing etc., deep change has taken place in the microbiological research field, and sensitive detects and accurate Bacteria Identification becomes possibility.Carry out the rapid detection of special microorganism and identify the important means that has become modern microorganism diagnosis and ecological study by molecular biology method.Since the end of the eighties in last century to the nineties, Protocols in Molecular Biology begins to be widely used in the microorganism species structural analysis, and development rapidly, and the research focus mainly concentrates on the 16S rRNA with conserved sequence.Research method comprises molecular hybridization, PCR method, SSCP method, DGGE method, RFLP and clone library method etc., has very high susceptibility, compare obvious superiority with traditional cultural method or other method that does not rely on culture technique, promoted the fast development of microorganism species structural research.But, the method of these PCR-based may be introduced error in amplified reaction, reduce the tolerance range of resultant information, and in the real work, can not reach its intended purposes fully with single molecular ecology method, and each method all there is the deficiency of self, usually will be in conjunction with the multiple molecular ecology method of utilization, sometimes even will can comprehensively analyze the microecosystem of complex environment in conjunction with traditional method.
Microbial ecology and microecology result of study make people recognize that beneficial microorganism has vital role to improving water quality, control fishes and shrimps disease and improving output in the water body.Susan E, Pryde, Anyhony J.Richardson, Colin S.Stewart, and Harry J.Flint.1999.Molecular Anyalysia of the Microbial Diversity Present in the Colonic Wall, Colonic Lumen, and Cecal Lumen of a Pig.Appl.Environ.Microbiol, the DNA information that 65:5372-5377 is used for the microorganism species structural analysis comprises 16SrDNA (small subunit ribosome gene), the phylogenetic systematics status information that has comprised corresponding bacterial species is the structure composition that object can be analyzed microorganism species exactly with 16S rDNA.A kind of commonly used be exactly to make up gene library, the sequence of clone's by analyzing some amount and their frequency of occurrences can be understood the main flora in the environmental sample to a certain extent and population structure information such as abundance occur.But the work of levels such as this method need be cloned, order-checking, labor intensive, financial resources and time are all bigger, are difficult to use in needs of production.In addition, found dependence 16S rDNA-DGGE technology commonly used again: Muyzer G, de Waal E C, Uitterlinden A G.Profiling of complexmicrobial population by denaturing gradient gel electrophoresia analysis of polymerase chainreaction-amplified genes encoding for 16S rRNA..Applied Environmental Microbiology, 1993,59 (3): 695-700.But this molecular detecting method generally can only be analyzed the following dna fragmentation of 500bp base pair, the phyletic evolution relevant information that obtains seldom, its collection of illustrative plates generally has only a ten or twenty band, the weak tendency bacterium can not be detected in the system, conditional request is harsh relatively, the DGGE deposition condition is different, even the resulting collection of illustrative plates of identical sample is also variant, reclaim glue to the later stage cutting and set up the gene clone library, check order then, therefore neither be quick, labor intensive, material resources, and when microbe species was too much, same band can comprise the DNA information of a plurality of kinds, therefore, can not accomplish under higher resolving power, to analyze the relatively difference of microbial bacteria group structure.Present RFLP technology in addition based on 16S rRNA amplification, from environmental sample, extract total DNA, with universal primer amplification 16S rDNA, with various restriction enzymes amplified production is carried out restriction analysis, because microbe species is formed different in the different floras, its restriction enzyme mapping difference, so just can obtain reflecting the dna gel electrophoretogram of microorganism species structure composition, but this method needs plurality of enzymes to cut process, the clone, more time-consuming, and be a kind of sxemiquantitative or qualitative analysis, for the similar flora of collection of illustrative plates, its composition is not necessarily identical.
Summary of the invention:
The objective of the invention is to overcome above the deficiencies in the prior art, a kind of improvement T-RFLP-PCR combined with fluorescent hybridization in situ technique prawn culturing microorganisms in water flora structure check and analysis method fast is provided, make its detection have quick, accurate, specific characteristics more, and its condition of fluorescence in situ hybridization of detection by quantitative shrimp pool water dominant microflora NOB is optimized.
The present invention will improve T-RFLP and be applied in the research shrimp aquaculture water body complex environment sample in conjunction with the FISH technology, directly extract total genomic dna of microorganism in the shrimp aquaculture water body example without pure culture, design specificity T-RFLP-PCR universal primer (sequence: upstream primer: 5 '-AGA GTT TGA TCC TGG CTC AG-3 ', downstream primer: 5 '-CCG TCA ATT CCTTTR AGT TT-3 '), its 5 ' end 6-fam fluorescent mark, the RCR circulating reaction, amplification obtains various microorganism target dna fragment one ends and just has fluorescent mark, HaeIII digests dna fragmentation with the specificity Restriction Enzyme, different microorganisms target dna fragment there are differences because of its nucleotide sequence difference can cause restriction enzyme site, produced the restriction fragment of different lengths, electrophoretic separation and fluoroscopic examination, having only end to have fluorescently-labeled fragment can be detected.Because nucleotide sequence has diversity, the nucleotide sequence of the same gene of different microorganisms there are differences, after cutting, same enzyme may obtain the T-RF of different lengths, being reflected in the T-RFLP detection is exactly different fluorescent signals, by generating the analysis of T-RFLP collection of illustrative plates, disclose kind, quantity and the population size etc. of microorganism in the sample, thereby resolve structure, function and the dynamic change thereof of microorganism species.But rely on the T-RFLP technology separately, its defective is also arranged, the PCR-based amplification technique, will be subjected to the otherness amplification of flora different strain DNA, the difference of target dna copy number between bacterial classification, the length distribution that the deviation of round pcr itself and enzyme are cut back T-RF also can cause complicated multifarious factor affecting such as underestimate, and the deficiency of the molecular detection technology of FISH technology to be a kind of analysis of molecules technology that does not rely on PCR can finely remedy above single dependence PCR, combine molecular biological accuracy and microscopical visual information, can in nature or artificial microbial environment, monitor and identify the different microorganisms individuality, simultaneously microbe colony be estimated.Therefore use T-RFLP in conjunction with the FISH technology, fine combination advantage separately and avoid mutual deficiency, qualitative and quantitative analysis shrimp aquaculture water body is along with the time changes the dynamic change that microbial ecological diversity and dominant microflora are formed structure.
The present invention optimizes in conjunction with the fish analysis technical qualification T-RFLP, mainly sample total DNA is extracted, T-RFLP-PCR has been designed Auele Specific Primer and determined circulating reaction system and reaction parameter, optimized the HaeIII enzyme and cut system, dominant microflora FISH condition optimizing.
In order to obtain high-quality total DNA, repeat CTAB extracting and chloroform/primary isoamyl alcohol extracting, till cannot see the interface, going out the macromole of polysaccharide and other pollutions, purify DNA, the agarose gel electrophoresis with 1% detects.
To dominant microflora FISH condition optimizing, mainly be that the autofluorescence to sample slackens, the hybridization of non-specific property is eliminated, and has determined suitable hybridization time and elutriant concentration.
In order to eliminate the interference of sample autofluorescence, obtain best effect, after sample dominant bacteria NOB is fixed with 4% Paraformaldehyde 96 again 46 ℃ add heat setting, handle PH6.5, bacteria suspension extension rate 10 with 0.2mol/L HCI
4, make its thalline degree of scatter better, hybridize again afterwards.
For the eliminating of non-specific hybridization, adopted the HEX label probe to solve.Experiment is found, can well eliminate false positive, sends fluorescent red-orange under the 490nm exciting light.Therefore, after the sample hybridization, and carry out the fluorescence microscopy simultaneously.
The specificity that accuracy that FISH detects and reliability mainly depend on oligonucleotide probe for similar probe with base mismatch to target sequence, has added competitive probe in therefore testing, and makes that target sequence soon can be detected.The general probe NIT3 of ite oxidation bacterium 16SrRNA design, it is 5 '-CCTGTGCTCCATGCTCCG-3 ' that its 5 ' end is used HEX fluorescent mark, sequence; Its competitive probe CNIT3 sequence is 5 '-CCTGTGCTCCAGGCTCCG-3 '.CNIT probe and NIT probe use simultaneously, to guarantee the specificity of NIT probe.
Purpose of the present invention specifically is achieved through the following technical solutions:
A kind of method that detects shrimp aquaculture microorganisms in water polymorphism of flora comprises the steps and processing condition:
(1) extraction of mixing microorganisms total genomic dna in the shrimp aquaculture water body example;
(2) design specificity T-RFLP-PCR universal primer carries out circulating reaction; The upstream primer of described specificity T-RFLP-PCR universal primer is 5 '-AGA GTT TGA TCC TGG CTC AG-3 ', downstream primer is 5 '-CCG TCA ATT CCT TTRAGT TT-3 ', 5 ' end 6-fam fluorescent mark; The PCR reaction system is 23mM MgCl
22.0-2.5uL, the dNTP1-2uL of 2.5mM; Annealing temperature is 57-58 ℃;
(3) product purification is cut DNA with specificity restriction enzyme HaeIII enzyme; Enzyme is cut system DNA≤1ug; Product purification is meant the PCR product after DyNA quant200 concentration determination, gets the 5ugPCR product through MO BIO UItraClean PCRClean-up DNA Purification kit purifying;
(4) microorganism species polymorphism structural analysis; After the HaeIII enzyme is cut dna fragmentation and the fluorescently-labeled standard DNA object of reference of tool is mixed, separate through 1% agarose gel electrophoresis, fluorescent scanning detects after silver dyes, it is detected to have fluorescently-labeled segment, be converted into each peak in the T-RFLP fluorescence pattern, each peak carries out species abundance (the remarkable sum at peak in the S=T-RFLP collection of illustrative plates, think roughly the corresponding different species in a peak), diversity index (Shannon-Weiner index: H=-∑ P as an OUT (terminal fluorescent mark fragment or activity classification unit)
iLnP
iWith the Simpson index: D=1-∑ P
i 2, wherein, P
iRepresent that the peak height at certain peak accounts for the ratio on total peak) and species all spend (E=H '/H
Max, wherein, H
Max=ln S) analyzes; Then the finger printing band of the T-RFLP of DNA is cut glue and reclaim order-checking, sequence identifies whether be to form chimeric fragment by RDP database CHECK-CHIMERA software analysis, and Classification compares definite the classification in the RDP database, utilize the Blast software of NCBA and the 16S rDNA sequence in the GenBank database to carry out the similarity comparison sequence of identifying, choose homology greater than 97%, grow tree with Neighbor-Joining method constructing system; Described 1% sepharose is meant that the 1g agarose is dissolved in 100ml distilled water;
(5) dominant bacteria fluorescence in situ hybridization technique detection by quantitative;
A handles testing required slide
Slide is cleaned siliconizing, and the preparation of gelatin smear;
B sample collecting and processing
Get original bacterium liquid, add the fresh preparation substratum of equivalent, bubbling air activates 1-2 days, is diluted to 10 with deionized water
-4, centrifugal subsequently, abandon supernatant liquor, add PBS, fully shake up dispersion, regulate pH to 6.0-6.5, the purpose of regulating pH is that the nitrobacteria that flocks together can better be disperseed; Slide is placed into heat setting in the baking oven, the time is 1-3h again; With fixing 10-20min under the 4% Paraformaldehyde 96 room temperature, wash air-dry again; Sample ethanol dehydration 3-5min after will fixing at last; Described 1% sepharose is meant that the 1g agarose is dissolved in 100ml distilled water;
C hybridization
With probe NIT3 and probe CNIT3 HEX mark, probe that mark is good and hybridization buffer are mixed in the airtight hybridizing box; Hybridization temperature is controlled at 46 ℃, and hybridization time is 2-3h, the hybridization back clear probe of elutriant, and NaCl concentration is 60mmol/L in the elutriant; By the densitometer of component at hybridization buffer, described hybridization buffer is by 0.01%SDS, 40% deionized formamide, 120mmolL
-1Tris-HC and 900mmolL
-1NaCl forms, pH=7.2;
The d fluorescence microscope
Slide is observed under fluorescent microscope, and caused fluorescent red-orange reaction comes quantitative shrimp aquaculture water body dominant microflora NOB under 490nm laser, and the eyepiece magnification is 10, and object lens magnification Mob is 20;
For further realizing the object of the invention, the extraction of mixing microorganisms total genomic dna comprises the steps: in the shrimp aquaculture water body example of described step (1)
A filters water sample with the vacuum filtration device, and filtrate is transferred to the centrifuge tube of 100ml, and the centrifugal 5-10min of 10000-13000rpm inhales and removes supernatant liquor, with the resuspended precipitation of sterilized water, transfers to the 1.5ml centrifuge tube;
B adds 567 μ l TE, and blow and beat repeatedly with suction pipe and make it resuspended. add 30-40 μ l 10%SDS and 3-5 μ l 20mg/ml Proteinase K, mixing, 37 ℃ of temperature are bathed 1h;
C adds 100 μ l 5mol/L NaCl, and abundant mixing adds the 5%CTAB/NaCl solution of 80-100 μ l again, mix and put 65 ℃ being incubated 10-15min, add isopyknic chloroform/primary isoamyl alcohol, mixing, 4 ℃ of centrifugal 10-15min under 4000-6000rpm change supernatant liquor in another centrifuge tube over to;
D adds the Virahol of 0.6 times of volume, and deposit D NA washs in the cold ethanol of 1ml 70% (the cold ethanol of 100ml95% adds the 36ml distilled water diluting), dry air 10-15min, the resuspended DNA precipitation of 100 μ l TE damping fluids.
Described step (3) is cut DNA with special restriction enzyme HaeIII enzyme and is meant that the enzyme system of cutting contains HaeIII 1ul, 10xMBuffer 2ul, DNA≤1ug, aqua sterilisa up to 20ul, 37 ℃ of waters enzymes are cut 2-3h, recognition sequence GG|CC (G: guanine, C: cytosine(Cyt)).
Described step (5) probe NIT3 is the general probe of Nitrobacter α-proteobacteria, and sequence is 5 '-CCTGTGCTCCATGCTCCG-3 ', at 5 '-end HEX mark; Described probe CNIT3 sequence is 5 '-CCTGTGCTCCAGGCTCCG-3 '.
With respect to prior art, the improved T-RFLP-PCR combined with fluorescent hybridization in situ technique of the present invention is applied to microorganism species polymorphism in the research microbial ecosystem, have easy, quick, sensitive, comprehensive characteristics, qualitative and quantitative analysis shrimp aquaculture water body changes the dynamic change of microbial ecological diversity and dominant microflora composition structure along with the time effectively.
Description of drawings
Fig. 1 is embodiment 1 (April a 1) sampling microorganism species T-RFLP fluorescence pattern;
Fig. 2-5 is four (April 1, April 16, May 1, May 16) samplings of embodiment 2 shrimp aquaculture seedling field water bodys microorganism species T-RFLP fluorescence pattern;
Fig. 6-7 is embodiment 3 shrimp aquaculture seedling field water body different depthss (40cm and 150cm) sampling microorganism species T-RFLP fluorescence pattern.
Embodiment
The present invention is further illustrated below in conjunction with embodiment, need to prove, these embodiment do not constitute limiting the scope of the invention.
Aquaculture is the mainstay industry of China's Coastal Areas, and shrimp culture industry is consequence therein, and the prawn culturing area of China reaches 2,400,000 mu.Yet because disease frequently takes place, in recent years the prawn culturing output of China is lower than low ebb, more and more scholars thinks that the basic reason of prawn disease is the deterioration of breeding ecological environment, and the environment of pollution and the vicious cycle between the aquaculture organism have caused the lasting outburst of disease.Only but some can be cultivated traditional separation bacterial classification method, when carrying out the microbial diversity analysis, can not reflect the composition and the diversity of mixed bacterial exactly.This experiment is a research object with the shrimp aquaculture seedling field of Dongsheng Town, Zhongshan City, Guangdong Province, changes in conjunction with the direct detection of dynamic microorganism species of FISH technology with T-RFLP, and step and processing condition that prawn culturing water body polymorphism of flora detects are as follows:
Get the water sample in No. 1 pond, 6 meters * 10 meters in No. 1 pond, about 2 meters of the depth of water.Throw in Penaeus vannamei seedling 150,000 tails mid-March, the same ordinary production of management during the breed, April 1 once sampling, multiple spot is gathered top layer 30cm with interior water sample 2000ml, takes back the laboratory vacuum filtration, filtrate-20 ℃ preservation.
(1) extraction of microorganism total DNA in the water body example of shrimp aquaculture seedling field:
A. filtrate is transferred to the centrifuge tube of 100ml, and the centrifugal 5min of 10000rpm inhales and removes supernatant liquor, with the resuspended precipitation of sterilized water, transfers to the 1.5ml centrifuge tube;
B. add 567 μ l TE, blow and beat repeatedly with suction pipe and make it resuspended, add 30 μ l 10%SDS and 3 μ l 20mg/ml Proteinase Ks, mixing, 37 ℃ of temperature are bathed 1h;
C. add 100 μ l 5mol/L NaCl, abundant mixing adds the 5%CTAB/NaCl solution of 80 μ l again, mix and put 65 ℃ being incubated 10min, add isopyknic chloroform/primary isoamyl alcohol, mixing, 4 ℃ of centrifugal 15min under 4000rpm change supernatant liquor in another centrifuge tube over to;
D. the Virahol that adds 0.6 times of volume, deposit D NA washs in the cold ethanol of 1ml 70% (the cold ethanol of 100ml95% adds the 36ml distilled water diluting), dry air 15min, the resuspended DNA precipitation of 100 μ l TE damping fluids.
(2) specificity T-RFLP-PCR universal primer, the PCR circulating reaction:
The a.T-RFLP-PCR reaction system is 25uL, contains template 80ng, Taq archaeal dna polymerase 1uL, 23mM MgCl 2.5uL, the dNTP 1.5uL of 2.5mM;
B. reaction parameter: 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 58 ℃ of sex change 30s, 72 ℃ of sex change 2min, 28 circulations; 72 ℃ are extended 10min;
(3) product purification, cut DNA with specificity restriction enzyme HaeIII enzyme:
The a.PCR product is got the 5ugPCR product through MO BIO UItraClean PCRClean-up DNA Purification kit purifying after DyNA quant200 concentration determination;
The b.HaeIII enzyme is cut DNA, and its enzyme system of cutting contains HaeIII 1ul, 10xM Buffer 2ul, and DNA0.5ug, aqua sterilisa upto 20ul, 37 ℃ of waters enzymes are cut 3h;
(4) microorganism species polymorphism structural analysis:
After the a.HaeIII enzyme is cut dna fragmentation and the fluorescently-labeled standard DNA object of reference of tool is mixed, separate through 1% agarose gel electrophoresis, fluorescent scanning detects after silver dyes, and it is detected to have fluorescently-labeled segment, is converted into each " peak " in the T-RFLP fluorescence pattern.Each peak is analyzed as an OUT: species abundance (richness), diversity index, species are all spent (evenness);
The finger printing band of the T-RFLP of b.DNA is cut glue and is reclaimed order-checking, sequence identifies whether be to form chimeric fragment at RDP (ribosomal database project) database CHECK-CHIMERA software analysis, and Classification compares definite the classification in the RDP database, then the sequence of identifying is utilized Blast software and the 16S rDNA sequence in the GenBank database of NCBA (National Center forBiotechnology Information) to carry out the similarity comparison, choose homology greater than 97%, grow tree with Neighbor-Joining method constructing system;
(5) dominant bacteria FISH detection by quantitative:
The A slide is handled
The a slide cleans hot suds and scrubs, and tap water washes repeatedly, and 96% is alcohol-pickled, and calcination is soaked 24h then in 1% (massfraction) hydrochloric acid, and tap water washes repeatedly, deionization washing 3 times;
B siliconizing slide and cover glass boil 10min in 1% (massfraction) hydrochloric acid, deionization washing 3 times, and 50 ℃ of oven dry, it is standby that tinfoil is wrapped 4 ℃ of preservations.Slide has the bundle water-based through after the siliconizing, can effectively prevent cell loss;
B sample collecting and pre-treatment
A gets original bacterium liquid 500ml, places to make its precipitation half an hour, outwells supernatant liquid, adds the fresh preparation substratum of equivalent, bubbling air, activation 1d;
B gets bacterium liquid and dilutes with deionized water, gets 5ml dilution back sample to 10
-4, centrifugal under the 4000r/min condition, abandon supernatant liquor, add 5mlPBS, fully shake up dispersion.The bacterium liquid of getting after 10 μ l disperse is applied on the slide, regulates pH to 6.5;
C heat setting: slide is placed into heat setting 2h in 50 ℃ of baking ovens;
The d Paraformaldehyde 96 is fixed: behind the heat setting, 4% (the 4g Paraformaldehyde 96 is dissolved in 100ml distilled water) Paraformaldehyde 96 (pareforaldehyde, PFA) fixing 15min under the room temperature is with the flushing of PBS room temperature, natural air drying;
Sample after the e dehydration will be fixed, the 4min that in 96% ethanol, dewaters under the room temperature, natural air drying;
The C hybridization
NIT3 is the general probe of Nitrobacter α-proteobacteria, and sequence is 5 '-CCTGTGCTCCATGCTCCG-3 ', and probe is used the HEX mark at 5 '-end, and competitive probe CNIT3 sequence is 5 '-CCTGTGCTCCAGGCTCCG-3 '.The hybridization solution of each 0.5 μ L of NIT3 and CNIT3 probe and 9.6 μ L carries out hybridization in hybridizing box..Reaction times is 2h, 46 ℃ of hybridization temperatures;
The cleaning of D probe
Take out slide glass in the hybridizing box and put into 48 ℃ of hybridization elution liquid of 50ml, NaCl concentration is controlled to be 60mmol/L in the elutriant;
The E microscopy
Slide is observed under fluorescent microscope, and caused fluorescent red-orange reaction comes quantitative shrimp aquaculture water body dominant microflora NOB under 490nm laser, and the eyepiece magnification is 10, and object lens magnification Mob is 20;
The result shows on the genescan figure of water sample and tens peaks occurred, each outstanding slightly part can be considered to a kind of T-RF, show the bacterium that has tens kinds of monoids in the water sample at least, being distributed in electrophoresis proceeds in the scope of 15min-30min, the peak E that occurs at 19min-25min wherein, F, I, the area maximum that J accounts for, their area sum has surpassed 80% of all peak total areas, be the dominant bacteria in the bacteria flora, wherein especially with peak 1 area maximum, the quantity of bacterium in bacterium colony that shows this T-RF representative is maximum, the advantage band is cut glue and is reclaimed order-checking, carry out the systematics analysis with the 16S rDNA sequence in the GenBank database, do not find the high known array of any similarity, these fragments may derive from the unknown bacterium that we also do not understand, understanding for the shrimp aquaculture microorganisms in water can be more comprehensive like this, but and traditional separation and concentration cultural method can only be cultivated culturing micro-organisms, the kind of microorganism, quantity and function all can't reflect the truth of shrimp aquaculture water body.
Embodiment 2
Carry out the flora check and analysis of different time points with embodiment 1 shrimp aquaculture water body environment, all once sampling in April to Mays per two (April 1, April 16, May 1, May 16).Multiple spot is gathered top layer 30cm with interior water sample 4 * 2000ml, takes back the laboratory vacuum filtration, filtrate-20 ℃ preservation, and step and processing condition that prawn culturing water body polymorphism of flora detects are as follows:
(1) extraction of microorganism total DNA in the water body example of shrimp aquaculture seedling field:
A. filtrate is transferred to the centrifuge tube of 100ml, and the centrifugal 10min of 13000rpm inhales and removes supernatant liquor, with the resuspended precipitation of sterilized water, transfers to the 1.5ml centrifuge tube;
B. add 567 μ l TE, blow and beat repeatedly with suction pipe and make it resuspended. add 40 μ l 10%SDS and 5 μ l 20mg/ml Proteinase Ks, mixing, 37 ℃ of temperature are bathed 1h;
C. add 100 μ l 5mol/L NaCl, abundant mixing adds the 5%CTAB/NaCl solution of 90 μ l again, mix and put 65 ℃ being incubated 10min, add isopyknic chloroform/primary isoamyl alcohol, mixing, 4 ℃ of centrifugal 10min under 5000rpm change supernatant liquor in another centrifuge tube over to;
D. the Virahol that adds 0.6 times of volume, deposit D NA washs in the cold ethanol of 1ml 70% (the cold ethanol of 100ml95% adds the 36ml distilled water diluting), dry air 10min, the resuspended DNA precipitation of 100 μ l TE damping fluids;
(2) specificity T-RFLP-PCR universal primer, the PCR circulating reaction:
The a.T-RFLP-PCR reaction system is 25uL, contains template 80ng, Taq archaeal dna polymerase 1uL, 23mM MgCl 2.0uL, the dNTP 1uL of 2.5mM;
B. reaction parameter: 94 ℃ of sex change 3min; 94 ℃ of sex change 30s, 57 ℃ of sex change 30s, 72 ℃ of sex change 2min, 28 circulations; 72 ℃ are extended 10min;
(3) product purification, cut DNA with specificity restriction enzyme HaeIII enzyme:
The a.PCR product is got the 5ugPCR product through MO BIO UItraClean PCRClean-up DNA Purification kit purifying after DyNA quant200 concentration determination;
The b.HaeIII enzyme is cut DNA, and its enzyme system of cutting contains HaeIII 1ul, 10xM Buffer 2ul, and DNA1ug, aqua sterilisa up to20ul, 37 ℃ of waters enzymes are cut 2h;
(4) microorganism species polymorphism structural analysis:
After the a.HaeIII enzyme is cut dna fragmentation and the fluorescently-labeled standard DNA object of reference of tool is mixed, separate through 1% agarose gel electrophoresis, fluorescent scanning detects after silver dyes, and it is detected to have fluorescently-labeled segment, is converted into each " peak " in the T-RFLP fluorescence pattern.Each peak can be used as an OUT and analyzes: species abundance (richness), diversity index, species are all spent (evenness);
The finger printing band of the T-RFLP of b.DNA is cut glue and is reclaimed order-checking, sequence identifies whether be to form chimeric fragment at RDP (ribosomal database project) database CHECK-CHIMERA software analysis, and Classification compares definite the classification in the RDP database, then the sequence of identifying is utilized Blast software and the 16S rDNA sequence in the GenBank database of NCBA (National Center forBiotechnology Information) to carry out the similarity comparison, choose homology greater than 97%, grow tree with Neighbor-Joining method constructing system;
(5) dominant bacteria FISH detection by quantitative:
A. slide is handled
A. slide cleans hot suds and scrubs, and tap water washes repeatedly, and 96% is alcohol-pickled, and calcination is soaked 24h then in 1% (massfraction) hydrochloric acid, and tap water washes repeatedly, deionization washing 3 times;
B. siliconizing slide and cover glass boil 10min in 1% hydrochloric acid, deionization washing 3 times, and 50 ℃ of oven dry, it is standby that tinfoil is wrapped 4 ℃ of preservations.Slide has the bundle water-based through after the siliconizing, can effectively prevent cell loss;
B. sample collecting and pre-treatment
A. get original bacterium liquid 500ml, place and make its precipitation half an hour, outwell supernatant liquid, add the fresh preparation substratum of equivalent, bubbling air, activation 1d;
B. get bacterium liquid and dilute, get 5ml dilution back sample to 10 with deionized water
-4, centrifugal under the 4000r/min condition, abandon supernatant liquor, add 5mlPBS, fully shake up dispersion.The bacterium liquid of getting after 10 μ l disperse is applied on the slide, regulates pH to 6.0;
C. heat setting is placed into heat setting 1h in 50 ℃ of baking ovens with slide;
D. the Paraformaldehyde 96 stationary heat fixing after, 4% (the 4g Paraformaldehyde 96 is dissolved in 100ml distilled water) Paraformaldehyde 96 (pareforaldehyde, PFA) fixing 10min under the room temperature is with the flushing of PBS room temperature, natural air drying;
E. the sample after dehydration will be fixed, the 5min that in 96% ethanol, dewaters under the room temperature, natural air drying;
C. hybridization
NIT3 is the general probe of Nitrobacter α-proteobacteria, and sequence is 5 '-CCTGTGCTCCATGCTCCG-3 ', and probe is used the HEX mark at 5 '-end, and competitive probe CNIT3 sequence is 5 '-CCTGTGCTCCAGGCTCCG-3 '.The hybridization solution of each 0.5 μ L of NIT3 and CNIT3 probe and 9.6 μ L carries out hybridization in hybridizing box..Reaction times is 2.5h, 46 ℃ of hybridization temperatures;
D. the cleaning of probe
Take out slide glass in the hybridizing box and put into 48 ℃ of hybridization elution liquid of 50ml, NaCl concentration is controlled to be 60mmol/L in the elutriant;
E. microscopy
Slide is observed under fluorescent microscope, and caused fluorescent red-orange reaction comes quantitative shrimp aquaculture water body dominant microflora NOB under 490nm laser, and the eyepiece magnification is 10, and object lens magnification Mob is 20.
The result show T-RFLP in conjunction with FISH technology dynamic monitoring shrimp aquaculture water body along with the time changes, variations such as the kind of microorganism, quantity and population size, and the DNA of T-RFLP fluorescence band correspondence is cut glue recovery amplification again, fluorescence pattern has good reproducibility and repeatability, contrasting each collection of illustrative plates finds, peak I, II, III, IV have on each figure, particularly the area sum of peak II, III, IV has been occupied 80% of all peak total areas, constitutes the main population (as table 1) of this flora structure.
The bacterium quantity of four kinds of T-RF representatives of table 1
As can be seen from Table 1, the absolute quantity of the bacterium of every section fragment representative also is in the dynamic change.With regard to a certain fragment, the bacterium of fragment I representative reached maximum value April 1, and the bacterium of fragment II, III and IV representative reached maximum value May 1.The bacterium absolute quantity rangeability of fragment II, IV representative reaches an order of magnitude, and the absolute quantity of the bacterium of fragment I, III representative is slight fluctuations on the same order of magnitude then.
Embodiment 3:
With embodiment 1 shrimp aquaculture water body environment, T-RFLP directly detects vertical (degree of depth 40cm and the 150cm) changes in distribution of shrimp pool microorganisms in water flora in conjunction with the FISH technology, on June 5th, 2009 is (40cm) and deep layer (150cm) 2 * 2000ml of multidraw at random on the top layer respectively, take back the laboratory vacuum filtration, filtrate-20 ℃ preservation, step and processing condition that prawn culturing water body polymorphism of flora detects are as follows:
(1) extraction of microorganism total DNA in the water body example of shrimp aquaculture seedling field;
A. filtrate is transferred to the centrifuge tube of 100ml, and the centrifugal 8min of 12000rpm inhales and removes supernatant liquor, with the resuspended precipitation of sterilized water, transfers to the 1.5ml centrifuge tube;
B. add 567 μ l TE, blow and beat repeatedly with suction pipe and make it resuspended. add the 20mg/ml Proteinase K of 35 μ l 10%SDS and 4 μ l, mixing, 37 ℃ of temperature are bathed 1h;
C. add 100 μ l 5mol/LNaCl, abundant mixing adds the 5%CTAB/NaCl solution of 100 μ l again, mix and put 65 ℃ being incubated 10min, add isopyknic chloroform/primary isoamyl alcohol, mixing, 4 ℃ of centrifugal 15min under 6000rpm change supernatant liquor in another centrifuge tube over to;
D. the Virahol that adds 0.6 times of volume, deposit D NA washs in the cold ethanol of 1ml 70% (the cold ethanol of 100ml95% adds the 36ml distilled water diluting), dry air 15min, the resuspended DNA precipitation of 100 μ l TE damping fluids;
(2) specificity T-RFLP-PCR universal primer, the PCR circulating reaction:
The a.T-RFLP-PCR reaction system is 25uL, contains template 80ng, Taq archaeal dna polymerase 1uL, 23mM MgCl 2.0uL, the dNTP 1uL of 2.5mM;
B. reaction parameter: 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 57 ℃ of sex change 30s, 72 ℃ of sex change 2min, 28 circulations; 72 ℃ are extended 10min;
(3) product purification is cut DNA with specificity restriction enzyme HaeIII enzyme;
The a.PCR product is got the 5ugPCR product through MO BIO UItraClean PCRClean-up DNA Purification kit purifying after DyNA quant200 concentration determination;
The b.HaeIII enzyme is cut DNA, and its enzyme system of cutting contains HaeIII 1ul, 10xM Buffer 2ul, and DNA0.8ug, aqua sterilisa upto 20ul, 37 ℃ of waters enzymes are cut 3h;
(4) microorganism species polymorphism structural analysis:
After a HaeIII enzyme is cut dna fragmentation and the fluorescently-labeled standard DNA object of reference of tool is mixed, separate through 1% agarose gel electrophoresis, fluorescent scanning detects after silver dyes, and it is detected to have fluorescently-labeled segment, is converted into each " peak " in the T-RFLP fluorescence pattern.Each peak can be used as an OUT and analyzes: species abundance (richness), diversity index, species are all spent (evenness);
The finger printing band of the T-RFLP of b DNA is cut glue and is reclaimed order-checking, sequence identifies whether be to form chimeric fragment at RDP (ribosomal database project) database CHECK-CHIMERA software analysis, and Classification compares definite the classification in the RDP database, then the sequence of identifying is utilized Blast software and the 16S rDNA sequence in the GenBank database of NCBA (National Center forBiotechnology Information) to carry out the similarity comparison, choose homology greater than 97%, grow tree with Neighbor-Joining method constructing system;
(5) dominant bacteria FISH detection by quantitative:
The A slide is handled
The a slide cleans hot suds and scrubs, and tap water washes repeatedly, and 96% is alcohol-pickled, and calcination is soaked 24h then in 1% (massfraction) hydrochloric acid, and tap water washes repeatedly, deionization washing 3 times;
B siliconizing slide and cover glass boil 10min in 1% (massfraction) hydrochloric acid, deionization washing 3 times, and 50 ℃ of oven dry, it is standby that tinfoil is wrapped 4 ℃ of preservations.Slide has the bundle water-based through after the siliconizing, can effectively prevent cell loss;
B sample collecting and pre-treatment
A gets original bacterium liquid 500ml, places to make its precipitation half an hour, outwells supernatant liquid, adds the fresh preparation substratum of equivalent, bubbling air, activation 1d;
B gets bacterium liquid and dilutes with deionized water, gets 5ml dilution back sample to 10
-4, centrifugal under the 4000r/min condition, abandon supernatant liquor, add 5mlPBS, fully shake up dispersion.The bacterium liquid of getting after 10 μ l disperse is applied on the slide, regulates pH to 6.3;
C heat setting: slide is placed into heat setting 2h in 50 ℃ of baking ovens;
The d Paraformaldehyde 96 is fixed: behind the heat setting, 4% (the 4g Paraformaldehyde 96 is dissolved in 100ml distilled water) Paraformaldehyde 96 (pareforaldehyde, PFA) fixing 15min under the room temperature is with the flushing of PBS room temperature, natural air drying;
E dehydration: the sample after will fixing, the 3min that in 96% ethanol, dewaters under the room temperature, natural air drying;
C. hybridization
NIT3 is the general probe of Nitrobacter α-proteobacteria, and sequence is 5 '-CCTGTGCTCCATGCTCCG-3,, probe is used the HEX mark at 5 '-end, and competitive probe CNIT3 sequence is 5 '-CCTGTGCTCCAGGCTCCG-3 '.The hybridization solution of each 0.5 μ L of NIT3 and CNIT3 probe and 9.6 μ L carries out hybridization in hybridizing box..Reaction times is 3h, 46 ℃ of hybridization temperatures;
The cleaning of D probe
Take out slide glass in the hybridizing box and put into 48 ℃ of hybridization elution liquid of 50ml, NaCl concentration is controlled to be 60mmol/L in the elutriant;
The E microscopy
Slide is observed under fluorescent microscope, and caused fluorescent red-orange reaction comes quantitative shrimp aquaculture water body dominant microflora NOB under 490nm laser, and the eyepiece magnification is 10, and object lens magnification Mob is 20.
The result shows the calculating of T-RFLP collection of illustrative plates 6 flora diversity: species abundance (richness) S=4, and Shannon-Weiner index: H=1.26, species all spend (evenness): E=0.07; T-RFLP collection of illustrative plates 7 flora diversity are calculated: species abundance (richness): S=9.05, Shannon-Weiner index: H=3.36, species are all spent (evenness): E=0.47, great changes have taken place for the microorganism of different depths, this may with the artificial little ecosystem of aquaculture water, the ability of bearing load, biomass is many, and biological species is few, and environmental factor easily changes, cause the bacterial number fluctuation easily, wherein the band of 500bp size contains 5 kinds of sequence fragments, composition situation 33%, 25%, 4%, 28% and 10%; Wherein the band of 800bp size contains 9 kinds of sequence fragments, composition situation 64%, 23%, 3%, 3%, 2%, 1%, 1%, 1% and 1%; Wherein the band of 1000bp size contains 4 kinds of sequence fragments, the composition situation is 84.2%, 10.2%, 4.6% and 0.9%, and calculated that the minimum similarity of Sorenson paired comparisons likeness coefficient (pairwise similarity coefficient Cs) is 86% and 83% in collection of illustrative plates 6 and the collection of illustrative plates 7, illustrate that the flora in the collection of illustrative plates has good continuity and variability.
Above experimental result proves absolutely T-RFLP in conjunction with the FISH method, can be used for monitoring the constitutional features of the complicated microorganism species of shrimp aquaculture water body and advantage NOB group's dynamic change.
Claims (4)
1. method that detects shrimp aquaculture microorganisms in water polymorphism of flora is characterized in that comprising the steps and processing condition:
(1) extraction of mixing microorganisms total genomic dna in the shrimp aquaculture water body example;
(2) design specificity T-RFLP-PCR universal primer, carry out circulating reaction: the upstream primer of described specificity T-RFLP-PCR universal primer is 5 '-AGA GTT TGA TCC TGG CTC AG-3 ', downstream primer is 5 '-CCG TCA ATT CCT TTRAGT TT-3 ', 5 ' end 6-fam fluorescent mark; The PCR reaction system is 23mM MgCl
22.0-2.5uL, the dNTP1-2uL of 2.5mM; Annealing temperature is 57-58 ℃;
(3) product purification, cut DNA with specificity restriction enzyme HaeIII enzyme: enzyme is cut system DNA≤1ug; Product purification is meant the PCR product after DyNA quant200 concentration determination, gets the 5ugPCR product through MO BIO UItraClean PCRClean-up DNA Purification kit purifying;
(4) microorganism species polymorphism structural analysis: after the HaeIII enzyme is cut dna fragmentation and the fluorescently-labeled standard DNA object of reference of tool is mixed, separate through 1% agarose gel electrophoresis, silver dyes the back fluorescent scanning and detects, it is detected to have fluorescently-labeled segment, be converted into each peak in the T-RFLP fluorescence pattern, each peak carries out species abundance as a terminal fluorescent mark fragment or activity classification unit, diversity index and species are all spent analysis, then the finger printing band of the T-RFLP of DNA is cut glue and reclaim order-checking, sequence identifies whether be to form chimeric fragment by RDP database CHECK-CHIMERA software analysis, and Classification compares definite the classification in the RDP database, utilize the Blast software of NCBA and the 16S rDNA sequence in the GenBank database to carry out the similarity comparison sequence of identifying, choose homology greater than 97%, grow tree with Neighbor-Joining method constructing system; Described 1% sepharose is meant that the 1g agarose is dissolved in 100ml distilled water;
(5) dominant bacteria fluorescence in situ hybridization technique detection by quantitative:
A handles testing required slide
Slide is cleaned siliconizing, and the preparation of gelatin smear;
B sample collecting and processing
Get original bacterium liquid, add the fresh preparation substratum of equivalent, bubbling air activates 1-2 days, is diluted to 10 with deionized water
-4, centrifugal subsequently, abandon supernatant liquor, add PBS, fully shake up dispersion, regulate pH to 6.0-6.5, the purpose of regulating pH is that the nitrobacteria that flocks together can better be disperseed; Slide is placed into heat setting in the baking oven, the time is 1-3h again, again with fixing 10-20min under the 4% Paraformaldehyde 96 room temperature, wash air-dry, the sample ethanol dehydration 3-5min after will fixing at last; Described 4% Paraformaldehyde 96 is dissolved in 100ml distilled water for the 4g Paraformaldehyde 96;
C hybridization
With probe NIT3 and probe CNIT3 HEX mark, probe that mark is good and hybridization buffer (0.01%SDS, 40% deionized formamide (DAF), Tris-HCl (pH=7.2) 20mmolL
-1, NaCl 900mmolL
-1, pH=7.2) being mixed in the airtight hybridizing box, hybridization temperature is controlled at 46 ℃, and hybridization time is 2-3h, the hybridization back clear probe of elutriant, NaCl concentration is 60mmol/L in the elutriant; By the densitometer of component at hybridization buffer, described hybridization buffer is by 0.01%SDS, 40% deionized formamide, 120mmolL
-1Tris-HC and 900mmolL
-1NaCl forms, pH=7.2;
D. fluorescence microscope
Slide is observed under fluorescent microscope, and caused fluorescent red-orange reaction comes quantitative shrimp aquaculture water body dominant microflora NOB under 490nm laser, and the eyepiece magnification is 10, and object lens magnification Mob is 20;
2. the method for detection shrimp aquaculture microorganisms in water polymorphism of flora according to claim 1 is characterized in that: the extraction of mixing microorganisms total genomic dna comprises the steps: in the shrimp aquaculture water body example of described step (1)
A filters water sample with the vacuum filtration device, and filtrate is transferred to the centrifuge tube of 100ml, and the centrifugal 5-10min of 10000-13000rpm inhales and removes supernatant liquor, with the resuspended precipitation of sterilized water, transfers to the 1.5ml centrifuge tube;
B adds 567 μ l TE, blows and beats repeatedly with suction pipe and makes it resuspended, adds 30-40 μ l 10%SDS and 3-5 μ l 20mg/ml Proteinase K, mixing, and 37 ℃ of temperature are bathed 1h;
C adds 100 μ l 5mol/L NaCl, and abundant mixing adds the 5%CTAB/NaCl solution of 80-100 μ l again, mix and put 65 ℃ being incubated 10-15min, add isopyknic chloroform/primary isoamyl alcohol, mixing, 4 ℃ of centrifugal 10-15min under 4000-6000rpm change supernatant liquor in another centrifuge tube over to;
D adds the Virahol of 0.6 times of volume, and deposit D NA washs in the 1ml 70% cold ethanol, dry air 10-15min, the resuspended DNA precipitation of 100 μ l TE damping fluids;
3. the method for detection shrimp aquaculture microorganisms in water polymorphism of flora according to claim 1, it is characterized in that: described step (3) is cut DNA with special restriction enzyme HaeIII enzyme and is meant that the enzyme system of cutting contains HaeIII 1ul, 10x M Buffer 2ul, DNA≤1ug, aqua sterilisa up to 20ul, 37 ℃ of waters enzymes are cut 2-3h, recognition sequence GG|CC (G: guanine, C: cytosine(Cyt));
4. the method for detection shrimp aquaculture microorganisms in water polymorphism of flora according to claim 1, it is characterized in that: described step (5) probe NIT3 is the general probe of Nitrobacter α-proteobacteria, sequence is 5 '-CCTGTGCTCCATGCTCCG-3 ', at 5 '-end HEX mark; Described probe CNIT3 sequence is 5 '-CCTGTGCTCCAGGCTCCG-3 '.
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