CN104946627A - Method for total DNA extraction and diversity analysis of ship ballast water microorganisms - Google Patents
Method for total DNA extraction and diversity analysis of ship ballast water microorganisms Download PDFInfo
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Abstract
The invention discloses a method for total DNA extraction and diversity analysis of ship ballast water microorganisms. The method comprises the steps that according to the characteristics of ship ballast water, two-level filtering and refrigerating fixation is performed by using millipore filtration membranes of 3 micrometers and 0.22 micrometer, and adhering of the microorganisms to the filtration membranes is reinforced; the microorganisms are processed by using cetyltrimethyl ammonium bromide (CTAB) to enable the total DNA purity of the obtained microorganisms to meet the requirements of directly performing subsequent molecular biology study; 16SrDNA amplification and denaturing gradient gel electrophoresis (DGGE) can be performed on extracted DNA fragments, and the diversity degree of ship ballast water microorganism populations can be judged after digitalization analysis is performed on electrophoretogram; the varieties of dominant bacterial populations of the ballast water is obtained by performing rubber tapping recovering, clone sequencing and BLAST sequence alignment on special bands, and therefore the structual components of the microorganism populations are determined. According to the method, a basis is laid for studying the diversity and ecological function of the microorganisms in the ship ballast water and sediment.
Description
Technical field
The invention belongs to environmental microorganism molecular ecology field, be specifically related to the method for the extraction of a kind of ballast water for ship microorganism total DNA and diversity analysis.
Background technology
Ballast water for ship be boats and ships when zero load in order to keep stability of hull, when setting sail, ballast enters the seawater of ballast tank of ship, is then discharged ballast tank when arriving at harbour loading.Water ballast and sedimental discharge are the main paties that marine organisms are propagated between the port of departure and port, point of destination, and that has been regarded as that ocean faces by world's environmental protection fund (GEF) four one of threatens greatly.
Boats and ships will unload water ballast while freighting to port, ballast tank is cleared up simultaneously, due to the special construction of ballast tank, thorough cleaning can not be reached, with the local fresh seawater of each ballast of boats and ships, biology and the abiotic composition of bringing ballast tank into are various, and water ballast settling enters the sedimentation of discharge long term accumulation along with water ballast.Ballast tank deposits the special cabin of water ballast as boats and ships, its complex structure, unglazed in ballast tank, anoxic, have also may contain objectionable impurities, totally unfavorable to the existence of biology, major part biology cannot stand severe environment in ballast tank and dead, after can not adapting to the biological death of ballast tank environment in a large number, its corpse sinks to falling into the bilge, provides food source to saprovore.Because boats and ships have high surface-to-volume ratio, large-area microorganic adhesion is on its surface, so ballast tank has become the sanctuary of microorganism, this situation is referred to as " hull interior dirt ".At present by ballast water for ship and sedimental discharge, harmful microorganism may be propagated between Port State, pathogenic microorganism may threaten to biological in environment and people, in present stage ballast for cruising water and sediment, microbe survival state is not probed into completely, and the diversity of microorganism does not also understand.
The research mainly Hygienic indicator bacteria such as total plate count, coliform and some pathogenic bacterium in harbour investigation dock vessels water ballast of current domestic water ballast microbial diversity.Utilize molecular biology method to study microorganism in ballast water for ship also not launch completely, and the first step that microbial DNA extraction is carried out analyzing as molecular biology method is also a most important step, the research of its method have not been reported.
Denaturing gradient gel electrophoresis (DGGE) is used to the abrupt climatic change of gene at first, the separation principle of denaturing gradient gel electrophoresis to different DNA fragmentation be in denaturing gradient gel electrophoresis (DGGE) same length but there is not homotactic DNA fragmentation can be separated.There is not homotactic DNA molecular and have different behaviors of unwinding, the different positions at gel is stopped migration.Denaturing gradient gel electrophoresis (DGGE) is based upon PCR reaction on the basis of the amplification of the DNA segment in the conservative gene intervals such as 16SrRNA and 18SrRNA of all microorganisms in environmental sample, these DNA segments be amplified out can represent the information of all different microorganisms, the DNA segment of these different microorganisms using same primer pair amplifies to go out has identical base number, and they cannot separate by therefore general electrophoresis.Because the DNA fragmentation of the formed objects of Different Alkali base sequence can be separated by denaturing gradient gel electrophoresis (DGGE), use PCR-DGGE technology can to study the microbial diversity in environmental sample.
Ballast tank structure and water ballast dispensing rule cause the physics of ballast water for ship, chemical property and river, seawater and environmental wastewater to there is difference in height, microbe population and Species structure are also obviously different, and this sample just determined under varying environment there are differences when carrying out microflora's component analysis.There is no the relevant report about using PCR-DGGE technology to carry out ballast water for ship Microbial diversity Journal of Sex Research at present both at home and abroad.
Summary of the invention
The object of the present invention is to provide the method for the extraction of a kind of ballast water for ship microorganism total DNA and diversity analysis, the present invention is according to the characteristic of ballast water for ship, use two-stage filtration and the cryofixation of 3 μm and 0.22 μm millipore filtrations, strengthen the attachment of microorganism on filter membrane, use cetyl trimethylammonium bromide (CTAB) process, remove the material that DNA extraction is impacted, the purity of microorganism total DNA is made to reach the needs directly carrying out subsequent molecular, the DNA fragmentation extracted can carry out amplification and the denaturing gradient gel electrophoresis of 16SrDNA, QuantityOne software is used to carry out digital assay to denaturing gradient gel electrophoresis separation electrophoresis figure, judge ballast water for ship microbial population diversity level, reclaim by cutting glue to specific band, cloning and sequencing also carries out BLAST sequence alignment, the kind of water ballast dominant microflora can be drawn, thus determine that biological community structure forms.
The object of the invention is to be achieved through the following technical solutions:
The invention provides the method for the extraction of a kind of ballast water for ship microorganism total DNA and diversity analysis, the method comprises the following steps:
1) boats and ships different ballast tank water ballast sample is collected;
2) pre-treatment is carried out to ballast for cruising water sample;
3) microorganism total DNA in sample is extracted after pre-treatment;
4) carrying out agarose gel electrophoresis detection to slightly carrying DNA, obtaining thick pressure-raising and carrying water microorganism STb gene agarose gel electrophoresis figure, judge that whether total DNA extraction is successful;
5) with the STb gene extracted for template, under the guiding of primer special, carry out pcr amplification; The sequence of described primer special is as shown in SEQ ID NO.1 and SEQ ID NO.2;
Upstream primer 357F-GC (SEQ IDNO.1):
5′-CGCCCGCCGCGCGCGGCGGGCGGGGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3′，
With downstream primer 518R (SEQ ID NO.2): 5 '-ATTACCGCGGCTGCTGG-3 ';
6) pcr amplification product is carried out agarose gel electrophoresis detection, obtain pcr amplification product agarose gel electrophoresis figure, and judge whether STb gene fragment increases successfully under the guiding of primer special;
7) pcr amplification product is carried out denaturing gradient gel electrophoresis, obtain denaturing gradient gel electrophoresis separation electrophoresis figure, judge the kind of ballast water for ship microbial population diversity level and water ballast dominant microflora, analyze ballast water for ship microbial diversity.
Preferably, step 1) described in different ballast tanks be: the ballast tank of boats and ships different spatial.
Preferably, step 2) described in pre-treatment comprise: filter and extract microorganism, cryofixation.Be specially: use 3 μm and 0.22 μm of millipore filtration carry out two-stage filtration extraction microorganism, cryofixation successively.
Preferably, step 3) described in extracting method as follows: use after 2% (m/v) cetyl trimethylammonium bromide (CTAB) process pretreated sample, use 3S DNA Isolation to extract test kit to extract, operation steps is carried out according to the explanation of test kit.
Preferably, step 4) described in slightly carry DNA carry out agarose gel electrophoresis detect comprise: with 1% agarose gel electrophoresis detection slightly carry DNA; Dyeing is taken pictures.
More preferably, step 4) described in carry out agarose gel electrophoresis be detected as slightly carrying DNA: with the applied sample amount of each sample 5 μ L, 1 μ L 6 × Loadding buffer electrophoresis detection pcr amplified fragment on the sepharose of 1%, electrophoretic buffer is 1 × TAE buffering; Voltage 140V, time 20min; Standard molecular weight object of reference is made with DL500DNA Maker; Electrophoresis terminates rear EB lucifuge dyeing 40min, takes pictures, obtain thick pressure-raising and carry water microorganism STb gene agarose gel electrophoresis figure by gel imaging system observations.
Preferably, step 5) described in the PCR reaction system that adopts of pcr amplification be: 35 μ LddH
2o, 5uL contain the 10* reaction buffer of 15mmol/L magnesium ion, 1 μ L upstream primer 357F-GC (SEQ ID NO.1), 1 μ L downstream primer 518R (SEQ ID NO.2), 5 μ L thymus nucleic acids, 1 μ LTaq enzyme, 2 μ L templates; Pcr amplification program is: initial 95 DEG C of denaturation 5min, 94 DEG C of sex change 1min, 65 DEG C of annealing 1min (each cycle annealing temperature reduces by 0.5 DEG C), 72 DEG C of primer extension 30s, 20 circulations; 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C of primer extension 30s, 15 circulations; 72 DEG C of ends extend 8min; Preserve pcr amplification product for 4 DEG C.
Preferably, step 6) described in agarose gel electrophoresis detect as follows: with 1% agarose gel electrophoresis detection pcr amplification product; Dyeing is taken pictures.
More preferably, step 6) described in agarose gel electrophoresis carried out to pcr amplification product be detected as: with the applied sample amount of each sample 5 μ L, 1 μ L 6 × Loadding buffer electrophoresis detection pcr amplified fragment on the sepharose of 1%, electrophoretic buffer is 1 × TAE buffering; Voltage 140V, electrophoresis time is 20min; Electrophoresis terminates rear EB lucifuge dyeing 40min, takes pictures, obtain pcr amplification product agarose gel electrophoresis figure by gel imaging system observations.
Preferably, step 7) described in denaturing gradient gel electrophoresis comprise: in detection in Gene Mutation system, electrophoretic separation is carried out to PCR reaction product; Electrophoresis terminates poststaining and takes pictures.
More preferably, step 7) described in pcr amplification product carried out denaturing gradient gel electrophoresis be: make that denaturing agent gradient is 35% ~ 65%, acrylamide intensity is the gel of 8%; Assemble the electrophoresis chamber put into containing 1 × TAE damping fluid, be preheating to 60 DEG C; Every hole adds the PCR reaction product of 30 μ L and 6 × Loadding buffer of 15 μ L; Connect electrophoresis power, electrophoresis 16h under 160V, 60 DEG C of conditions; After electrophoresis terminates, carefully take out gel, EB lucifuge dyeing 20min, moves back dye 20min, then takes pictures by gel imaging system observations, obtain denaturing gradient gel electrophoresis separation electrophoresis figure in clear water.
Preferably, step 7) in also comprise: use Quantity One software to carry out digital assay to denaturing gradient gel electrophoresis separation electrophoresis figure, obtain denatured gradient gel electrophoresis strip intensity map, versus grayscale according to each band calculates bacterial quorum sensing diversity index H ' and evenness index J, and then judges ballast water for ship microbial population diversity level.
Preferably, step 7) in also comprise: the specific band of denaturing gradient gel electrophoresis separation electrophoresis figure electrophoretic band obtained is cut that glue reclaims, cloning and sequencing carry out BLAST sequence alignment, the kind of water ballast dominant microflora can be drawn, thus determine that biological community structure forms.
More preferably, the method for ballast water for ship microorganism total DNA extraction of the present invention and diversity analysis comprises following concrete steps:
1) collect boats and ships different ballast tank water ballast sample: in the different ballast tanks of boats and ships, use water sampler to gather water ballast, collect 2L sample with the blue mouth vial of sterilizing, 4 DEG C are saved to sample pretreatment;
2) carrying out pre-treatment to ballast for cruising water sample: water sample is carried in pressure, after 3 μm and 0.22 μm of millipore filtration (the prior sterilising treatment of filter membrane) two-stage filtration, discard 3 μm of millipore filtrations, 0.22 μm of millipore filtration is water ballast microorganism; Use sterilizing operation surgical forceps, 0.22 μm of filter membrane is transferred in 1.5mL centrifuge tube, puts into-80 DEG C of refrigerator and cooled immediately and freeze 4h; Take out freezing after be placed with the centrifuge tube of microorganism filter membrane, use sterilizing surgical scissors and tweezers, 0.22 μm of filter membrane shredded and transfers in 1.5mL sterile centrifugation tube;
3) microorganism total DNA in sample is extracted after pre-treatment: in the centrifuge tube that filter membrane is housed, add 200 μ L 2% (m/v) cetyl trimethylammonium bromides (CTAB), 65 DEG C of water-bath 1h; Use 3S DNA Isolation to extract test kit to extract microbial DNA, in pretreated sample, add 200 μ L Solution SUS, sample is suspended; Add 400 μ L Solution LYS, 300mg quartzite, covers centrifuge tube lid, high speed concuss 30 minutes on turbula shaker; With desk centrifuge, 12000r/min, the centrifugal 5min of room temperature; Supernatant liquor is transferred to completely in sterilizing 1.5mL centrifuge tube, adds 120 μ L Solution BID, cover centrifuge tube lid, mixing of turning upside down; All be transferred in 3S post by solution 1mL TIP, 2mL centrifuge tube put into by pillar, not lid centrifuge tube lid, and room temperature places 5min; Cover centrifuge tube lid, 12000r/min, the centrifugal 5min of room temperature, takes off 3S post, discards the waste liquid in centrifuge tube; Put back to by pillar in same centrifuge tube, add 600 μ L Wash Solution, 10000r/min, the centrifugal 2min of room temperature, takes off 3S post, discards the waste liquid in centrifuge tube; Repeat, put back to by pillar in same centrifuge tube, add 600 μ L Wash Solution, 10000r/min, the centrifugal 2min of room temperature, takes off 3S post, discards the waste liquid in centrifuge tube; Pillar is put back in same centrifuge tube, 10000r/min, the centrifugal 2min of room temperature, to remove residual Wash Solution; TE is preheating to 50-55 DEG C, pillar is put into new 1.5mL sterile centrifugation tube, add 30 μ L TE in pillar central authorities, pillar and collection tube are placed in 50-55 DEG C of baking oven, place 2min, 12000r/min, the centrifugal 1min of room temperature after taking out; Repeat, open centrifuge tube lid, add 30 μ L TE in same pillar central authorities, be placed in 50-55 DEG C of baking oven, place 2min, 12000r/min, the centrifugal 1min of room temperature after taking out; Liquid in collection tube is microorganism total DNA;
4) carry out agarose gel electrophoresis detection to slightly carrying DNA: with the applied sample amount of each sample 5 μ L, 1 μ L 6 × Loadding buffer electrophoresis detection pcr amplified fragment on the sepharose of 1%, electrophoretic buffer is 1 × TAE buffering; Voltage 140V, time 20min; Standard molecular weight object of reference is made with DL500DNA Maker; Electrophoresis terminates rear EB lucifuge dyeing 40min, takes pictures, obtain thick pressure-raising and carry water microorganism STb gene agarose gel electrophoresis figure by gel imaging system observations;
5) with the STb gene extracted for template, under the guiding of primer special, carry out pcr amplification: primer special is 357F-GC:
5 '-CGCCCGCCGCGCGCGGCGGGCGGGGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3 ' (SEQ ID NO.1) and 518R:5 '-ATTACCGCGGCTGCTGG-3 ' (SEQ ID NO.2); PCR reaction system is: 35 μ LddH
2o, 5 μ L contain the 10* reaction buffer of 15mmol/L magnesium ion, 1 μ L upstream primer 357F-GC, 1 μ L downstream primer 518R, 5 μ L thymus nucleic acids, 1 μ LTaq enzyme, 2 μ L templates; Pcr amplification program is: initial 95 DEG C of denaturation 5min, 94 DEG C of sex change 1min, 65 DEG C of annealing 1min (each cycle annealing temperature reduces by 0.5 DEG C), 72 DEG C of primer extension 30s, 20 circulations; 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C of primer extension 30s, 15 circulations; 72 DEG C of ends extend 8min; Preserve pcr amplification product for 4 DEG C;
6) carry out agarose gel electrophoresis detection to pcr amplification product: with the applied sample amount of each sample 5 μ L, 1 μ L6 × Loadding buffer electrophoresis detection pcr amplified fragment on the sepharose of 1%, electrophoretic buffer is 1 × TAE buffering; Voltage 140V, time 20min; Applied sample amount is 5 μ L pcr amplification products, 1 μ L 6 × Loadding buffer; Standard molecular weight object of reference is made with DL500DNA Maker; Electrophoresis terminates rear EB lucifuge dyeing 40min, takes pictures, obtain pcr amplification product agarose gel electrophoresis figure by gel imaging system observations;
7) pcr amplification product is carried out denaturing gradient gel electrophoresis: making denaturing agent gradient is 35% ~ 65%, acrylamide intensity is 8% gel; Assemble the electrophoresis chamber put into containing 1 × TAE damping fluid, be preheating to 60 DEG C; Every hole adds PCR reaction product and 15 μ L6 × Loadding buffer of 30 μ L; Connect electrophoresis power, electrophoresis 16h under 160V, 60 DEG C of conditions; After electrophoresis terminates, carefully take out gel, EB lucifuge dyeing 20min, moves back dye 20min, then takes pictures by gel imaging system observations, obtain denaturing gradient gel electrophoresis separation electrophoresis figure in clear water.QuantityOne software is used to carry out digital assay to denaturing gradient gel electrophoresis separation electrophoresis figure, judge ballast water for ship microbial population diversity level, by cutting specific band, glue reclaims, cloning and sequencing carry out BLAST sequence alignment, the kind of water ballast dominant microflora can be drawn, thus determine that biological community structure forms.
The present invention has following beneficial effect:
1, for the singularity of ballast water for ship, utilize two-stage filtration and the cryofixation of use 3 μm and 0.22 μm millipore filtrations, strengthen the attachment of microorganism on filter membrane, and use cetyl trimethylammonium bromide (CTAB) to process, CTAB not only can be used for lysis, also assist in removing humic acid, the DNA purity obtained is higher, and A260/A230 can reach 1.32 ± 0.02.Method therefor is simple and convenient, does not need purified follow-up pcr analysis and the DGGE of namely can be used for analyze.
2, for the singularity of ballast water for ship, method of the present invention eliminates tediously long, loaded down with trivial details DNA washing step, saves time and avoids DNA to lose, and determines the method being suitable for water ballast microbial DNA and extracting, makes easy and simple to handle.
3, the DNA fragmentation utilizing the inventive method to extract can carry out amplification and the denaturing gradient gel electrophoresis of 16SrDNA, Quantity One software is used to carry out digital assay to denaturing gradient gel electrophoresis separation electrophoresis figure, obtain denatured gradient gel electrophoresis strip intensity map, versus grayscale according to each band calculates bacterial quorum sensing diversity index H ' and evenness index J, and then judge ballast water for ship microbial population diversity level, and glue recovery can be cut to the specific band of denaturing gradient gel electrophoresis bands of a spectrum, cloning and sequencing, carry out BLAST sequence alignment, thus determine that biological community structure forms.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is that thick pressure-raising carries water microorganism STb gene agarose gel electrophoresis figure;
Fig. 2 is pcr amplification product agarose gel electrophoresis figure;
Fig. 3 is denaturing gradient gel electrophoresis separation electrophoresis figure (a) and band intensity schematic diagram (b);
Fig. 4 is bacterial quorum sensing diversity index (H ') and evenness index (J);
Fig. 5 is denaturing gradient gel electrophoresis specific band sequence alignment result.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make certain adjustments and improvements.These all belong to protection scope of the present invention.
embodiment
Choose boats and ships, sample, get the water ballast of the ballast tank of boats and ships different spatial, to the population structure analysis of bacterium and the determining step of dominant microflora as follows
1) water ballast microorganism total DNA isolation and determination:
In the different ballast tanks of boats and ships, use water sampler to gather water ballast, collect 2L sample, 4 DEG C of preservations with the blue mouth vial of sterilizing; Water sample is carried in pressure, after 3 μm and 0.22 μm of millipore filtration (the prior sterilising treatment of filter membrane) two-stage filtration, discards 3 μm of millipore filtrations, 0.22 μm of millipore filtration is water ballast microorganism; Use sterilizing operation surgical forceps, 0.22 μm of filter membrane is transferred in 1.5mL centrifuge tube, puts into-80 DEG C of refrigerator and cooled immediately and freeze 4h; Take out freezing after be placed with the centrifuge tube of microorganism filter membrane, use sterilizing surgical scissors and tweezers, 0.22 μm of filter membrane shredded and transfers in 1.5mL sterile centrifugation tube; 200 μ L 2% (m/v) cetyl trimethylammonium bromides (CTAB) are added, 65 DEG C of water-bath 1h in the centrifuge tube that filter membrane is housed; Use 3S DNA Isolation to extract test kit to extract microbial DNA, in the sample after CTAB process, add 200 μ L Solution SUS, sample is suspended; Add 400 μ L Solution LYS and 300mg quartzites, cover centrifuge tube lid, high speed concuss 30 minutes on turbula shaker; With desk centrifuge, 12000r/min, the centrifugal 5min of room temperature; Supernatant liquor is transferred to completely in sterilizing 1.5mL centrifuge tube, adds 120 μ LSolution BID, cover centrifuge tube lid, mixing of turning upside down; All be transferred in 3S post by solution 1mL TIP, 2mL centrifuge tube put into by pillar, not lid centrifuge tube lid, and room temperature places 5min; Cover centrifuge tube lid, 12000r/min, the centrifugal 5min of room temperature, takes off 3S post, discards the waste liquid in centrifuge tube; Put back to by pillar in same centrifuge tube, add 600 μ L Wash Solution, 10000r/min, the centrifugal 2min of room temperature, takes off 3S post, discards the waste liquid in centrifuge tube; Repeat, put back to by pillar in same centrifuge tube, add 600 μ L Wash Solution, 10000r/min, the centrifugal 2min of room temperature, takes off 3S post, discards the waste liquid in centrifuge tube; Pillar is put back in same centrifuge tube, 10000r/min, the centrifugal 2min of room temperature, to remove residual Wash Solution; TE is preheating to 50-55 DEG C, pillar is put into new 1.5mL sterile centrifugation tube, add 30 μ L TE in pillar central authorities, pillar and collection tube are placed in 50-55 DEG C of baking oven, place 2min, 12000r/min, the centrifugal 1min of room temperature after taking out; Repeat, open centrifuge tube lid, add 30 μ L TE in same pillar central authorities, be placed in 50-55 DEG C of baking oven, place 2min, 12000r/min, the centrifugal 1min of room temperature after taking out; Liquid in collection tube is microorganism total DNA.
With the applied sample amount of each sample 5 μ L, 1 μ L 6 × Loadding buffer electrophoresis detection pcr amplified fragment on the sepharose of 1%, electrophoretic buffer is 1 × TAE buffering; Voltage 140V, time 20min; Standard molecular weight object of reference is made with DL500DNA Maker; Electrophoresis terminates rear EB lucifuge dyeing 40min, takes pictures, obtain thick pressure-raising and carry water microorganism STb gene agarose gel electrophoresis figure (see Fig. 1) by gel imaging system observations.As shown in Figure 1, microorganism total DNA fragment presents light after dyeing, and DNA extraction success is described.
2) pcr amplification and product detect:
With
357F-GC:5 '-CGCCCGCCGCGCGCGGCGGGCGGGGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3 ' (SEQ ID NO.1) and 518R:5 '-ATTACCGCGGCTGCTGG-3 ' (SEQ IDNO.2) is primer; PCR reaction system: 35 μ LddH
20,5uL 10* reaction buffer (magnesium ion containing 15mmol/L), 1 μ L upstream primer 357F-GC, 1 μ L downstream primer 518R, 5 μ L thymus nucleic acids, 1 μ LTaq enzyme, 2 μ L templates; Pcr amplification program is: initial 95 DEG C of denaturation 5min, 94 DEG C of sex change 1min, 65 DEG C of annealing 1min (each cycle annealing temperature reduces by 0.5 DEG C), 72 DEG C of primer extension 30s, 20 circulations; 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C of primer extension 30s, 15 circulations; 72 DEG C of ends extend 8min; Preserve pcr amplification product for 4 DEG C; With the applied sample amount of each sample 5 μ L, 1 μ L6 × Loadding buffer electrophoresis detection pcr amplified fragment on the sepharose of 1%, electrophoretic buffer is 1 × TAE buffering; Voltage 140V, electrophoresis time is 20min; Applied sample amount is 5 μ LPCR amplified productions, 1 μ Llording buffer; Standard molecular weight object of reference is made with DL500DNA Maker; Electrophoresis terminates rear EB lucifuge dyeing 40min, takes pictures, obtain pcr amplification product agarose gel electrophoresis figure (see Fig. 2) by gel imaging system observations.As shown in Figure 2, PCR primer size, between 200kb-300kb, shows that DNA completes the amplification of 16SrDNA under primer special guides, can be used for follow-up denaturing gradient gel electrophoresis.
3) denaturing gradient gel electrophoresis separation is carried out and result represents:
Making denaturing agent gradient is 35% ~ 65%, acrylamide intensity is the gel of 8%; Assemble the electrophoresis chamber put into containing 1 × TAE damping fluid, be preheating to 60 DEG C; Every hole adds the PCR reaction product of 30 μ L and 6 × Loadding buffer of 15 μ L; Connect electrophoresis power, voltage 160V, temperature 60 C, time 16h; After electrophoresis terminates, careful taking-up gel, EB lucifuge dyeing 20min, dye 20min is moved back in clear water, then take pictures by gel imaging system observations, obtain denaturing gradient gel electrophoresis separation electrophoresis figure (see Fig. 3 a), to use Quantity One software to carry out digital assay to denaturing gradient gel electrophoresis separation electrophoresis figure, obtain denatured gradient gel electrophoresis strip intensity schematic diagram (see Fig. 3 b).From Fig. 3 a and Fig. 3 b, 6 water ballast samples are through DNA extraction and specific amplification, and after carrying out denaturing gradient gel electrophoresis, the DNA fragmentation of the formed objects of Different Alkali base sequence is separated, and obtains 17 specific bands altogether.
4) judge ballast water for ship microbial population diversity level and determine that biological community structure forms:
Use Quantity One software to carry out digital assay to denaturing gradient gel electrophoresis separation electrophoresis figure, the versus grayscale according to each band calculates bacterial quorum sensing diversity index (H ') and evenness index (J) (see Fig. 4).As shown in Figure 4, community diversity index and evenness index present reverse proportionality, and the bacterial flora diversity index of sample 2 is the highest, and evenness index is minimum.After analysis microbial population diversity level, sterilizing scalpel is used to be cut off by each specific band, be positioned in 1.5mL sterile centrifugation tube respectively, in each centrifuge tube, add 30 μ L distilled waters, 5000r/min, the centrifugal 1min of room temperature, hold over night, delivers to biotech firm and carries out cloning and sequencing, base sequence is carried out BLAST sequence alignment, to microbe species corresponding to each specific band be obtained, thus determine that biological community structure forms.As shown in Figure 5, the microbe species of each specific band representative have found corresponding species name through BLAST sequence alignment.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (8)
1. a method for the extraction of ballast water for ship microorganism total DNA and diversity analysis, it is characterized in that, described method comprises the steps:
1) boats and ships different ballast tank water ballast sample is collected;
2) pre-treatment is carried out to ballast for cruising water sample;
3) microorganism total DNA in sample is extracted after pre-treatment;
4) carrying out agarose gel electrophoresis detection to slightly carrying DNA, obtaining thick pressure-raising and carrying water microorganism STb gene agarose gel electrophoresis figure, judge that whether total DNA extraction is successful;
5) with the STb gene extracted for template, under the guiding of primer special, carry out pcr amplification; The sequence of described primer special is as shown in SEQ ID NO.1 and SEQ ID NO.2;
6) agarose gel electrophoresis detection is carried out to pcr amplification product, obtain pcr amplification product agarose gel electrophoresis figure, and judge whether STb gene fragment increases successfully under the guiding of primer special;
7) pcr amplification product is carried out denaturing gradient gel electrophoresis, obtain denaturing gradient gel electrophoresis separation electrophoresis figure, judge the kind of ballast water for ship microbial population diversity level and water ballast dominant microflora, analyze ballast water for ship microbial diversity.
2. method according to claim 1, is characterized in that, step 1) described in different ballast tank be the ballast tank of boats and ships different spatial.
3. method according to claim 1, is characterized in that, step 2) described in pre-treatment comprise and use 3 μm and 0.22 μm of millipore filtration to carry out two-stage filtration extraction microorganism, cryofixation successively.
4. method according to claim 1, is characterized in that, step 3) described in extraction be use volume mass ratio to be after 2% cetyl trimethylammonium bromide processing sample, use 3S DNA Isolation to extract test kit and extract.
5. method according to claim 1, is characterized in that, step 5) described in the PCR reaction system that adopts of pcr amplification be: 35 μ LddH
2o, 5 μ L contain the 10* reaction buffer of 15mmol/L magnesium ion, the upstream primer of 1 μ L as shown in SEQ ID NO.1, the downstream primer of 1 μ L as shown in SEQ ID NO.2,5 μ L thymus nucleic acids, 1 μ L Taq enzyme, 2 μ L templates; Pcr amplification program is: initial 95 DEG C of denaturation 5min, 94 DEG C of sex change 1min, 65 DEG C of annealing 1min, 72 DEG C of primer extension 30s, 20 circulations; 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C of primer extension 30s, 15 circulations; 72 DEG C of ends extend 8min, obtain microorganism pcr amplification product.
6. method according to claim 1, is characterized in that, step 7) described in denaturing gradient gel electrophoresis comprise: in detection in Gene Mutation system, carry out electrophoretic separation to PCR reaction product, electrophoresis terminates poststaining and takes pictures.
7. method according to claim 1, it is characterized in that, step 7) in also comprise: use Quantity One software to carry out digital assay to denaturing gradient gel electrophoresis separation electrophoresis figure, obtain denatured gradient gel electrophoresis strip intensity map, versus grayscale according to each band calculates bacterial quorum sensing diversity index H ' and evenness index J, and then judges ballast water for ship microbial population diversity level.
8. method according to claim 1, it is characterized in that, step 7) in also comprise: the specific band of denaturing gradient gel electrophoresis separation electrophoresis figure electrophoretic band obtained is cut that glue reclaims, cloning and sequencing carry out BLAST sequence alignment, the kind of water ballast dominant microflora can be drawn, thus determine that biological community structure forms.
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| CN107058607A (en) * | 2017-06-29 | 2017-08-18 | 上海海洋大学 | A kind of multifarious method of drug-fast bacteria in quick detection aquatic products |
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| CN101883738A (en) * | 2007-12-04 | 2010-11-10 | 比尔吉尔·尼尔森 | Apparatus and method for ballast water treatment |
| WO2013192567A1 (en) * | 2012-06-22 | 2013-12-27 | Wayne State University | Automated viability testing system |
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| CN101883738A (en) * | 2007-12-04 | 2010-11-10 | 比尔吉尔·尼尔森 | Apparatus and method for ballast water treatment |
| WO2013192567A1 (en) * | 2012-06-22 | 2013-12-27 | Wayne State University | Automated viability testing system |
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| CN107058607A (en) * | 2017-06-29 | 2017-08-18 | 上海海洋大学 | A kind of multifarious method of drug-fast bacteria in quick detection aquatic products |
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