CN104946627B - The extraction of ballast water for ship microorganism total DNA and the method for diversity analysis - Google Patents
The extraction of ballast water for ship microorganism total DNA and the method for diversity analysis Download PDFInfo
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Abstract
The invention discloses a kind of extraction of ballast water for ship microorganism total DNA and the method for diversity analysis；Characteristic of the invention according to ballast water for ship, use the double-filtration and cryofixation of 3 μm and 0.22 μm miillpore filters, strengthen attachment of the microorganism on filter membrane, the need for the microorganism total DNA purity obtained using cetyl trimethylammonium bromide (CTAB) processing reaches directly progress subsequent molecular, the DNA fragmentation of extraction can carry out 16SrDNA amplification and denaturing gradient gel electrophoresis (DGGE), ballast water for ship microbial population diversity level is can determine whether after electrophoretogram is digitized into analysis, by by specific band gel extraction, cloning and sequencing simultaneously carries out BLAST sequence alignments, obtain the species of ballast water dominant microflora, so that it is determined that biological community structure is constituted, laid the foundation for microbial diversity in ships ballast water from now on and deposit with ecological functions.
Description
Technical field
The invention belongs to environmental microorganism molecular ecology field, and in particular to a kind of ballast water for ship microorganism total DNA
Extraction and the method for diversity analysis.
Background technology
Ballast water for ship be ship in zero load in order to keep stability of hull, when setting sail, ballast enters ballast tank of ship
Seawater, ballast tank is then discharged when arriving at harbour loading.The discharge of ballast water and deposit is marine organisms in the port of departure and mesh
Ground port between the main path propagated, four big one of threatened by what the environmentally friendly fund (GEF) in the world regarded as that ocean faces.
Ship will unload ballast water while loading to port, while ballast tank is cleared up, due to the special construction of ballast tank,
Thorough cleaning can not be reached, with the local fresh seawater of each ballast of ship, the biological and abiotic composition for bringing ballast tank into is various,
Ballast water sediment is discharged into discharge long term accumulation sedimentation with ballast water.Ballast tank deposits the special cabin of ballast water as ship
Room, its is complicated, unglazed in ballast tank, anoxic, being also possible to containing harmful substance of having, is totally unfavorable to biological existence
, most of biology can not stand the adverse circumstances in ballast tank and dead, and the biology for not adapting to ballast tank environment largely is dead
After dying, its corpse sinks to falling into the bilge, and food source is provided to saprophage.Because ship has high surface area and volume
Than the microorganism of large area is attached to its surface, so sanctuary of the ballast tank into microorganism, such case is referred to as " ship
Internal portion dirt ".At present by the discharge of ballast water for ship and deposit, harmful microorganism may be propagated between Port State,
Pathogenic microorganisms may threaten to biological in environment and people, at this stage microbe survival in ballast water for ship and deposit
State is not probed into completely, and the diversity of microorganism does not understand also.
The bacterium mainly in harbour investigation dock vessels ballast water of the research of country's ballast water microbial diversity at present
The Hygienic indicator bacterias such as sum, coliform and some pathogenic bacteria.Using molecular biology method to microorganism in ballast water for ship
Research is carried out to have not yet fully deployed, and it is also most that microbial DNA, which extracts the first step analyzed as molecular biology method,
An important step, the research of its method has not been reported.
Denaturing gradient gel electrophoresis (DGGE) is initially used to the abrupt climatic change of gene, and denaturing gradient gel electrophoresis is to difference
The separation principle of DNA fragmentation is same length but with not homotactic DNA fragmentation energy in denaturing gradient gel electrophoresis (DGGE)
It is enough to be separated.There are different behaviors of unwinding with not homotactic DNA molecular, will stop migrating in the diverse location of gel.Become
Property gradient gel electrophoresis (DGGE) be built upon PCR reactions to the 16SrRNA and 18SrRNA of all microorganisms in environmental sample
On the basis of amplification Deng the interval DNA segment of conservative gene, it is micro- that these DNA segments being amplified out can represent all differences
Biological information, the DNA segment of these different microorganisms gone out using same primer pair amplifies has identical base number, therefore
General electrophoresis can not separate them.Because denaturing gradient gel electrophoresis (DGGE) can be by the identical of different base sequences
The DNA fragmentation of size is separated, and uses PCR-DGGE technologies to be ground to the microbial diversity in environmental sample
Study carefully.
Ballast cabin structure and ballast water dispensing rule cause physics, chemical property and the river of ballast water for ship, seawater and
There is difference in height in environmental wastewater, micro organism quantity and Species structure are also significantly different, and this just determines the sample under varying environment
Product have differences when carrying out microbiologic population's component analysis.It there is no both at home and abroad on entering to navigate with PCR-DGGE technologies at present
The relevant report of ship ballast water Microbial diversity Journal of Sex Research.
The content of the invention
It is an object of the invention to provide a kind of extraction of ballast water for ship microorganism total DNA and the method for diversity analysis；
The present invention, using the double-filtration and cryofixation of 3 μm and 0.22 μm miillpore filters, is strengthened according to the characteristic of ballast water for ship
Attachment of the microorganism on filter membrane, is handled using cetyl trimethylammonium bromide (CTAB), removes and DNA extractions are impacted
Material so that the purity of microorganism total DNA reach directly carry out subsequent molecular the need for, the DNA pieces of extraction
Section can carry out 16SrDNA amplification and denaturing gradient gel electrophoresis, using QuantityOne softwares to denaturing gradient gel electrophoresis
Separation electrophoresis figure is digitized analysis, ballast water for ship microbial population diversity level is judged, by being cut to specific band
Glue reclaim, cloning and sequencing simultaneously carry out BLAST sequence alignments, it can be deduced that the species of ballast water dominant microflora, so that it is determined that micro- life
Thing structure of community is constituted.
The purpose of the present invention is achieved through the following technical solutions：
The invention provides a kind of extraction of ballast water for ship microorganism total DNA and the method for diversity analysis, this method bag
Include following steps：
1) the different ballast tank ballast water samples of ship are collected；
2) ballast for cruising water sample is pre-processed；
3) microorganism total DNA in sample after extraction is pre-processed；
4) row agarose gel electrophoresis detection is entered to the thick DNA that carries, obtains thick pressure-raising and carry water microorganism STb gene Ago-Gel electricity
Swimming figure, judges whether Genome DNA extraction succeeds；
5) using the STb gene of extraction as template, performing PCR amplification is entered under the guiding of primer special；The sequence of the primer special
Row are as shown in SEQ ID NO.1 and SEQ ID NO.2；
Sense primer 357F-GC (SEQ IDNO.1)：
5 '-CGCCCGCCGCGCGCGGCGGGCGGGGGGGGCACGGGGGGCCTACGGGAGGCAGCAG- 3 ',
With anti-sense primer 518R (SEQ ID NO.2)：5′-ATTACCGCGGCTGCTGG-3′；
6) pcr amplification product is entered into row agarose gel electrophoresis detection, obtains pcr amplification product agarose gel electrophoresis figure,
And judge whether STb gene fragment expands success under the guiding of primer special；
7) pcr amplification product is subjected to denaturing gradient gel electrophoresis, obtains denaturing gradient gel electrophoresis separation electrophoresis figure, judged
The species of ballast water for ship microbial population diversity level and ballast water dominant microflora, analyzes ballast water for ship Microbial diversity
Property.
Preferably, step 1) described in different ballast tanks be：The ballast tank of ship different spatial.
Preferably, step 2) described in pre-process and include：Microorganism, cryofixation are extracted in filtering.Specially：Use successively
3 μm and 0.22 μm of miillpore filter progress double-filtration extraction microorganism, cryofixation.
Preferably, step 3) described in extracting method it is as follows：Use 2% (m/v) cetyl trimethylammonium bromide
(CTAB) handle after pretreated sample, extracted using 3S DNA Isolation extracts kits, operating procedure root
Carried out according to the explanation of kit.
Preferably, step 4) described in it is thick carry DNA and enter row agarose gel electrophoresis detection include：With 1% agarose
Detected through gel electrophoresis slightly carries DNA；Dyeing is taken pictures.
It is highly preferred that step 4) described in it is thick carry DNA and enter row agarose gel electrophoresis be detected as：With each μ of sample 5
L applied sample amount, 1 μ 6 × Loadding of L buffer electrophoresis detection pcr amplified fragments on 1% Ago-Gel, electrophoresis delays
Fliud flushing buffers for 1 × TAE；Voltage 140V, time 20min；Make standard molecular weight object of reference with DL500DNA Maker；Electrophoresis knot
Shu Houyong EB lucifuges dye 40min, observe result with gel imaging system and take pictures, and obtain thick pressure-raising and carry water microorganism STb gene fine jade
Sepharose electrophoretogram.
Preferably, step 5) described in PCR expand used PCR reaction systems and be：35μLddH2O, 5uL contain
The 10* reaction buffers of 15mmol/L magnesium ions, 1 μ L sense primers 357F-GC (SEQ ID NO.1), 1 μ L anti-sense primers 518R
(SEQ ID NO.2), 5 μ L DNAs, 1 μ LTaq enzymes, 2 μ L templates；PCR amplification programs are：Originate 95 DEG C of pre-degenerations
5min, 94 DEG C of denaturation 1min, 65 DEG C of annealing 1min (each cycle annealing temperature reduce by 0.5 DEG C), 72 DEG C of primer extend 30s, 20
Individual circulation；94 DEG C of denaturation 1min, 55 DEG C of annealing 1min, 72 DEG C of primer extend 30s, 15 circulations；72 DEG C extend 8min eventually；4℃
Preserve pcr amplification product.
Preferably, step 6) described in agarose gel electrophoresis detection it is as follows：Detected with 1% agarose gel electrophoresis
Pcr amplification product；Dyeing is taken pictures.
It is highly preferred that step 6) described in row agarose gel electrophoresis entered to pcr amplification product be detected as：With each sample
The μ L of product 5 applied sample amount, 1 μ 6 × Loadding of L buffer electrophoresis detection pcr amplified fragments on 1% Ago-Gel, electricity
Buffer solution of swimming buffers for 1 × TAE；Voltage 140V, electrophoresis time is 20min；Electrophoresis dyes 40min after terminating with EB lucifuges, uses
Gel imaging system is observed result and taken pictures, and obtains pcr amplification product agarose gel electrophoresis figure.
Preferably, step 7) described in denaturing gradient gel electrophoresis include：It is anti-to PCR in detection in Gene Mutation system
Product is answered to be separated by electrophoresis；Electrophoresis terminates poststaining and taken pictures.
It is highly preferred that step 7) described in by pcr amplification product progress denaturing gradient gel electrophoresis be：Make denaturant ladder
Degree is the gel that 35%~65%, acrylamide intensity is 8%；Assembling is put into the electrophoresis tank containing 1 × TAE buffer solutions, in advance
Heat is to 60 DEG C；30 μ L PCR reaction products and 15 μ L 6 × Loadding buffer are added per hole；Electrophoresis power is connected,
160V, electrophoresis 16h under the conditions of 60 DEG C；After electrophoresis terminates, gel is carefully taken out, EB lucifuges dyeing 20min moves back dye in clear water
20min, then observes result with gel imaging system and takes pictures, obtain denaturing gradient gel electrophoresis separation electrophoresis figure.
Preferably, step 7) in also include：Using Quantity One softwares to denaturing gradient gel electrophoresis separation electrophoresis
Figure is digitized analysis, obtains denatured gradient gel electrophoresis strip intensity map, calculates thin according to the versus grayscale of each band
Flora falls various structures sex index H ' and evenness index J, and then judges ballast water for ship microbial population diversity level.
Preferably, step 7) in also include：To the spy of the denaturing gradient gel electrophoresis separation electrophoresis figure electrophoretic band of acquisition
Different band gel extraction, cloning and sequencing simultaneously carry out BLAST sequence alignments, it can be deduced that the species of ballast water dominant microflora, so that
Determine that biological community structure is constituted.
It is highly preferred that the ballast water for ship microorganism total DNA extraction of the present invention and the method for diversity analysis are including following
Specific steps：
1) the different ballast tank ballast water samples of ship are collected：In the different ballast tanks of ship ballast is gathered using hydrophore
Water, collects 2L samples, 4 DEG C preserve to sample pretreatment with the blue mouth vial of sterilizing；
2) ballast for cruising water sample is pre-processed：Pressure carries water sample, through 3 μm and 0.22 μm of miillpore filter (filter membrane
Prior sterilization treatment) after double-filtration, it is ballast water microorganism to discard on 3 μm of miillpore filters, 0.22 μm of miillpore filter；Using going out
Bacterium operation surgical forceps, 0.22 μm of filter membrane is transferred in 1.5mL centrifuge tubes, is immediately placed in -80 DEG C of refrigerators and freezes 4h；Take out freezing
The centrifuge tube of microorganism filter membrane is placed with afterwards, using sterilizing surgical scissors and tweezers, 0.22 μm of filter membrane is shredded and is transferred to 1.5mL and goes out
In bacterium centrifuge tube；
3) microorganism total DNA in sample after extraction is pre-processed：(the m/ of 200 μ L 2% are added into the centrifuge tube equipped with filter membrane
V) cetyl trimethylammonium bromide (CTAB), 65 DEG C of water-bath 1h；Using 3S DNA Isolation extracts kits to micro- life
Thing DNA is extracted, and 200 μ L Solution SUS are added into pretreated sample, and sample is suspended；Add 400
μ L Solution LYS, 300mg quartzs, cover centrifugation lid, are acutely shaken 30 minutes at a high speed on turbula shaker；With
Desk centrifuge, 12000r/min, room temperature centrifugation 5min；Supernatant is transferred to completely in sterilizing 1.5mL centrifuge tubes, added
120 μ L Solution BID, cover centrifugation lid, mixing of turning upside down；Solution is fully transferred in 3S posts with 1mL TIP,
Pillar is put into 2mL centrifuge tubes, and centrifugation lid is not covered, and room temperature places 5min；Cover centrifugation lid, 12000r/min, room temperature from
Heart 5min, removes 3S posts, discards the waste liquid in centrifuge tube；Pillar is put back in same centrifuge tube, 600 μ L Wash are added
Solution, 10000r/min, room temperature centrifugation 2min, remove 3S posts, discard the waste liquid in centrifuge tube；Repeat, pillar is put back to
In same centrifuge tube, 600 μ L Wash Solution, 10000r/min, room temperature centrifugation 2min are added, 3S posts is removed, discards
Waste liquid in centrifuge tube；Pillar is put back in same centrifuge tube, 10000r/min, room temperature centrifugation 2min, to remove residual
Wash Solution；TE is preheating to 50-55 DEG C, pillar is put into new 1.5mL sterile centrifugation tubes, is added in pillar center
Enter 30 μ L TE, pillar and collecting pipe be placed in 50-55 DEG C of baking oven, place 2min, 12000r/min after taking-up, room temperature from
Heart 1min；Repeat, open centrifugation lid, add 30 μ L TE in same pillar center, be placed in 50-55 DEG C of baking oven, place
12000r/min after 2min, taking-up, room temperature centrifugation 1min；Liquid in collecting pipe is microorganism total DNA；
4) row agarose gel electrophoresis detection is entered to the thick DNA that carries：With each μ L of sample 5 applied sample amount, 1 μ L 6 ×
Loadding buffer electrophoresis detection pcr amplified fragments on 1% Ago-Gel, electrophoretic buffer buffers for 1 × TAE；
Voltage 140V, time 20min；Make standard molecular weight object of reference with DL500DNA Maker；Electrophoresis is dyed after terminating with EB lucifuges
40min, observes result with gel imaging system and takes pictures, and obtains thick pressure-raising and carries water microorganism STb gene agarose gel electrophoresis figure；
5) using the STb gene of extraction as template, performing PCR amplification is entered under the guiding of primer special：Primer special is 357F-
GC：
5′-CGCCCGCCGCGCGCGGCGGGCGGGGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3′(SEQ ID
) and 518R NO.1：5′-ATTACCGCGGCTGCTGG-3′(SEQ ID NO.2)；PCR reaction systems are：35μLddH2O, 5 μ L
The 10* reaction buffers of the magnesium ion containing 15mmol/L, 1 μ L sense primers 357F-GC, 1 μ L anti-sense primers 518R, 5 μ L deoxidation cores
Ribosomal ribonucleic acid, 1 μ LTaq enzymes, 2 μ L templates；PCR amplification programs are：Starting 95 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 1min, 65 DEG C are moved back
Fiery 1min (each cycle annealing temperature reduces by 0.5 DEG C), 72 DEG C of primer extend 30s, 20 circulations；94 DEG C are denatured 1min, 55 DEG C
Anneal 1min, 72 DEG C of primer extend 30s, 15 circulations；72 DEG C extend 8min eventually；4 DEG C of preservation pcr amplification products；
6) row agarose gel electrophoresis detection is entered to pcr amplification product：With each μ L of sample 5 applied sample amount, 1 μ L6 ×
Loadding buffer electrophoresis detection pcr amplified fragments on 1% Ago-Gel, electrophoretic buffer buffers for 1 × TAE；
Voltage 140V, time 20min；Applied sample amount is 5 μ L pcr amplification products, 1 μ 6 × Loadding of L buffer；Use DL500DNA
Maker makees standard molecular weight object of reference；Electrophoresis dyes 40min after terminating with EB lucifuges, and result is observed simultaneously with gel imaging system
Take pictures, obtain pcr amplification product agarose gel electrophoresis figure；
7) pcr amplification product is subjected to denaturing gradient gel electrophoresis：It is 35%~65%, acryloyl to make denaturing agent gradient
Amine intensity is 8% gel；Assembling is put into the electrophoresis tank containing 1 × TAE buffer solutions, is preheating to 60 DEG C；Add 30 μ L's per hole
PCR reaction products and 15 μ L6 × Loadding buffer；Connect electrophoresis power, the electrophoresis 16h under the conditions of 160V, 60 DEG C；Electricity
After swimming terminates, gel is carefully taken out, EB lucifuges dyeing 20min moves back dye 20min in clear water, then seen with gel imaging system
Examine result and take pictures, obtain denaturing gradient gel electrophoresis separation electrophoresis figure.Using QuantityOne softwares to denaturing gradient gel electricity
Swimming separation electrophoresis figure is digitized analysis, ballast water for ship microbial population diversity level is judged, by specific band
Gel extraction, cloning and sequencing simultaneously carry out BLAST sequence alignments, it can be deduced that the species of ballast water dominant microflora, so that it is determined that micro-
Biology community structure is constituted.
The present invention has the advantages that：
1st, for the particularity of ballast water for ship, double-filtration and freezing using 3 μm and 0.22 μm miillpore filters of use
It is fixed, attachment of the microorganism on filter membrane is strengthened, and using cetyl trimethylammonium bromide (CTAB) processing, CTAB is not only
Available for cell cracking, removal humic acid is additionally aided, obtained DNA purity is higher, and A260/A230 is up to 1.32 ± 0.02.
Method therefor is simple and convenient, is not required to purified can be used to follow-up PCR analyses and DGGE analyses.
2nd, for the particularity of ballast water for ship, method of the invention eliminates tediously long, cumbersome DNA washing steps, section
Save time and avoid DNA from losing, it is determined that be suitable for the method for ballast water microbial DNA extraction so that easy to operate.
3rd, the DNA fragmentation extracted using the inventive method can carry out 16SrDNA amplification and denaturing gradient gel electrophoresis, make
Analysis is digitized to denaturing gradient gel electrophoresis separation electrophoresis figure with Quantity One softwares, denaturing gradient gel is obtained
Electrophoretic band intensity map, bacterial quorum sensing diversity indices H ' and evenness index are calculated according to the versus grayscale of each band
J, and then judge ballast water for ship microbial population diversity level, and can be to the specific bands of denaturing gradient gel electrophoresis bands of a spectrum
Gel extraction, cloning and sequencing, carry out BLAST sequence alignments, so that it is determined that biological community structure is constituted.
Brief description of the drawings
By reading the detailed description made with reference to the following drawings to non-limiting example, further feature of the invention,
Objects and advantages will become more apparent upon：
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 indices (H ') and evenness index (J)；
Fig. 5 is denaturing gradient gel electrophoresis specific band sequence alignment result.
Embodiment
With reference to embodiment, the present invention is described in detail.Following examples will be helpful to those skilled in the art
The present invention is further understood, but the invention is not limited in any way.It should be pointed out that to one of ordinary skill in the art
For, without departing from the inventive concept of the premise, it can also make certain adjustments and improvements.These belong to the guarantor of the present invention
Protect scope.
Embodiment
Ship is chosen, is sampled, takes the ballast water of the ballast tank of ship different spatial, to the population structure of bacterium
The determination step of analysis and dominant microflora is as follows
1) ballast water microorganism total DNA is extracted and detected：
Ballast water is gathered using hydrophore in the different ballast tanks of ship, 2L samples, 4 are collected with the blue mouth vial of sterilizing
DEG C preserve；Pressure carries water sample, after 3 μm and 0.22 μm of miillpore filter (the prior sterilization treatment of filter membrane) double-filtration, discards 3 μm
It is ballast water microorganism on miillpore filter, 0.22 μm of miillpore filter；Using sterilizing operation surgical forceps, 0.22 μm of filter membrane is transferred to
In 1.5mL centrifuge tubes, it is immediately placed in -80 DEG C of refrigerators and freezes 4h；The centrifuge tube that microorganism filter membrane is placed with after freezing is taken out, is used
Sterilized surgical scissors and tweezers, and 0.22 μm of filter membrane is shredded and is transferred in 1.5mL sterile centrifugation tubes；To the centrifuge tube equipped with filter membrane
200 μ L 2% (m/v) cetyl trimethylammonium bromides (CTAB) of interior addition, 65 DEG C of water-bath 1h；Use 3S DNA
Isolation extracts kits are extracted to microbial DNA, and 200 μ L are added in the sample to after being handled through CTAB
Solution SUS, sample is suspended；400 μ L Solution LYS and 300mg quartzs are added, centrifugation lid is covered,
Acutely shaken 30 minutes at a high speed on turbula shaker；With desk centrifuge, 12000r/min, room temperature centrifugation 5min；By supernatant
Liquid is transferred in sterilizing 1.5mL centrifuge tubes completely, is added 120 μ LSolution BID, is covered centrifugation lid, turn upside down mixed
It is even；Solution is fully transferred in 3S posts with 1mL TIP, pillar is put into 2mL centrifuge tubes, centrifugation lid is not covered, room temperature is placed
5min；Centrifugation lid is covered, 12000r/min, room temperature centrifugation 5min remove 3S posts, discard the waste liquid in centrifuge tube；By pillar
Put back in same centrifuge tube, add 600 μ L Wash Solution, 10000r/min, room temperature centrifugation 2min, remove 3S posts,
Discard the waste liquid in centrifuge tube；Repeat, pillar put back in same centrifuge tube, add 600 μ L Wash Solution,
10000r/min, room temperature centrifugation 2min, removes 3S posts, discards the waste liquid in centrifuge tube；Pillar is put back in same centrifuge tube,
10000r/min, room temperature centrifugation 2min, to remove the Wash Solution of residual；TE is preheating to 50-55 DEG C, pillar is put
Enter in new 1.5mL sterile centrifugation tubes, add 30 μ L TE in pillar center, pillar and collecting pipe are placed on 50-55 DEG C of baking oven
In, place 2min, 12000r/min after taking-up, room temperature centrifugation 1min；Repeat, centrifugation lid is opened, in same pillar center
30 μ L TE are added, are placed in 50-55 DEG C of baking oven, 2min, 12000r/min after taking-up, room temperature centrifugation 1min is placed；Collecting pipe
In liquid be microorganism total DNA.
With each μ L of sample 5 applied sample amount, 1 μ 6 × Loadding of L buffer electrophoresis on 1% Ago-Gel is examined
Pcr amplified fragment is surveyed, electrophoretic buffer buffers for 1 × TAE；Voltage 140V, time 20min；Marked with DL500DNA Maker
Quasi-molecule amount object of reference；Electrophoresis dyes 40min after terminating with EB lucifuges, observes result with gel imaging system and takes pictures, obtains slightly
Pressure-raising carries water microorganism STb gene agarose gel electrophoresis figure (see Fig. 1).As shown in Figure 1, microorganism total DNA fragment is after dyeing
Light is presented, illustrates that DNA is extracted successfully.
2) PCR amplifications and product detection：
With
357F-GC：5′-CGCCCGCCGCGCGCGGCGGGCGGGGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3′
(SEQ ID NO.1) and 518R：5 '-ATTACCGCGGCTGCTGG-3 ' (SEQ IDNO.2) are primer；PCR reaction systems：35μ
LddH20,5uL 10* reaction buffers (magnesium ion containing 15mmol/L), 1 μ L sense primers 357F-GC, 1 μ L anti-sense primers
518R, 5 μ L DNAs, 1 μ LTaq enzymes, 2 μ L templates；PCR amplification programs are：95 DEG C of pre-degeneration 5min of starting, 94 DEG C of changes
Property 1min, 65 DEG C of annealing 1min (each cycle annealing temperature reduce by 0.5 DEG C), 72 DEG C of primer extend 30s, 20 circulations；94℃
It is denatured 1min, 55 DEG C of annealing 1min, 72 DEG C of primer extend 30s, 15 circulations；72 DEG C extend 8min eventually；4 DEG C preserve PCR amplifications
Product；With each μ L of sample 5 applied sample amount, 1 μ L6 × Loadding buffer electrophoresis detections on 1% Ago-Gel
Pcr amplified fragment, electrophoretic buffer buffers for 1 × TAE；Voltage 140V, electrophoresis time is 20min；Applied sample amount expands for 5 μ LPCR
Increase production thing, 1 μ Llording buffer；Make standard molecular weight object of reference with DL500DNA Maker；Electrophoresis is kept away after terminating with EB
Light dyes 40min, observes result with gel imaging system and takes pictures, obtains pcr amplification product agarose gel electrophoresis figure (see figure
2).As shown in Figure 2, PCR primer size shows that DNA is completed under primer special guiding between 200kb-300kb
16SrDNA amplification, available for follow-up denaturing gradient gel electrophoresis.
3) carry out denaturing gradient gel electrophoresis separation and result is represented：
It is the gel that 35%~65%, acrylamide intensity is 8% to make denaturing agent gradient；Assembling is put into containing 1 × TAE
In the electrophoresis tank of buffer solution, 60 DEG C are preheating to；30 μ L PCR reaction products and 15 μ L 6 × Loadding are added per hole
buffer；Connect electrophoresis power, voltage 160V, temperature 60 C, time 16h；After electrophoresis terminates, gel, EB lucifuges are carefully taken out
20min is dyed, dye 20min is moved back in clear water, then result is observed with gel imaging system and takes pictures, denaturing gradient gel electricity is obtained
Swimming separation electrophoresis figure (see Fig. 3 a), numeral is carried out using Quantity One softwares to denaturing gradient gel electrophoresis separation electrophoresis figure
Change analysis, obtain denatured gradient gel electrophoresis strip intensity schematic diagram (see Fig. 3 b).From Fig. 3 a and Fig. 3 b, 6 ballast waters
Sample is being extracted and specific amplification by DNA, is carried out after denaturing gradient gel electrophoresis, the formed objects of different base sequences
DNA fragmentation is separated, and 17 specific bands are obtained.
4) judge ballast water for ship microbial population diversity level and determine biological community structure composition：
Analysis is digitized to denaturing gradient gel electrophoresis separation electrophoresis figure using Quantity One softwares, according to every
The versus grayscale of individual band calculates bacterial quorum sensing diversity indices (H ') and evenness index (J) (see Fig. 4).Can by Fig. 4
Know, inverse proportion, the bacterial community diversity indices highest of sample 2, the uniformity is presented with evenness index in community diversity index
Index is minimum.After analysis microbial population diversity level, each specific band is cut off using sterilizing scalpel, respectively
It is positioned in 1.5mL sterile centrifugation tubes, 30 μ L distilled waters is added into each centrifuge tube, 5000r/min, room temperature centrifuges 1min,
Stand overnight, be sent to biotech firm and carry out cloning and sequencing, base sequence is subjected to BLAST sequence alignments, will obtain each special
The corresponding microbe species of band, so that it is determined that biological community structure is constituted.As shown in Figure 5, each specific band is represented
Microbe species all have found corresponding species name by BLAST sequence alignments.
The specific embodiment of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substantive content of the present invention.
Claims (6)
1. a kind of ballast water for ship microorganism total DNA extraction and the method for diversity analysis, it is characterised in that methods described includes
Following steps：
1) the different ballast tank ballast water samples of ship are collected；
2) ballast for cruising water sample is pre-processed；
3) microorganism total DNA in sample after extraction is pre-processed；
4) row agarose gel electrophoresis detection is entered to the thick DNA that carries, obtains thick pressure-raising and carry water microorganism STb gene agarose gel electrophoresis
Figure, judges whether Genome DNA extraction succeeds；
5) using the STb gene of extraction as template, performing PCR amplification is entered under the guiding of primer special；The sequence of the primer special is such as
Shown in SEQ ID NO.1 and SEQ ID NO.2；
6) enter row agarose gel electrophoresis detection to pcr amplification product, obtain pcr amplification product agarose gel electrophoresis figure, and sentence
Whether disconnected STb gene fragment expands success under the guiding of primer special；
7) pcr amplification product is subjected to denaturing gradient gel electrophoresis, obtains denaturing gradient gel electrophoresis separation electrophoresis figure, judge ship
The species of ballast water microbial population diversity level and ballast water dominant microflora, analyzes ballast water for ship microbial diversity；
Step 2) described in pretreatment include successively using 3 μm and 0.22 μm of miillpore filter carry out double-filtration extraction microorganism,
Cryofixation；
Step 3) described in extraction be using volume mass ratio be 2% cetyl trimethylammonium bromide processing sample after, make
Extracted with 3S DNAIsolation extracts kits.
2. different ballast tanks are ship different spaces described according to the method described in claim 1, it is characterised in that step 1)
The ballast tank of position.
3. PCR described according to the method described in claim 1, it is characterised in that step 5) expands used PCR reactants
It is to be：35μLddH2O, the 10* reaction buffers of 5 μ L magnesium ions containing 15mmol/L, upstreams of the 1 μ L as shown in SEQ ID NO.1
Primer, anti-sense primers of the 1 μ L as shown in SEQ ID NO.2,5 μ L DNAs, 1 μ L Taq enzymes, 2 μ L templates；PCR is expanded
Program is：Starting 95 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 1min, 65 DEG C of annealing 1min, 72 DEG C of primer extend 30s, 20 circulations；
94 DEG C of denaturation 1min, 55 DEG C of annealing 1min, 72 DEG C of primer extend 30s, 15 circulations；72 DEG C extend 8min eventually, obtain microorganism
Pcr amplification product.
4. the denaturing gradient gel electrophoresis described according to the method described in claim 1, it is characterised in that step 7) includes：
PCR reaction products are separated by electrophoresis in detection in Gene Mutation system, electrophoresis terminates poststaining and taken pictures.
5. also include according to the method described in claim 1, it is characterised in that step 7)：Use Quantity One softwares
Analysis is digitized to denaturing gradient gel electrophoresis separation electrophoresis figure, denatured gradient gel electrophoresis strip intensity map, root is obtained
Bacterial quorum sensing diversity indices H ' and evenness index J is calculated according to the versus grayscale of each band, and then judges ship pressure
Carry water microorganism population diversity degree.
6. also include according to the method described in claim 1, it is characterised in that step 7)：To the denaturing gradient gel of acquisition
The specific band gel extraction of electrophoretogram electrophoretic band, cloning and sequencing is separated by electrophoresis and BLAST sequence alignments are carried out, it can be deduced that
The species of ballast water dominant microflora, so that it is determined that biological community structure is constituted.
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| WO2013192567A1 (en) * | 2012-06-22 | 2013-12-27 | Wayne State University | Automated viability testing system |
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| DGGE用于两种不同类型生境的微生物群落结构的研究;张明月;《中国优秀硕士学位论文全文数据库 基础科学辑》;20130315;第1页、第18-24页第二章部分、第27页1.5部分 * |
| PCR 技术检测海运船舶压载水病原弧菌初探;霍颖异等;《浙江大学学报（理学版）》;20100531;第37卷(第3期);第331右栏第1段至第333页右栏最后一段 * |
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