CN102115740A - Method for extracting genomic DNA (Deoxyribose Nucleic Acid) from fish myxosporean - Google Patents
Method for extracting genomic DNA (Deoxyribose Nucleic Acid) from fish myxosporean Download PDFInfo
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Abstract
The invention discloses a method for extracting genomic DNA (Deoxyribose Nucleic Acid) from fish myxosporean. The method for separating myxosporean spores, which is provided by the invention, comprises the following steps of: (a) separating myxosporean spore sacs from a myxosporean-infected host; (b) flushing myxosporean spore sacs obtained in the step (a) with a 10 g/l PBS (Phosphate Buffer Solution) to obtain a myxosporean spore solution; (c) centrifuging the myxosporean spore solution obtained in the step (b) and collecting precipitates; and (d) adding the 10 g/l PBS and 10 g/l SDS (Sodium Dodecyl Sulfate) into the precipitates obtained in the step (c), mixing, centrifuging and collecting precipitates. As an experiment proves, a genome extracted by the method has high genomic DNA purity, does not contain any gene of the host, and has high extracting content. Meanwhile, the method can be applied to identification of myxosporean spores.
Description
Technical field
The present invention relates to a kind of extraction fish myxospore molitor genomic dna method.
Background technology
The myxospore worm refers to a big class protozoon of Myxosporea (Myxosporea), is most species, a most common sporozooid in seawater and freshwater fish parasite.The myxospore worm produces the spore of various shapes and different sizes, unlimitedly so be called the myxospore worm in growth course.
Both at home and abroad, myxosporidiasis popular quite general and serious do not have more effective control method so far yet.The common method of preventing and treating adopts unslaked lime and lime nitrogen to make disinfecting utensils more.In the China area of respectively breeding fish, popular multiple myxosporidiasis, for example silver carp iodine spore worm is popular in provinces such as Zhejiang Hangzhou area and Jiangsu, Hubei, Hunan, it invades neural system and the sensory organ of silver carp, makes disease fish body thin, and tail upwarps, spin up and down, promptly dead soon; The viscera tissue and the gill of cake type myxobolus Chang Daliang invasion and attack grass carp seedling mainly are enteron aisles, the production of serious harm grass carp seedling, and this interior syndrome state is the most serious with Guangdong; " the burying the bank disease " of being popular in Guangdong and Guangxi, its main pathogens are wild carp myxobolus.Often appear at the dace one pole worm on carp, the crucian skin, often make the fish body be covered with white point-like or block sporangiocyst, have a strong impact on growing of fish, even cause death.
Each myxospore worm has its parasitic site in the host.In freshwater fish, the gill, intestines and gall-bladder are the most normal stymied organs; Gall-bladder of marine fishes and bladder often parasitic a kind or multiple myxospore worm.Can form trophont when myxospore insect infection fish body, trophont continues to grow, and the division of karyon repeated multiple times forms several spore parents (sporonts).The continued growth of spore parent is grown, and its karyon also divides several times, forms 6～18 daughter nucleus, forms spore at last.The trophont continued growth, the spore number of formation also increases thereupon.Host tissue around trophont because of continuous irriate takes place to degenerate and change, produces a skim trophont is surrounded, and macroscopic white myxospore cysts occurs.
Also imperfectly understand the life history of myxospore worm.To whole life cycle center variation, infection method and have or not aspects such as vector, also exist more arguement.Spore that there are some researches show Myxobolus cerebralis, Ceratomyxa shasta can not the direct infection salmon, the worm that needs Tubificidae is as the intermediate host, but also exist between fish and the fish can direct infection myxospore worm kind, as Enteromyxum etc.
Separating myxospore worm spore and extract purified genomic dna from the fish host is basis to myxospore worm classification identification research; Simultaneously also be important research basis and the developing direction that the control sporozoite infects.
Summary of the invention
An object of the present invention is to provide a kind of extraction myxospore molitor genomic dna method.
Extraction myxospore molitor genomic dna method provided by the invention comprises the steps:
1) myxospore worm spore is pulverized, add the extracting solution mixing, obtain mixed solution;
2) in the mixed solution of step 1), add chloroform isoamyl alcohol mixed solution mixing, centrifugal, collect supernatant liquor;
3) to step 2) add chloroform isoamyl alcohol mixed solution mixing in the supernatant liquor that obtains, centrifugal, collect supernatant liquor;
4) in the supernatant liquor that step 3) obtains, add the Virahol mixing, leave standstill collecting precipitation;
5) precipitation that step 4) is obtained is carried out rinsing, drying, dissolving, RNA digestion successively, obtains digestion product;
6) digestion product that step 5) is obtained carries out extracting with phenol chloroform isoamyl alcohol mixed solution and chloroform isoamyl alcohol mixed solution successively, centrifugal, collection supernatant liquor;
7) add the 3MNaAc aqueous solution and dehydrated alcohol mixing in the supernatant liquor that step 6) obtains, leave standstill, centrifugal, collecting precipitation obtains DNA.
In the step 1), the proportioning of described extracting solution and described myxospore worm spore is (10ml-50ml): (1.5g-2g), the proportioning of described extracting solution and described myxospore worm spore is specially (10ml, 40ml or 50ml): (1.5g, 1.8g or 2g);
Step 2) in, the volume ratio of the mixed solution of described step 1) and described chloroform isoamyl alcohol mixed solution is 1: (1-2), the volume ratio of the mixed solution of described step 1) and described chloroform isoamyl alcohol mixed solution was specially 1: 1,1: 1.5 or 1: 2,
In the step 3), described step 2) the supernatant liquor and the volume ratio of described chloroform isoamyl alcohol mixed solution are 1: (1-2), described step 2) supernatant liquor and the volume ratio of described chloroform isoamyl alcohol mixed solution be specially 1: 1,1: 1.5 or 1: 2,
In the step 4), the supernatant liquor that described step 3) obtains and the volume ratio of described Virahol are (1-3): (1-2); The supernatant liquor that described step 3) obtains and the volume ratio of described Virahol were specially 1: 1,3: 2 or 3: 1; The temperature of described Virahol is-20 ℃;
In the step 5), described rinsing is carried out rinsing for using 75% (volumn concentration) aqueous ethanolic solution, 75% (volumn concentration) aqueous ethanolic solution and dehydrated alcohol successively;
Described dissolved method obtains solution A for dissolving described precipitation with the TE damping fluid,
The TE damping fluid is by solute and solvent composition, and described solute is Tris alkali, EDTA, and described solvent is a water, and the concentration of described Tris alkali in described TE damping fluid is 10mM, and the concentration of described EDTA in described TE damping fluid is 1mM, transfers pH value to 8.0 with hydrochloric acid.
The method of described digestion obtains digestion product for add RNaseA in described solution A, and the RNaseA add-on is the 20mg/l solution A;
Step 6) comprises the steps:
A) digestion product that step 5) is obtained and be the phenol chloroform isoamyl alcohol mixed solution mixing of its 1 times-3 times of volumes, centrifugal, collect supernatant A,
The described digestion product that step 5) is obtained and be that the phenol chloroform isoamyl alcohol mixed solution of its 1 times-3 times of volumes is specially the digestion product that step 5) is obtained and is the phenol chloroform isoamyl alcohol mixed solution of its 1 times, 2 times or 3 times volume;
B) supernatant A that steps A is obtained and be the chloroform isoamyl alcohol mixed solution mixing of its 1 times-3 times of volumes, centrifugal, collect supernatant liquor,
Described with steps A) supernatant A that obtains and be that the chloroform isoamyl alcohol mixed solution of its 1 times-3 times of volumes is specially steps A) supernatant A that obtains and be the chloroform isoamyl alcohol mixed solution of its 1 times, 2 times or 3 times volume;
In the step 7), the volume ratio of the supernatant liquor that described step 6) obtains, the described 3MNaAc aqueous solution and described dehydrated alcohol is: 1: (0.05-0.2): (2-3), be specially 1: (0.005,0.1 or 0.2): (2,2.5 or 3).
In the step 1), described extracting solution is by solute and solvent composition, described solute is NaCl, EDTA, SDS, Tris, described solvent is a water, the concentration of described Tris in described extracting solution is 0.1M-0.5M, the concentration of described NaCl in described extracting solution is 0.4M-0.6M, and the concentration of described EDTA in described extracting solution is 0.005M-0.015M; The concentration of described SDS in described extracting solution is 10g/l-20g/l;
The concentration of described Tri s in described extracting solution is specially 0.1M, 0.2M or 0.5M, the concentration of described NaCl in described extracting solution is specially 0.4M, 0.5M or 0.6M, and the concentration of described EDTA in described extracting solution is specially 0.005M, 0.01M or 0.015M; The concentration of described SDS in described extracting solution is specially 10g/l, 15g/l or 20g/l;
The pH value of described extracting solution is 7.5;
Step 2), in step 3) and the step 6), described chloroform isoamyl alcohol mixed solution is formed by chloroform and primary isoamyl alcohol, the volume ratio of described chloroform and primary isoamyl alcohol is 24: 1;
In the step 4), described time of repose is 20min-40min, and described time of repose is specially 20min, 30min or 40min;
The described temperature that leaves standstill is-20 ℃-4 ℃, and the described temperature that leaves standstill is specially-20 ℃, 0 ℃ or 4 ℃;
In the step 5), the temperature of described digestion is 35 ℃-38 ℃, and the temperature of described digestion is specially 35 ℃, 37 ℃ or 38 ℃, and the time of described digestion is 0.5h-1.5h, and described digestion time is specially 0.5h, 1h or 1.5h;
In the step 6), described centrifugal temperature is 0 ℃-4 ℃, described centrifugal temperature is specially 0 ℃, 1 ℃ or 4 ℃, described centrifugal centrifugal force is 10000g-13000g, described centrifugal centrifugal force is specially 10000g, 12000g or 13000g, the described centrifugal time is 5min-15min, and the described centrifugal time is specially 5min, 10min or 15min;
Described phenol chloroform isoamyl alcohol mixed solution is made up of phenol, chloroform and primary isoamyl alcohol, and the volume ratio of described phenol, chloroform and primary isoamyl alcohol is 25: 24: 1; The pH value of described phenol chloroform isoamyl alcohol mixed solution is 8.0;
In the step 7), described centrifugal temperature is 0 ℃-4 ℃, described centrifugal temperature is specially 0 ℃, 1 ℃ or 4 ℃, described centrifugal centrifugal force is 10000g-13000g, described centrifugal centrifugal force is specially 10000g, 12000g or 13000g, the described centrifugal time is 5min-15min, and the described centrifugal time is specially 5min, 10min or 15min.
In the step 1), described grinding is for to grind in liquid nitrogen;
Step 2) and in the step 3), described centrifugal temperature is 0 ℃-4 ℃, described centrifugal temperature all is specially 0 ℃, 1 ℃ or 4 ℃, described centrifugal centrifugal force is 10000g-13000g, described centrifugal centrifugal force all is specially 10000g, 12000g or 13000g, the described centrifugal time is 5min-15min, and the described centrifugal time all is specially 5min, 10min or 15min;
In the step 7), described time of repose is 20min-60min, and described time of repose is specially 20min, 30min or 60min; Described dwell temperature is-70 ℃-20 ℃, and described dwell temperature is specially-70 ℃, 0 ℃, 20 ℃.
Described myxospore worm is Thelohania, lucky pottery one pole worm (Thelohanellus kitauei).
Another object of the present invention provides a kind of method of separating myxospore worm spore.
The method of separation myxospore worm spore provided by the invention comprises the steps:
A) with PBS damping fluid flushing myxospore worm sporocyst, obtain myxospore worm spore solution;
B) the myxospore worm spore solution centrifugal, the collecting precipitation that step a) are obtained;
C) in the precipitation that step b) obtains, add the PBS damping fluid and the SDS aqueous solution, mix, centrifugal, collecting precipitation;
D) add the PBS damping fluid and the SDS aqueous solution in the precipitation that step c) obtains, mix, centrifugal, collecting precipitation is myxospore worm spore.
In the step b), described centrifugal centrifugal force is 5000g-8000g, is specially 5000g, 6000g or 8000g, and the described centrifugal time is 5min-15min, is specially 5min, 10min or 15min,
In step c) and the step d), the volume ratio of the described PBS damping fluid and the described SDS aqueous solution is: 10: 0.5.
Step a), c) and d) in, described PBS damping fluid is prepared as follows: with NaCl, Na
2HPO
4, NaH
2PO
4Mixing obtains the PBS damping fluid with water, and the concentration of NaCl in the PBS damping fluid is 8g/l, Na
2HPO
4Concentration at the PBS damping fluid is 2.2g/l, Na
2HPO
4Concentration at the PBS damping fluid is 0.4g/l; The pH value of described PBS damping fluid is 7.0;
Step c) and d) in, described blended temperature is 24 ℃-28 ℃, and described blended temperature all is specially 24 ℃, 25 ℃ or 28 ℃, and the described blended time is 5min-15min, the described blended time all is specially 5min, 10min or 15min, and described blended mode is puts upside down mixing; The concentration of the described SDS aqueous solution is 200g/l.
Step c) and d) in, described centrifugal centrifugal force is 6000g-10000g, described centrifugal centrifugal force all is specially 6000g, 8000g or 10000g, and the described centrifugal time is 5min-15min, and the described centrifugal time all is specially 5min, 10min or 15min.
Described myxospore worm is Thelohania, lucky pottery one pole worm (Thelohanellus kitauei).
The application of described method in the classification of myxospore worm is identified also is the scope of protection of the invention.
The genome that extracts with present method that experiment showed, of the present invention, through detecting, the purity height of genomic dna do not contain host's gene, and extracted amount is many, therefore can be used for identifying myxospore worm spore.
Description of drawings
Fig. 1 is a DM3 myxospore worm genome sample P CR amplification
Fig. 2 is a Henneguya spore shape feature
Fig. 3 belongs to the spore shape feature for myxobolus
Fig. 4 is a Thelohania spore shape feature
Fig. 5 is dyeing back Thelohania spore shape feature
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Embodiment 1, extraction myxospore molitor genomic dna
Method one:
One, the myxospore molitor genomic dna extracts
1, separates sporocyst from infecting myxospore worm host
Infection of myxospore worm and sporangial growth site have very high specificity, therefore only get the sporocyst of same position growth when sampling.
Anatomical isolation goes out 6 myxospore worm sporocysts from dead crucian (Cyprinus carpio) the enteron aisle position of infecting the myxospore worm, is numbered DM1-DM6 respectively.
2, from sporocyst, separate myxospore worm spore particle
PH value with sterilization is that 7.0 PBS carefully flushes out the spore particle from 1 sporocyst (DM1), and is careful as far as possible in the operation, prevents that the histocyte of spore cyst wall from sneaking in the spore particle, promptly obtains containing the sample of myxospore worm spore.
With sample transfer to 4 ℃ of preservations of 50ml centrifuge tube.
The PBS damping fluid is prepared as follows: with NaCl, Na
2HPO
4, NaH
2PO
4Mix with water, the concentration of NaCl in the PBS damping fluid is 8g/l, Na
2HPO
4Concentration at the PBS damping fluid is 2.2g/l, Na
2HPO
4Concentration at the PBS damping fluid is 0.4g/l.
3, remove the host cell that the myxospore worm is mixed with
The sample that contains myxospore worm spore that obtains with the centrifugal 10min of 6000 * g, is carefully outwelled supernatant liquor; Add 10ml sterilization pH value and be 7.0 PBS in precipitation, adding 0.5ml concentration again is the SDS aqueous solution of 200g/l, reaches 10g/l to its final concentration, puts upside down mixing at 25 ℃ during handling 10min, carefully outwells supernatant liquor with the centrifugal 10min of 8000 * g again; Add sterilization PBS in centrifuge tube, resuspended spore repeats SDS again and handles twice of operation; Constantly carry out microscopy in the operating process, observe spore precipitation situation and spore purity, the myxospore worm spore precipitation that obtains is placed-20 ℃ of preservations.
4, the method for using liquid nitrogen grinding is extracted genomic dna
Getting above-mentioned 2.0g myxospore worm spore is deposited in the liquid nitrogen and pulverizes rapidly; Add the 10mL extracting solution, the quick oscillation mixing; Add isopyknic chloroform isoamyl alcohol mixed solution (volume ratio of chloroform isoamyl alcohol is 24: 1) mixing, with 4 ℃, 12,000g, centrifugal 10min.Supernatant liquor is used isopyknic chloroform isoamyl alcohol mixed solution (volume ratio of chloroform isoamyl alcohol is 24: 1) again, and extracting once.With 4 ℃, 12,000g, centrifugal 10min.Get supernatant liquor, add the isopropanol precipitating of-20 ℃ of precoolings of 2/3 times of volume, mixing leaves standstill about 30min.Choose flocks with the capillary glass rod, with 75% ethanol repeatedly rinsing for several times use the dehydrated alcohol rinsing again 1 time, dry up, be resuspended among the 500ulTE, add 1 μ lRNaseA (10mg/mL), handle 1h down for 37 ℃, the digestion product that obtains and be its 1 times of volume phenol chloroform isoamyl alcohol mixed solution (pH8.0, the volume ratio of phenol chloroform, primary isoamyl alcohol is 25: 24: 1) mix, with 4 ℃, 12,000g, centrifugal 10min.Get supernatant A, with supernatant A and be its 1 times of volume the chloroform isoamyl alcohol mixed solution (volume ratio of chloroform isoamyl alcohol is 24: 1 mixings, with 4 ℃, 12,000g, centrifugal 10min.Get supernatant liquor, add the 3MNaAc aqueous solution of 1/10 times of volume, the dehydrated alcohol of 2.5 times of volumes ,-70 ℃ staticly settle 30min.Precipitation is with 75% ethanol rinsing, and is air-dry, is dissolved among the 200ulTE, obtains the DNA sample, called after DM1 ,-20 ℃ of preservations.
DNA extraction liquid is by solute and solvent composition, described solute is NaCl, EDTA, SDS, Tris, and described solvent is a water, and the concentration of described Tris in described extracting solution is 0.2M, the concentration of described NaCl in described extracting solution is 0.5M, and the concentration of described EDTA in described extracting solution is 0.01M; The concentration of described SDS in described extracting solution is 15g/l; The pH value of DNA extraction liquid is 7.5.
The TE damping fluid is by solute and solvent composition, and described solute is Tris alkali, EDTA, and described solvent is a water, and the concentration of described Tris alkali in described Tris alkali is 10mM, and the concentration of described EDTA in described TE damping fluid is 1mM, transfers pH value to 8.0 with hydrochloric acid.
Method two:
One, the myxospore molitor genomic dna extracts
1, separates sporocyst from infecting myxospore worm host
Infection of myxospore worm and sporangial growth site have very high specificity, therefore only get the sporocyst of same position growth when sampling.
From dead crucian (Cyprinus carpio) the enteron aisle position of infecting the myxospore worm, isolate myxospore worm sporocyst.
2, from sporocyst, separate myxospore worm spore particle
PH value with sterilization is that 7.0 PBS carefully flushes out the spore particle from 1 sporocyst, and is careful as far as possible in the operation, prevents that the histocyte of spore cyst wall from sneaking in the spore particle, promptly obtains containing the sample of myxospore worm spore.
With sample transfer to 4 ℃ of preservations of 50ml centrifuge tube.
The PBS damping fluid is prepared as follows: with NaCl, Na
2HPO
4, NaH
2PO
4Mix with water, the concentration of NaCl in the PBS damping fluid is 8g/l, Na
2HPO
4Concentration at the PBS damping fluid is 2.2g/l, Na
2HPO
4Concentration at the PBS damping fluid is 0.4g/l.
3, remove the host cell that the myxospore worm is mixed with
The sample that contains myxospore worm spore that obtains with the centrifugal 5min of 5000 * g, is carefully outwelled supernatant liquor; Add 10ml sterilization PBS in precipitation, adding 0.5ml concentration again is the SDS aqueous solution of 200g/l, reaches 10g/L to its final concentration, puts upside down mixing at 24 ℃ during handling 5min, carefully outwells supernatant liquor with the centrifugal 5min of 6000 * g again; Add sterilization PBS in centrifuge tube, resuspended spore repeats SDS again and handles twice of operation; Constantly carry out microscopy in the operating process, observe spore precipitation situation and spore purity, the myxospore worm spore precipitation that obtains is placed-20 ℃ of preservations.
4, the method for using liquid nitrogen grinding is extracted genomic dna
Getting above-mentioned 1.5g myxospore worm spore is deposited in the liquid nitrogen and pulverizes rapidly; Add the 40mL extracting solution, the quick oscillation mixing; Add 1.5 times of volume chloroform isoamyl alcohol mixed solutions (volume ratio of chloroform isoamyl alcohol is 24: 1) mixing, with 0 ℃, 10,000g, centrifugal 5min.Supernatant liquor is used the chloroform isoamyl alcohol mixed solution (volume ratio of chloroform isoamyl alcohol is 24: 1) of 1.5 times of volumes again, and extracting once.With 0 ℃, 10,000g, centrifugal 5min.Get supernatant liquor, add the isopropanol precipitating of isopyknic-20 ℃ of precoolings, mixing leaves standstill about 20min.Choose flocks with the capillary glass rod, with 75% ethanol repeatedly rinsing for several times use the dehydrated alcohol rinsing again 1 time, dry up, be resuspended among the 500ulTE, add 1 μ lRNaseA (10mg/mL), handle 0.5h down for 35 ℃.The digestion product that obtains and be that its 2 times of volume phenol chloroform isoamyl alcohol mixed solutions (pH8.0, the volume ratio of phenol chloroform, primary isoamyl alcohol is 25: 24: 1) mix, with 0 ℃, 10,000g, centrifugal 5min.Get supernatant A, with supernatant A and be its 2 times of volumes the chloroform isoamyl alcohol mixed solution (volume ratio of chloroform isoamyl alcohol is 24: 1 mixings, with 0 ℃, 10,000g, centrifugal 5min.Get supernatant liquor, add the 3MNaAc aqueous solution of 0.005 times of volume, the dehydrated alcohol of 2 times of volumes, 0 ℃ staticly settles 20min.Precipitation is with 75% ethanol rinsing, and is air-dry, is dissolved among the 200ulTE, obtains the DNA sample, called after DM1 ,-20 ℃ of preservations.
DNA extraction liquid is by solute and solvent composition, described solute is NaCl, EDTA, SDS, Tris, and described solvent is a water, and the concentration of described Tris in described extracting solution is 0.1M, the concentration of described NaCl in described extracting solution is 0.4M, and the concentration of described EDTA in described extracting solution is 0.005M; The concentration of described SDS in described extracting solution is 10g/l; The pH value of DNA extraction liquid is 7.5.
The TE damping fluid is by solute and solvent composition, and described solute is Tris alkali, EDTA, and described solvent is a water, and the concentration of described Tris alkali in described Tris alkali is 10mM, and the concentration of described EDTA in described TE damping fluid is 1mM, transfers pH value to 8.0 with hydrochloric acid.
Method three:
One, the myxospore molitor genomic dna extracts
1, separates sporocyst from infecting myxospore worm host
Infection of myxospore worm and sporangial growth site have very high specificity, therefore only get the sporocyst of same position growth when sampling.
From dead crucian (Cyprinus carpio) the enteron aisle position of infecting the myxospore worm, isolate myxospore worm sporocyst.
2, from sporocyst, separate myxospore worm spore particle
PH value with sterilization is that 7.0 PBS carefully flushes out the spore particle from 1 sporocyst, and is careful as far as possible in the operation, prevents that the histocyte of spore cyst wall from sneaking in the spore particle, promptly obtains containing the sample of myxospore worm spore.
With sample transfer to 4 ℃ of preservations of 50ml centrifuge tube.
The PBS damping fluid is prepared as follows: with NaCl, Na
2HPO
4, NaH
2PO
4Mix with water, the concentration of NaCl in the PBS damping fluid is 8g/l, Na
2HPO
4Concentration at the PBS damping fluid is 2.2g/l, Na
2HPO
4Concentration at the PBS damping fluid is 0.4g/l.
3, remove the host cell that the myxospore worm is mixed with
The sample that contains myxospore worm spore that obtains with the centrifugal 15min of 8000 * g, is carefully outwelled supernatant liquor; Add 10ml sterilization PBS in precipitation, adding 0.5ml concentration again is the SDS aqueous solution of 200g/l, reaches 10g/l to its final concentration, puts upside down mixing at 28 ℃ during handling 15min, carefully outwells supernatant liquor with the centrifugal 15min of 10000 * g again; Add sterilization PBS in centrifuge tube, resuspended spore repeats SDS again and handles twice of operation; Constantly carry out microscopy in the operating process, observe spore precipitation situation and spore purity, the myxospore worm spore precipitation that obtains is placed-20 ℃ of preservations.
4, the method for using liquid nitrogen grinding is extracted genomic dna
Getting above-mentioned 1.8g myxospore worm spore is deposited in the liquid nitrogen and pulverizes rapidly; Add the 50mL extracting solution, the quick oscillation mixing; Chloroform isoamyl alcohol mixed solution (volume ratio of chloroform isoamyl alcohol is 24: the 1) mixing that adds 2 times of volumes, with 1 ℃, 13,000g, centrifugal 15min.Supernatant liquor is used the chloroform isoamyl alcohol mixed solution (volume ratio of chloroform isoamyl alcohol is 24: 1) of 2 times of volumes again, and extracting once.With 1 ℃, 13,000g, centrifugal 15min.Get supernatant liquor, add the isopropanol precipitating of-20 ℃ of precoolings of 1/3 times of volume, mixing leaves standstill about 40min.Choose flocks with the capillary glass rod, with 75% ethanol repeatedly rinsing for several times use the dehydrated alcohol rinsing again 1 time, dry up, be resuspended among the 500ulTE, add 1 μ lRNaseA (10mg/mL), handle 1.5h down for 38 ℃, the digestion product that obtains and be its 3 times of volume phenol chloroform isoamyl alcohol mixed solution (pH8.0, the volume ratio of phenol chloroform, primary isoamyl alcohol is 25: 24: 1) mix, with 1 ℃, 13,000g, centrifugal 15min.Get supernatant A, with supernatant A and be its 3 times of volumes the chloroform isoamyl alcohol mixed solution (volume ratio of chloroform isoamyl alcohol is 24: 1 mixings, with 1 ℃, 13,000g, centrifugal 15min.Get supernatant liquor, add the 3MNaAc aqueous solution of 0.2 times of volume, the dehydrated alcohol of 3 times of volumes, 20 ℃ staticly settle 60min.Precipitation is with 75% ethanol rinsing, and is air-dry, is dissolved among the 200ulTE, obtains the DNA sample, called after DM1 ,-20 ℃ of preservations.
DNA extraction liquid is by solute and solvent composition, described solute is NaCl, EDTA, SDS, Tris, and described solvent is a water, and the concentration of described Tris in described extracting solution is 0.5M, the concentration of described NaCl in described extracting solution is 0.6M, and the concentration of described EDTA in described extracting solution is 0.015M; The concentration of described SDS in described extracting solution is 20g/l; Regulating pH value with hydrochloric acid is 7.5;
The TE damping fluid is by solute and solvent composition, and described solute is Tris alkali, EDTA, and described solvent is a water, and the concentration of described Tris alkali in described Tris alkali is 10mM, and the concentration of described EDTA in described TE damping fluid is 1mM, transfers pH value to 8.0 with hydrochloric acid.
Detect the DNA that aforesaid method one extracts DM2, DM3, DM4, DM5 and these 5 sporangiocyst miospores of DM6.
Two, detected result
1, detects the content of DNA
Extract purity and the content of the DNA of DM1, DM2, DM3, DM4, DM5 and DM6 with ultraviolet spectrometry measuring method one.When containing protein, phenol or other small molecules pollutents in the DNA sample, can influence the accurate mensuration of DNA absorbancy.0D260,0D280 and 0D230 by detecting same sample calculate the purity that its ratio comes working sample.
The 0D260/0D280 that the result extracts the DNA of DM1, DM2, DM3, DM4, DM5 and DM6 for method one is 1.8, and (＞1.9, showing has RNA to pollute;＜1.6, show protein is arranged, pollution such as phenol);
Extract the DNA sample of DM1, DM2, DM3, DM4, DM5 and DM6 for method one, can calculate content by the absorbance value of measuring 260nm and (sample be put into the correlation values that instrument can directly be read concentration and purity, the Thermo Scientific NanoDrop Nanodrop ND-2000 of company ultramicron nucleic acid-protein determinator), the experiment triplicate, results averaged.
The result is as shown in table 1:
Table 1 obtains the DNA sample size at present:
2, use the purity of the method validation genomic dna of PCR
Designed two pairs of primers:
Rag1:DNA recombination activation gene (Recombination activating genes RAG, genbank number is AY78704) is the key gene of vertebrates specific immune response, also is one of marker gene of vertebrates evolutionary analysis.Second intron of different fish Rag1 genes also is a high conservative, and about total length 718bp, the design primer is as follows:
Rag1-R：5’-GACACTATGGAGAAAGGGAGGTGGAGTT-3’
Rag1-F：5’-GGGAAGCAGAGGTCGCAGTTGGAGG-3’
S7: ribosomal protein gene (genbank number is AY103168) all shows higher similarity in relation vertebrates monoid far away; And fish S7 gene obviously is used as the valid model of host gene intron encoding function research than batrachians and human compact construction, total length 900bp, and the design primer is as follows:
S7-R：5’-TGAACTCGTCTGGCTTTTCGCC-3’
S7-F：5’-GCCTCTTCCTTGGCCGTC-3’
The DNA of the myxospore worm DM3 that extracts with aforesaid method one is a template, uses the primer (Rag1-R and Rag1-F) of Rag1 and the primer (S7-R and S7-F) of S7 respectively, carries out pcr amplification, is contrast with genomic dna of bright and beautiful carp and the genomic dna of carp.
The result as shown in Figure 1, wherein, F is bright and beautiful carp genomic dna pcr amplification result; L is carp genomic dna pcr amplification result; Arrow indicating bit 700bp.From figure, see gene, illustrate that the myxospore worm DM3DNA that extracts is purer, do not have the gene (Rag1 and S7) of infection host.Show at present, in F and L, all can detect the gene of Rag1 and S7, and in the myxospore worm DNA that extracts, do not have Rag1 and S7 extracting method can reach to detect and not have host gene with PCR.
Adopt the DNA that the detection method one that uses the same method is extracted DM1, DM2, DM4, DM5 and DM6, result and DM3 do not have significant difference.
3, order-checking sample classification
1), Molecular Identification
To the Molecular Identification of order-checking sample classification status by to 18SrRNA sequential analysis finish.About 18SrRNA sequence total length 2K.The 18SrRNA sequence of more different sporocysts source sample is if 5K left and right sides sequence can be in full accord then be thought the one pole worm of same kind.
The myxospore worm DNA (DM1) that extracts with aforesaid method one is a template, 18SrRNA primer 5 '-CTGCGGACGGCTCAGTAAATCAGT-3 '; 5 '-CCAGGACATCTTAGGGCATCACAGA-3 ' increases as primer, obtains 18SrRNA, and the 18SrRNA that obtains is checked order, and the result is that the dna sequence dna of 18SrRNA is shown in sequence in the sequence table 1.
18SrRNA is compared on genbank, find that the 18SrRNA sequence of this myxospore worm and registered lucky pottery one pole worm (Thelohanellus kitauei) (GQ396677) have the highest consistence, be 99%, can illustrate that this myxospore worm is a lucky pottery one pole worm (Thelohanellus kitauei).
The myxospore worm of the DM2-DM6 that employing aforesaid method detection method one obtains, result and DM1 do not have significant difference.
2), host type and parasitic site analysis
Observe parasitic host and the position of (DM1-DM6) myxospore worm, the result is for the host type of myxospore worm is crucian (Cyprinus carpio), the parasitic site is enteron aisle.
3), spore shape signature analysis
With the spore shape feature of microscopic examination myxospore worm, (Henneguya is documented in the parasitic myxospore worm of Sichuan Province fish: IV cercospora worm and one pole worm with many alunites thatch cercospora worm (Henneguya doneciSchulman, 1962); Chongqing Normal College's journal (natural science edition); Ma Chenglun, Dong new people, Wang Cisheng; 16 volumes, 1 phase, 12-15; 1999, the public can obtain from Institute of Feeds,China Academy of Agriculture Sciences.) and myxobolus rotundus nemeczek (Myxobolus rotundusNemeczek, 1911) (myxobolus belongs to, and is documented in the ultrastructural studies that the myxobolus rotundus nemeczek spore takes place; The hydrobiont journal; Wu Yingsong, Wang Jianguo; 25 volumes, 1 phase; 61-69; 2001, the public can obtain from Institute of Feeds,China Academy of Agriculture Sciences.) for contrasting, the results are shown in Figure 2-5, wherein Fig. 2 is Henneguya (many alunites thatch cercospora worm Henneguyadoneci Schulman, 1962), Fig. 3 belongs to (myxobolus rotundus nemeczek (Myxobolus rotundusNemeczek for myxobolus, 1911), Fig. 4 is the figure (amplifying 100 times) that method one obtains the myxospore worm of DM1, and Fig. 5 obtains the figure (amplifying 1000 times) of the myxospore worm of DM1 for dyeing back method one;
As can be seen from the figure:
Myxobolus belongs to: spore is avette or oval, and is flat, and front end has 2 utmost point capsules, waits greatly or do not wait, and 1 iodinophilous vacuole is arranged in the matter in the spore.
Henneguya: spore circle, avette or fusiform, 2 in utmost point capsule extends two urogomphus by the shell sheet to the rear portion.
Myxospore worm DM1: spore is generally bigger, resembles a pear in shape or scrotiform, and the shell face differs very few with seam face width degree, so several subcirculars in transverse section.Have only 1 utmost point capsule, generally bigger, often be 1/2 of spore length, polar filament often is the bandlet of distortion, and the light-emitting particles of oil droplet shape is still arranged in the matter in the spore, and 1 iodinophilous vacuole is arranged, and these are the notable feature of Thelohania myxobolus section.Therefore determine that the DM1 myxospore worm that method one obtains is a Thelohania.
The DM2-DM6 myxospore worm of adopting aforesaid method detection method one to obtain, result and DM1 do not have significant difference.
Myxobolus section comprises that mainly tail spore Eimeria, myxobolus belong to and Thelohania, wherein myxobolus belongs to sporozoite and studies the most extensive abroad, it mainly infects rainbow trout, and lucky pottery one pole worm is typical Enterozoa, promptly have only the packing of lucky pottery one pole worm to be grown in the enteron aisle position, its field planting position is the loose connective tissue of intestinal mucosa lower floor, can directly determine by host and tissue specificity, and not need careful morphological specificity to identify.Report shows that the spore that does not belong to the source together of myxobolus section has marked difference in profile at present.
Passable by above experiment, kind by 4 proof samples: host type, parasitic site, spore shape feature, 18S sequence similarity are analyzed, and therefore, the Myxostoma that DM1-DM6 is described is in myxobolus section, Thelohania, lucky pottery one pole worm (Thelohanellus kitauei).
Adopt the myxospore worm that use the same method detection method two and method three obtain, result and method one no significant difference.
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