CN103224982A - Detection and expelling method for coilia ectenes heteromazocraes lingmueni based on molecular marker technology - Google Patents
Detection and expelling method for coilia ectenes heteromazocraes lingmueni based on molecular marker technology Download PDFInfo
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Abstract
The invention discloses a detection and expelling method for coilia ectenes heteromazocraes lingmueni based on a molecular marker technology. Species identification for the heteromazocraes parasitizing in the coilia ectenes is carried out by using a method combining a common optical microscope and molecular genetics, and further a corresponding expelling method is adopted. Firstly, preliminary determination is carried out according to microscopic examination; then, 5'-termianl partial sequence of 28S rRNA genes is amplified by molecular genetics so as to determine 28s sequence of the heteromazocraes lingmueni; and a parasite can be accurately determined as the heteromazocraes lingmueni by comparison analysis. After the determination is finished, mebendazole with a concentration of 1 mg/L is used to spray the whole pool; polypides can be shed and dead in 72 hours; and at the same time, 50%-60% of water is exchanged in time to discharge shed parasite eggs so as to prevent later recurrence. The method provided by the invention is not restricted by seasons and environment, and can detect the heteromazocraes lingmueni in various tissues and growth periods of the parasites in terms of DNA. The detection technology is simple in method and low in cost; and the expelling method is simple and easy to master.
Description
Technical field
The present invention relates to rapid detection and the expelling method thereof of the different hook pincers of Zhong Linshi worm, specifically, be based on detection and the expelling method of the different hook pincers of the cutter long-tailed anchovy Lin Shi worm of molecular marking technique, belong to the fish farming technical field.
Background technology
The different hook pincers of Lin Shi worm (Heteromazocraes lingmueni) is a kind of advantage parasite that colonizes on the cutter long-tailed anchovy gill filament of the Changjiang river, and it utilizes opisthaptor to open and inhales gill small pieces on the pincers gill filament, destroy the epithelial cell of the gill filament, cause the invasion of other pathogenic bacterias, cause inflammation, cause the gill rot illness.The different hook pincers of the parasitic Lin Shi of cutter long-tailed anchovy worm is more, and infection rate is up to 74%, and lethality rate can reach 100%.This parasitic accurate evaluation and the expeling that takes appropriate measures become the key link in the breed of cutter long-tailed anchovy.
All be that the method that adopts form to differentiate is identified in the past, and because the finiteness of parasite morphological specificity, and the difference that causes of fixing means, the larva that perhaps has only worm's ovum or morphological specificity to distinguish, may there be certain fault rate from the morphological specificity evaluation, finally causes missing best occasion for the treatment.
Summary of the invention
The objective of the invention is to overcome above-mentioned weak point, thereby detection and the expelling method of a kind of different hook pincers of cutter long-tailed anchovy Lin Shi worm based on molecular marking technique are provided, on the basis of accurately identifying, provide a kind of method of treatment, ensure and culture surviving rate more than 95%.
The embodiment of the invention is achieved in that detection and the expelling method of a kind of different hook pincers of cutter long-tailed anchovy Lin Shi worm based on molecular marking technique, and this method is utilized the ordinary optical microscope method that combines with molecular genetic, the different hook pincers worm of parasitic cutter long-tailed anchovy is carried out kind identify;
At first, microscopy shows: worm's ovum stage ovum two ends have long polar filament;
The polypide stage is contained the opisthaptor of four right titles, the tool short handle, and wherein preceding 2 open chitins of a side are inhaled pincers obviously greater than the suction pincers of all the other 6 sealings, and the polypide afterbody has two pairs of sharp-pointed crotches;
Molecular genetic amplification 28S rRNA gene 5 ' end parts sequence is measured the different hook pincers of Lin Shi worm 28s sequence then, through compare of analysis, can identify accurately that this parasite is the different hook pincers of a Lin Shi worm;
After evaluation finished, with the Vermox full pool spilling head of 1mg/L concentration, after 72 hours, polypide was tear-away, dead; Simultaneously, also in time change water 50%-60%, discharge the worm's ovum that comes off, recurrence after preventing.
Further, the concrete steps of this detection method are:
Collected specimens host's cutter long-tailed anchovy, 30 cutter long-tailed anchovys of random inspection, average body is long 12 centimetres, and body weight 5 grams are examined under a microscope gill portion parasite and are added up its quantity, and part polypide flushing with clean water is totally preserved to 95% alcohol;
Carry out morphological structure and observe, visual inspection will have parasite cutter long-tailed anchovy and take out the fish gill and put on the slide glass, and anatomical lens is observed down and will be had the gill filament of polypide to choose, and put into to put microscopically after the centrifuge tube clear water cleans for several times again and observe and take pictures;
Carry out Molecular Identification, at first prepare template DNA, carry out pcr amplification and amplified fragments clone, after the dna sequence dna splicing, login NCBI carries out the blast compare of analysis, and downloads the higher sequence of similarity; Adjacent method has made up 28S rDNA molecular evolution tree in the ClustalX multisequencing comparison back employing MEGA software.
Further, collected specimens host's cutter long-tailed anchovy, 30 cutter long-tailed anchovys of random inspection, average body is long 12 centimetres, and body weight 5 grams are examined under a microscope gill portion parasite and are added up its quantity, and part polypide flushing with clean water is totally preserved to 95% alcohol.
Further, visual inspection will have parasite cutter long-tailed anchovy and take out the fish gill and put on the slide glass, and anatomical lens is observed down and will be had the gill filament of polypide to choose, and put into to put the Olympus0.5X microscopically after the centrifuge tube clear water cleans for several times again and observe and take pictures.
Further, the preparation method of template DNA is:
From-20 ℃ 95% alcohol preserve liquid take out body, place the aseptic centrifuge tube of 2ml, sterile water wash is also placed 4 ℃ of refrigerators displacements and is spent the night;
Cleaning and QIAGEN QIAamp DNA Mini Kit test kit carry out the extraction of DNA once more, and last constant volume is put in the 25ul sterilized water-20 ℃ of preservations.
Further, pcr amplification and amplified fragments cloning process are:
With
CD1：(GTAGGTGAACCTGCGGAAGGATCATT)
And CD2-(TTAGTTTCTTTTCCTCCGCT)
Be the different hook pincers of primer amplification Lin Shi worm 28S aligning primer; With 4ul parasite DNA is template, and NC5 and NC2 are primer amplification ITS fragment; Reaction system is 50 μ L, and reaction conditions is 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 60s, circulate 35 times; Be template with 4ul parasite DNA equally, CD1 and CD2 are primer amplification 28S fragment; Reaction system is 50 μ L, and reaction conditions is 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 60s, circulate 35 times; Amplified production is through 1% concentration agarose gel electrophoresis check and analysis;
To remain the PCR product and run back and receive glue and reclaim the test kit purifying with dna gel, be masterplate with the purified product, and the 28S fragment is increased in a large number, links to each other with the pMD18-T carrier to carry out TA and clone, and the sub-upgrading grain of screening positive clone checks order.
The present invention compares with existing other similar techniques and has the following advantages:
1, be not subjected to season, environmental restraint, directly the form with DNA all can detect at parasitic each tissue and developmental stage.
2, the detection technique method is easy, and is with low cost; The easy easy grasp of expelling method.
Description of drawings
Fig. 1 is the electrophoresis result of the different hook pincers of the Lin Shi worm PCR product that provides of the embodiment of the invention; M1: λ DNA/HindIII Marker; M2:DL1000; 1: the different hook pincers of Lin Shi Lin Shi worm;
Fig. 2 is that Lin Shi different hook pincers worm and the Genbank that the embodiment of the invention provides downloads worm kind 28S rDNA sequence NJ tree, digitized representation Bootstrap value among the figure;
Fig. 3 is the opticmicroscope figure of the different hook pincers of the Lin Shi worm that provides of the embodiment of the invention;
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer,, the present invention is further elaborated below in conjunction with accompanying drawing 1,2,3 and embodiment.Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in qualification the present invention.
The present invention is with opticmicroscope and the molecular genetic method that combines, and the different hook pincers worm of Yangtze valley cutter long-tailed anchovy parasitism carried out kind is identified and morphology is studied.Form result shows: polypide contains the opisthaptor of four right titles, the tool short handle, and wherein preceding 2 open chitins of a side are inhaled pincers obviously greater than the suction pincers of all the other 6 sealings, and the polypide afterbody has two pairs of sharp-pointed crotches, and the ovum two ends have long polar filament.Through identification and analysis, this parasite is the different hook pincers of a Lin Shi worm.Molecular genetic amplification 28S rRNA gene 5 ' end parts sequence, and adjacent method has made up 28S rDNA molecular evolution tree in the employing MEGA software.The result shows: Lin Shi different hook pincers worm is with the different hook pincers of six sour jujubes worm is in same branch and similarity is 100%, thus the further clear and definite classification position of the different hook pincers of Lin Shi worm, and measured the different hook pincers of Lin Shi worm 28s sequence first.
1. material and method
1.1 sample collection
Sample host cutter long-tailed anchovy catches from Jiangsu, Yangtze valley Jingjiang section, acquisition time in September, 2012.30 cutter long-tailed anchovys of random inspection, average body is long, and body weight is examined under a microscope gill portion parasite and is added up its quantity, and part polypide flushing with clean water is totally preserved to 95% alcohol.
1.2 morphological structure is observed
Visual inspection will have parasite cutter long-tailed anchovy and take out the fish gill and put on the slide glass, and anatomical lens is observed down and will be had the gill filament of polypide to choose, and put into to put the olympus0.5X microscopically after the centrifuge tube clear water cleans for several times again and observe and take pictures.
1.3 Molecular Identification
1.3.1 the preparation of template DNA
Preserve from-20 ℃ 95% alcohol and to take out 20 left and right sides polypides the liquid, place the aseptic centrifuge tube of 2ml, sterile water wash is also placed 4 ℃ of refrigerator displacements and is spent the night, cleaning also once more, QIAGEN QIAamp DNAMini Kit test kit carries out the extraction of DNA, last constant volume is put in the 25ul sterilized water-20 ℃ of preservations.1.3.2pcr amplification and amplified fragments clone
With CD1:(GTAGGTGAACCTGCGGAAGGATCATT) and CD2-(TTAGTTTCTTTTCCTCCGCT) be the different hook pincers of primer amplification Lin Shi worm 28S aligning primer, synthetic by Shanghai Jie Rui company limited.With 4ul parasite DNA is template, and NC5 and NC2 are primer amplification ITS fragment.Reaction system is 50 μ L, and reaction conditions is 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 60s, circulate 35 times.Be template with 4ul parasite DNA equally, CD1 and CD2 are primer amplification 28S fragment.Reaction system is 50 μ L, and reaction conditions is 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 60s, circulate 35 times.Amplified production is through 1% concentration agarose gel electrophoresis check and analysis.
To remain the PCR product runs back and receives glue and reclaim the test kit purifying with dna gel, with the purified product is masterplate, 28S and ITS sequence fragment are increased in a large number, directly order-checking of a part behind the PCR product purification, a part is connected to carries out the TA clone on the pMD18-T carrier, the sub-upgrading grain of screening positive clone is served the Hai Jierui order-checking.
1.3.3 sequential analysis
After the dna sequence dna splicing, login NCBI carries out the blast compare of analysis, and downloads the higher sequence of similarity.Adjacent method has made up 28S rDNA molecular evolution tree in the Clustal X multisequencing comparison back employing MEGA software,
2. result
2.1 form is described
Catch in the wild cutter long-tailed anchovy in the Yangtze valley, find its gill portion parasitic worm, identify that through morphologic observation this parasite is the different hook pincers of a Lin Shi worm (Heteromazocraes lingmueni).Polypide is bigger, closely spins taper, naked eyes as seen, the body surface stratum corneum is thicker and fine and closely woven ring grain arranged.The different hook pincers of Lin Shi worm telianthus has 7 testis, and the health both sides have flourishing caecum, and the yolk gland prosperity is covered with the intestines both sides.The blunt circle of polypide front end, narrower, broadening gradually backward.Body front end tool is the tang sucker forward, is an oval gonopore under the oral sucker, and pharynx is long cartridge type.Distinguish not obvious before the well differentiated opisthaptor of polypide outside of belly rear end tool, opisthaptor and body.Opisthaptor is asymmetric, has four pairs, and both sides of the edge are arranged in parallel separately, and wherein 2 of sides are that especially big open chitin is inhaled pincers, and 6 little dead front types of surplus person are inhaled pincers.The polypide afterbody has two pairs of sharp-pointed crotches, and wherein a pair of little hook in edge is more broad in the middle.
The different hook pincers of the Lin Shi worm back of the body, outside of belly body surface all have tangible transversal fold, find in the gauffer that nodosity, nodule surface contain branch than uniform small pores, have the function of sensation and substance metabolism exchange.This parasite only is found on the cutter long-tailed anchovy gill filament, and opisthaptor opens and inhale gill small pieces on the pincers gill filament, does not move after the absorption, and just the polypide front end is to front stretching.Destroy the epithelial cell of the gill filament, cause the invasion of other pathogenic bacterias, cause inflammation, cause the gill rot illness.September, the different hook pincers of the parasitic Lin Shi of cutter long-tailed anchovy worm is more, and infection rate is up to 74%, and parasitism was 7 when the cutter long-tailed anchovy was the highest.Happiness inhabits in competent fresh water of oxygen or the low salinity water environment, in suitable temperature, can impel it to lay eggs and hatch in the low salinity water territory.The ovum two ends have long polar filament, are easy to floating and propagation.
2.1 molecular systematics analysis
The present invention is a template with the different hook pincers of Lin Shi worm DNA, pcr amplification 28S rDNA5 ' terminal sequence, and product shows amplification gene clip size and expection consistent (as Fig. 1) through 1% agarose gel electrophoresis analysis.After the TA cloning and sequencing draws, the sequence total length is 949bp, G+C=53.32%.Cao Shanmei has measured the different hook pincers of six sour jujubes worm 28s sequence in the monogentic trematode research of South Sea fish hook pincers worm section, total length 890bp, and G+C=53.03% parasitizes on seven long-tailed anchovy gill filaments.Participate in 16 kinds of monogentic trematodes of comparison as can be seen from 28s molecular evolution tree and be divided into two on the whole, Lin Shi different hook pincers worm and the different hook pincers of six sour jujubes worm are in same branch and Bootstrap value 100 (as Fig. 2), Kimura2-parameter distance distance is 0.002 between the two simultaneously, and it is bigger with polypide genetic distances such as other little glass of worm section, Spanish mackerel pincers worm sections, the result shows: Lin Shi different hook pincers worm and the different hook pincers of six sour jujubes worm sibship are nearest, the further clear and definite classification position of the different hook pincers of Lin Shi worm.
Hook pincers worm section has 11 genus, preceding 2 suction pincers according to different hook pincers Eimeria haustorium one side in the key are big especially, polypide of the present invention contains the opisthaptor of four right titles, the tool short handle, wherein preceding 2 open chitins of a side are inhaled pincers obviously greater than the suction pincers of all the other 6 sealings, the polypide afterbody has two pairs of sharp-pointed crotches, meets the feature of different hook pincers Eimeria.The ovum two ends have long polar filament, and the husk shape ovum that does not have polar filament with the different hook pincers of six sour jujubes worm is different.
This experiment is by measuring 28S rRNA, and the sequence alignment analysis draws: Lin Shi different hook pincers worm and the different hook pincers of six sour jujubes worm sibship are nearest, and similarity is 100%, the further clear and definite classification position of the different hook pincers of Lin Shi worm.At present, the ITS sequence also is a molecule marker important in parasite system and the Study on Evolution, and evolutionary rate is very fast, and length is comparatively conservative.
3. the present invention detects the proof of effect
Someone is to river sea migration type, the structure of community of parasitic worm has carried out systematic study in fresh water migration type and the fresh water sedentariae cutter long-tailed anchovy, on the gill of cutter long-tailed anchovy and digestive tube, 10 kinds of parasitic worms have been found, the different hook pincers of the Lin Shi of discovery worm is wherein all arranged in three kinds of environmental cutter long-tailed anchovys, migration type Anqing section infection rate is up to 78%, similar with the different hook pincers of the Lin Shi insect infection rate 74% that Jingjiang section of the present invention is found, someone has studied parasite seasonal variation rule in the cutter long-tailed anchovy migration path, drawing the average infection rate of Chongming Island district Lin Shi different hook pincers worm is 42.82%, the December infection rate is also up to more than 70 percent, this experiment the Changjiang river hairtail is picked up from September Jingjiang section, migration monoid to the migration of Haikou, Chongming Island district matches with experimental result.
The above only is preferred embodiment of the present invention, not in order to restriction the present invention, all any modifications of being done within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. detection and expelling method based on the different hook pincers of the cutter long-tailed anchovy Lin Shi worm of molecular marking technique is characterized in that, this method is utilized the ordinary optical microscope method that combines with molecular genetic, the different hook pincers worm of parasitic cutter long-tailed anchovy is carried out kind identify;
At first, microscopy shows: worm's ovum stage ovum two ends have long polar filament;
The polypide stage is contained the opisthaptor of four right titles, the tool short handle, and wherein preceding 2 open chitins of a side are inhaled pincers obviously greater than the suction pincers of all the other 6 sealings, and the polypide afterbody has two pairs of sharp-pointed crotches;
Molecular genetic amplification 28S rRNA gene 5 ' end parts sequence is measured the different hook pincers of Lin Shi worm 28s sequence then, through compare of analysis, can identify accurately that this parasite is the different hook pincers of a Lin Shi worm;
After evaluation finished, with the Vermox full pool spilling head of lmg/L concentration, after 72 hours, polypide was tear-away, dead; Simultaneously, also in time change water 50%-60%, discharge the worm's ovum that comes off, recurrence after preventing.
2. detection and the expelling method of the different hook pincers of the cutter long-tailed anchovy Lin Shi worm based on molecular marking technique as claimed in claim 1 is characterized in that the concrete steps of this detection method are:
Collected specimens host's cutter long-tailed anchovy, 30 cutter long-tailed anchovys of random inspection, average body is long 12 centimetres, and body weight 5 grams are examined under a microscope gill portion parasite and are added up its quantity, and part polypide flushing with clean water is totally preserved to 95% alcohol;
Carry out morphological structure and observe, visual inspection will have parasite cutter long-tailed anchovy and take out the fish gill and put on the slide glass, and anatomical lens is observed down and will be had the gill filament of polypide to choose, and put into to put microscopically after the centrifuge tube clear water cleans for several times again and observe and take pictures;
Carry out Molecular Identification, at first prepare template DNA, carry out pcr amplification and amplified fragments clone, after the dna sequence dna splicing, login NCBI carries out the blast compare of analysis, and downloads the higher sequence of similarity; Adjacent method has made up 28S rDNA molecular evolution tree in the ClustalX multisequencing comparison back employing MEGA software.
3. detection and the expelling method of the different hook pincers of the cutter long-tailed anchovy Lin Shi worm based on molecular marking technique as claimed in claim 2, it is characterized in that, collected specimens host's cutter long-tailed anchovy, 30 cutter long-tailed anchovys of random inspection, long 12 centimetres of average body, body weight 5 gram is examined under a microscope gill portion parasite and is added up its quantity, and part polypide flushing with clean water is totally preserved to 95% alcohol.
4. detection and the expelling method of the different hook pincers of the cutter long-tailed anchovy Lin Shi worm based on molecular marking technique as claimed in claim 2, it is characterized in that, visual inspection, to have parasite cutter long-tailed anchovy takes out the fish gill and puts on the slide glass, anatomical lens is observed down and will be had the gill filament of polypide to choose, and puts into to put the Olympus0.5X microscopically after the centrifuge tube clear water cleans for several times again and observe and take pictures.
5. detection and the expelling method of the different hook pincers of the cutter long-tailed anchovy Lin Shi worm based on molecular marking technique as claimed in claim 2 is characterized in that the preparation method of template DNA is:
From-20 ℃ 95% alcohol preserve liquid take out body, place the aseptic centrifuge tube of 2ml, sterile water wash is also placed 4 ℃ of refrigerators displacements and is spent the night;
Cleaning and QIAGEN QIAamp DNA Mi ni Kit test kit carry out the extraction of DNA once more, and last constant volume is put in the 25 μ L sterilized waters-20 ℃ of preservations.
6. detection and the expelling method of the different hook pincers of the cutter long-tailed anchovy Lin Shi worm based on molecular marking technique as claimed in claim 2 is characterized in that, pcr amplification and amplified fragments cloning process are:
With
CD1：(GTAGGTGAACCTGCGGAAGGATCATT)
And CD2-(TTAGTTTCTTTTCCTCCGCT)
Be the different hook pincers of primer amplification Lin Shi worm 28S aligning primer; With 4ul parasite DNA is template, and NC5 and NC2 are primer amplification ITS fragment; Reaction system is 50 μ L, and reaction conditions is 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 60s, circulate 35 times; Be template with 4 μ L parasite DNA equally, CD1 and CD2 are primer amplification 28S fragment; Reaction system is 50 μ L, and reaction conditions is 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 60s, circulate 35 times; Amplified production is through 1% concentration agarose gel electrophoresis check and analysis;
To remain the PCR product and run back and receive glue and reclaim the test kit purifying with dna gel, be masterplate with the purified product, and the 28S fragment is increased in a large number, links to each other with the pMD18-T carrier to carry out TA and clone, and the sub-upgrading grain of screening positive clone checks order.
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