CN105524996B - A kind of method of small melonworm in quick detection water body - Google Patents
A kind of method of small melonworm in quick detection water body Download PDFInfo
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Abstract
The present invention relates to a kind of methods of small melonworm in quickly detection water body to acquire water sample according to Cultivated water size；After water sample is handled, the total DNA in water body is extracted；PCR amplification is carried out as primer using known nucleotide sequence IC-ITS-F 5'-GTTCCCCTTGAACGAGGAATTC-3' and IC-ITS-R 5'-TTAGTTTCTTTTCCTCCGC-3 ', obtain PCR product, it is cloned being transferred in bacillus coli DH 5 alpha after above-mentioned PCR product connection carrier, after sequencing result is compared using DNAMAN6.0 software, it searches the difference site between small melonworm and other detectable sibling species and designs the Chao Shi PCR primer for small melonworm, Chao Shi PCR is carried out using primer pair first time PCR product, electrophoresis detection is as the result is shown, there is the amplifiable shaping band of small melonworm, detection can be completed.Using this method very simple and effective have detected small melonworm in water body there are situations to prevent the outburst of small melonworm from providing effective way, great practical value is worthy to be popularized to carry out water body processing in time.
Description
Technical field
The present invention relates to molecular biology fields, in particular to acquisition, extraction, the amplification of small melonworm DNA sample in water body
And identify aspect, in particular to a kind of small melonworm rapid detection method of water body.
Background technique
Ichthyophthirius is a kind of worldwide distribution in protozoa, Ciliata, Hymenostomatida, recess section, Ichthyophthirius
Parasite of freshwater fishes, fish overwhelming infection is caused in high-density aquiculture, causes large quantities of fishes dead, causes huge
Economic loss.
In order to effectively control the generation of the disease, domestic and international researcher inquired into the disease cause of disease some biological characteristics,
The manufacture of pathological characteristic, physical chemistry and Chinese herbal medicine to the killing effect, small melonworm difference immune vaccine of in vitro small melonworm.Except base
Plinth study outer curative test result be not effect it is undesirable be exactly that application is poor.Integrated culture experience and experimental result are come
See, should rely mainly on prevention for the control strategy of ich at present, control supplemented by.It is deposited so understanding small melonworm in water body in time
In state, water body processing is carried out in advance or transfers cultivation fish body in time, is the optimal path for preventing ich from breaking out.
Summary of the invention
The contents of the present invention be in order to find in time small melonworm in water body there are situation, provide it is a kind of efficiently, it is quick, quasi-
The small melonworm detection method of true water body, to judge that a possibility that breaking out ich establishes eventually by small melonworm DNA concentration
Basis, to instruct actual cultivation to operate.
To achieve the above object, the present invention provides a kind of small melonworm methods in quickly detection water body, comprising the following steps:
Step 1: according to Cultivated water size, acquiring water sample；
Step 2: after water sample is handled, extracting the total DNA in water body；
Step 3: carrying out PCR amplification as primer using nucleotide sequence IC-ITS-F and IC-ITS-R, obtain PCR product
750bp, electrophoresis detection；
Step 4: by above-mentioned PCR product gel extraction, connecting to be transferred in bacillus coli DH 5 alpha after carrier and be cloned, and to the greatest extent
Picking monoclonal individual more than possible is sequenced；
Step 5: sequencing result is compared using DNAMAN6.0 software, find out small melonworm and the sibling species that measures it
Between difference site and design at least 2 pairs be directed to small melonworm Chao Shi PCR primers；
Step 6: carrying out Chao Shi PCR using the PCR product that above-mentioned primer pair step 3 obtains, it is known that electrophoresis detection result is aobvious
Show there is the amplifiable shaping band of small melonworm, the detection to melonworm small in water body can be completed.
The acquisition water sample of the step 1 is the aquaculture pond bottom of pond of required detection, water sample or pool row in pool wall different range
The isometric mixture of mouth of a river water sample in different time periods.
Water sample processing in the step 2 is the water sample is filtered by vacuum using 25 μm of filter membranes, by institute after suction filtration
The filter membrane stated is put into the 50ml centrifuge tube equipped with 35ml sterile water, is mixed in shaking on turbula shaker, 8000rpm centrifugation
5min, collection are deposited in -20 DEG C of preservations；Sediment is added new sterile water, after purging repeatedly, the centrifuge tube loaded on 1.5ml
In, 10000rpm is centrifuged 5min, and it is spare to abandon supernatant.
In the step 2 extraction of total DNA use kit method, detected through gel electrophoresis DNA extract situation after in -20 DEG C
It saves backup.
Primer nucleotide sequences IC-ITS-F and IC-ITS-R are respectively IC-ITS-F 5'- in the step 3
GTTCCCCTTGAACGAGGAATTC-3' and IC-ITS-R 5'-TTAGTTTCTTTTCCTCCGC-3 '.PCR amplification condition are as follows:
94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 recycle, 72 DEG C of extension 10min；
Pcr amplification reaction system is in every 25 μ l system: 10xbuffer 2.5 μ l, MgCl2(25mM) 3 μ l, dNTPs
(2.5mM) 2 μ l, concentration are each 0.5 μ l of positive anti-primer, 1 μ l of template DNA, the 0.25 μ l of Taq enzyme (5U/ μ l) of 100pmol/ μ l,
ddH2O 15.25μl；
The above-mentioned PCR product of electrophoresis detection is in 1% Ago-Gel, voltage 120V electrophoresis 15min, finally gel at
Electrophoresis detection can be completed as taking pictures in system.Chao Shi PCR primer for small melonworm is 2 pairs, respectively n-IC1-F and n-
IC1-R；N-IC2-F and n-IC1-R.PCR product 447bp is expanded through detection pair of primers (n-IC1-F and n-IC1-R), the
Two pairs of primer (n-IC2-F and n-IC1-R) amplification PCR products are 225bp.
The monoclonal individual of picking 20-40 in the step 4, it is 15 that finally sequencing, which obtains sequence,.
In the step 6, Chao Shi PCRPCR amplification condition are as follows: 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 30s, 55 DEG C are annealed
30s, 72 DEG C of extension 30s, 30 circulations, 72 DEG C of extension 10min；
Pcr amplification reaction system is in every 25 μ l system: 10xbuffer 2.5 μ l, MgCl2(25mM) 3 μ l, dNTPs
(2.5mM) 2 μ l, concentration are each 0.5 μ l of positive anti-primer, 1 μ l of template DNA, the 0.25 μ l of Taq enzyme (5U/ μ l) of 100pmol/ μ l,
ddH2O 15.25μl；
Environmental molecules biology techniques principle is applied to cultivation investigation by the present invention, need to be when obtaining small melonworm DNA
Target water body a certain range carries out water sampling in the time, quickly, simplicity, timely understood small melonworm in water body
There are situation, solve the problems, such as that ich Feed Discovery is difficult, to calculate ich eventually by the small melonworm DNA concentration of water body
Outburst probability lays the foundation, and treats hardly possible due to being in a bad way when solving the problems, such as ich discovery.Relative to traditional small worm
For the control method of disease, there is novelty, avoid drug treatment bring medicament residue, the effect of physical therapy modalities
Fruit is bad, and the problem of Biotherapy method time length prevents trouble before it happens, greatly reduces the outburst probability of ich.
Detailed description of the invention
Fig. 1 is the method flow diagram of the embodiment of the present invention.
Fig. 2 is the method for the present invention sensitivity technique, natural water breaks out small melonworm process, the small melonworm of different breeding water body is deposited
Result figure is extracted in the DNA of situation.
Fig. 3 be the embodiment of the present invention using IC-ITS-F and IC-ITS-R to the small melonworm DNA in the case of above-mentioned three kinds into
The result figure of row first time PCR.
Fig. 4 is that the embodiment of the present invention uses n-IC1-F and n-IC1-R to the small melonworm first time in the case of above-mentioned three kinds
The result figure of PCR product Chao Shi PCR.
Fig. 5 is that the embodiment of the present invention uses n-IC2-F and n-IC1-R to the small melonworm first time in the case of above-mentioned three kinds
The result figure of PCR product Chao Shi PCR.
Fig. 6 is the sequence of the small melonworm and sibling species that obtain after universal primer ITS-F/ITS-R first time PCR, side in figure
Frame is expressed as 2 couples of specific primer (n-IC1-F/n-IC1-R for the design of small melonworm specific site；n-IC2-F/n-IC1-
R)；Wherein, Fig. 6-1n-IC1-F is upstream primer, 6-2n-IC1-R is downstream primer；Fig. 6-3n-IC2-F is upstream primer, 6-
4n-IC1-R is downstream primer.
Specific embodiment
In order to be more clearly understood that technology contents of the invention, specific implementation method of the invention is made into one below
Walk explanation.
2,3,4,5 electrophoretogram swimming lane point sample sequence of attached drawing is successively are as follows: the small melonworm of 5/L, the small melonworm of 10/L, 15/L are small
Melonworm, the small melonworm of 25/L, the small melonworm of 40/L, make-up water source, copper fish, blank control, DNAMarker, first stage water sample,
Second stage water sample, phase III water sample, fourth stage water sample, the 5th stage water sample, blank control, DNAMarker are adopted and are fed
No. 6 water samples between maintaining the car, the pool water sample of tail beach 1, the pool water sample of tail beach 3, board house glass cylinder aquaculture pond water sample, the 4th workshop 5
Water body, blank control, DNAMarker.
DNAMrker stripe size is from the bottom up successively are as follows: 100bp；250bp；500bp；750bp；1000bp；2000bp；
3000bp；5000bp.
Attached drawing 6 obtains after being universal primer ITS-F/ITS-R first time PCR, the sequence of small melonworm and sibling species, and totally 10
A, sequence is shown in SEQUENCE LISTING.
Embodiment 1
Method sensitivity technique
In order to determine that the present invention can effectively extract the small melonworm DNA in water body, the fish body of the small melonworm of severe infections is taken
(there are many small particles on fish body surface), wash with distilled water fish body three times gently scrape the white packet on fish body surface with glass slide
Packing is collected into sterile petri dish by capsule together with the ichthyophthirius multifiliis in it, is stood, is drawn a large amount of mucus in another sterile
It purges in culture dish and repeatedly, the small melonworm being wrapped in mucus is separated, remove mucus.So far it is deposited in 2 culture dishes
Can largely choose from the single polypide of small melonworm (if mucus it is more can again repeat aforesaid operations with obtain it is further amounts of individually
Polypide).5,10,15,25,40 small melonworms are placed respectively with 1L water, is tested, is made by experiment flow as shown in Figure 1
Suction filtration processing is carried out to 5 water samples with the vacuum pump that filter sizes are 25 μm, filter membrane is put into the centrifuge tube of 50ml, in
It shakes and mixes on turbula shaker, 8000rpm is centrifuged 5min, removes supernatant, new sterile water is added, and is sucked into after purging repeatedly
In the centrifuge tube of 1.5ml, 10000rpm is centrifuged 5min, removes supernatant, extracts small melonworm using the kit that TIANGEN company produces
Total DNA is simultaneously dissolved in TE buffer, and -20 DEG C save backup.Fig. 2-A is DNA agarose gel electrophoresis testing result.
Gel electricity is carried out to PCR product after being expanded using IC-ITS-F and IC-ITS-R sequence as versatility primer
Swimming detection, as a result as shown in Fig. 3-A.By above-mentioned PCR product gel extraction, progress gram in bacillus coli DH 5 alpha is transferred to after connection
Grand, picking monoclonal individual is sequenced.It is compared, is searched and small melonworm difference site (difference using DNAMAN6.0 software
The search procedure in site is as shown in fig. 6, obtain 10 including small melonworm by universal primer IC-ITS-F and IC-ITS-R
Sequence, by the comparison of DNAMAN6.0, the sequence in voluntary election difference site such as box is as Chao Shi PCR primer, wherein
Fig. 6-1,6-2 are respectively 5 ', 3 ' Duan Xulie of pair of primers n-IC1-F, n-IC1-R；Fig. 6-3,6-4 are respectively second pair and draw
5 ', 3 ' Duan Xulie of object n-IC2-F, n-IC1-R.), design the specific primer n-IC1-F and n- for small melonworm DNA
IC1-R；N-IC2-F and n-IC1-R.Chao Shi PCR is carried out using above-mentioned 2 pairs of specific primers, as a result such as Fig. 4-A and Fig. 5-A institute
Show.
The results show that even the method for the present invention can still detect small melonworm polypide well under 5 polypide concentration
DNA fragmentation.
In addition, it (is respectively the 6th hole of Fig. 2-A that the test process, which is added to research institute cultivation water source as supplement water sample,
Sample, the 6th hole sample of 3-A, the 6th hole sample of 4-A, 5-A the 6th hole sample) and a tail health copper fish skin histology (i.e.
Respectively the 7th hole sample of the 7th hole sample of Fig. 2-A, the 7th hole sample of 3-A, the 7th hole sample of 4-A, 5-A) it is used as control,
This 2 samples tentatively judge research institute cultivation water source and the tail health copper fish without small melon without small melonworm DNA as the result is shown
Worm.
Embodiment 2
Natural water breaks out small melonworm process
Experiment flow is cleaned as shown in Figure 1, aquarium is sterilized, and is put into 2 tail copper fishes, is denoted as the first stage；It opens within second day
It is second stage that beginning, which feeds water earthworm postscript,；Fish body it is slow in action, do not feed actively, fish body has fragmentary single white point to be denoted as the
Three stages；To fish body surface, there are more obvious white points to be denoted as fourth stage；To fish body abdomeinal fin surface, the gill portion white in flakes and
Almost stationary state thing was denoted as the 5th stage to fish body.Respectively this 5 stages using siphonage the bottom of pond of culturing jar, pool wall not
Water sample 1L is taken with position mixing, suction filtration processing is carried out to water sample using the vacuum pump that filter sizes are 25 μm, by filter membrane
It is put into the centrifuge tube of 50ml, is mixed in being shaken on turbula shaker, 8000rpm is centrifuged 5min, removes supernatant, new nothing is added
Bacterium water is sucked into the centrifuge tube of 1.5ml after purging repeatedly, and 10000rpm is centrifuged 5min, is removed supernatant, is used TIANGEN company
The kit of production extracts the total environment DNA in filter membrane and is dissolved in TE buffer, and -20 DEG C save backup.Fig. 2-B is DNA fine jade
Sepharose electrophoresis detection result.
Gel electricity is carried out to PCR product after being expanded using IC-ITS-F and IC-ITS-R sequence as versatility primer
Swimming detection, as a result as shown in Fig. 3-B.Using specific primer n-IC1-F and n-IC1-R；N-IC2-F and n-IC1-R respectively into
Row Chao Shi PCR, as a result as shown in Fig. 4-B and Fig. 5-B.
The results show that infecting the 3 different time stages i.e. (third to the 5th at small melonworm initial stage to severe infections in fish body
Stage), small melonworm DNA fragmentation can be amplified from water body, and band brightness value is different.
Embodiment 3
There are situations for the small melonworm of different breeding water body
Experiment flow carries out water body detection, as a result as shown in Figure 1,5 different breeding places selected by studying in mandarin sturgeon
As shown in Fig. 2-C, 3-C, 4-C, 5-C, respectively indicates to adopt and feed the pond of workshop 6, No. 1 pool, No. 3 pools, the cultivation of board house glass cylinder
Pond, the water body situation in the 4th pond of workshop 5, only there is a small amount of small melonworm DNA in board house glass cylinder storage pond, holding pond as the result is shown, other
Water body is without small melonworm.In addition to board house glass cylinder aquaculture pond is temporarily feeding wild copper fish, other cultivation are pool cultivated right for investigation verifying
As being sturgeon, sturgeon belongs to the species of the not small melonworm of easy infection because of own biological feature, so, 1 supplement in conjunction with the embodiments
Water sample, embodiment 2 can tentatively learn that there is no small melonworms at the cultivation water source of mandarin sturgeon research institute, temporarily support the water of copper fish
Body and the fish body that small melonworm is broken out after feeding water earthworm may carry disease due to wild fish body itself or the water earthworm fed
Original causes ich to break out when environment is suitable in storage pond, holding pond.
。
Claims (7)
1. a kind of method of small melonworm in quickly detection water body, which comprises the following steps:
Step 1: according to Cultivated water size, acquiring water sample；
Step 2: after water sample is handled, extracting the total DNA in water body；
Step 3: carrying out PCR amplification as primer using nucleotide sequence IC-ITS-F and IC-ITS-R, obtain PCR product
750bp, electrophoresis detection；The primer nucleotide sequences IC-ITS-F and IC-ITS-R is respectively IC-ITS-F 5'-
GTTCCCCTTGAACGAGGAATTC-3' and IC-ITS-R 5'-TTAGTTTCTTTTCCTCCGC-3 '；
Step 4: by above-mentioned PCR product gel extraction, connecting to be transferred in bacillus coli DH 5 alpha after carrier and be cloned, and as far as possible
More picking monoclonal individuals are sequenced；
Step 5: sequencing result being compared using DNAMAN6.0 software, is found out between small melonworm and the sibling species measured
2 pairs of Chao Shi PCR primers for being directed to small melonworm are simultaneously designed in difference site, specific as follows:
N-IC1-F:5 '-GGTGAACCTTCTGGACCGAGGT-3 ',
N-IC1-R:5 '-ATTAAGATACATGATTCCTTTC-3 ',
N-IC2-F:5 '-AATAAAATACCTTCATATGTCT-3 ',
N-IC1-R:5 '-ATTAAGATACATGATTCCTTTC-3 '；
Step 6: Chao Shi PCR is carried out using the PCR product that above-mentioned primer pair step 3 obtains, until electrophoresis detection the results show that having
The small amplifiable shaping band of melonworm, can be completed the detection to melonworm small in water body.
2. the method for small melonworm in quick detection water body according to claim 1, which is characterized in that the step 1 is adopted
Collecting water sample is the aquaculture pond bottom of pond of required detection, water sample or pool discharge outlet water sample in different time periods etc. in pool wall different range
Volume mixture object.
3. the method for small melonworm in quick detection water body according to claim 1, which is characterized in that in the step 2
Water sample processing for the water sample is filtered by vacuum using 25 μm of filter membranes, after suction filtration by the filter membrane be put into equipped with 35ml without
It in the 50ml centrifuge tube of bacterium water, is mixed in being shaken on turbula shaker, 8000rpm is centrifuged 5min, and collection is deposited in -20 DEG C of guarantors
It deposits；Sediment is added new sterile water, after purging repeatedly, in the centrifuge tube loaded on 1.5ml, 10000rpm is centrifuged 5min, abandons
Supernatant is spare.
4. the method for small melonworm in quick detection water body according to claim 1, which is characterized in that total in the step 2
The extraction of DNA saves backup after using kit method, detected through gel electrophoresis DNA to extract situation in -20 DEG C.
5. the method for small melonworm in quick detection water body according to claim 1, which is characterized in that PCR in the step 3
Amplification condition are as follows: 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 circulations, 72 DEG C are prolonged
Stretch 10min；
Pcr amplification reaction system is in every 25 μ l system: 10 × buffer 2.5 μ l, MgCl23 μ l, dNTPs 2.5mM of 25mM
2 μ l, concentration are each 0.5 μ l of positive anti-primer, template DNA 1 μ l, Taq enzyme 5U/ μ l 0.25 μ l, ddH of 100pmol/ μ l2O
15.25μl；
The above-mentioned PCR product of electrophoresis detection is in 1% Ago-Gel, voltage 120V electrophoresis 15min, finally in gel imaging system
Taking pictures on system can be completed electrophoresis detection.
6. the method for small melonworm in quick detection water body according to claim 1, which is characterized in that chosen in the step 4
The monoclonal individual taken is 20-40.
7. the method for small melonworm in quick detection water body according to claim 1, which is characterized in that in the step 6, nest
The amplification condition of family name PCR are as follows: 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 are followed
Ring, 72 DEG C of extension 10min；
Pcr amplification reaction system is in every 25 μ l system: 10 × buffer 2.5 μ l, MgCl2 3 μ l, dNTPs 2.5mM of 25mM
2 μ l, concentration are each 0.5 μ l of positive anti-primer, template DNA 1 μ l, Taq enzyme 5U/ μ l 0.25 μ l, ddH of 100pmol/ μ l2O
15.25μl。
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| Non-invasive detection and quantification of the parasitic ciliate Ichthyophthirius multifiliis by real-time PCR;Olivier Jousson等;《DISEASES OF AQUATIC ORGANISMS》;20050718;第65卷;251-255 |
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