CN105506144A - Method for detecting early-stage attachment degree of barnacle larvae - Google Patents
Method for detecting early-stage attachment degree of barnacle larvae Download PDFInfo
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- CN105506144A CN105506144A CN201610047648.3A CN201610047648A CN105506144A CN 105506144 A CN105506144 A CN 105506144A CN 201610047648 A CN201610047648 A CN 201610047648A CN 105506144 A CN105506144 A CN 105506144A
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- barnacle
- mispairing
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- kentrogon
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
Abstract
The invention relates to a method for detecting the early-stage attachment degree of barnacle larvae. The method comprises the following steps that 1, specific primers of the barnacles are designed; 2, in the early stage of staining, before the naked eyes can see the attached barnacles, a sea water staining sample is collected, and a genome is extracted; 3, PCR amplification is performed on the genome sample through the specific primers of the barnacles to determine attachment of the barnacle larvae. The barnacle larva attachment determination method does not depend on a pure culture system and is not only suitable for barnacle larva attachment determination of the sea water site staining sample, but also suitable for barnacle larva attachment determination of different coating surfaces in the indoor pure culture system.
Description
Technical field
The invention belongs to biological technical field, particularly relate to the mensuration to stained early stage kentrogon attachment in main marime fouling eukaryote.
Background technology
Marine fouling organism also known as marine attaching organism, be grow alow, sewer line, oil platform, the netting gear of fishery and the harmful organism on all marine artificial facility surfaces.Wherein barnacle is one of topmost marime fouling, barnacle (Barnacle) is also called " gingival cyst of mucous gland in the newborn " or " Ke infuses son ", belong to Arthropoda (Arthropoda), Crustachia (Crustacea), Thoracica (Thoracica), cirriped, the barnacle of current discovery has 8 sections about 541 kinds, and wherein China about has 110 kinds.
Barnacle becomes one of topmost marime fouling with its special morphological structure, the life history and Population Ecological, mainly live in tideland and be with down, attachment is perched in hard object fixing or floating in the seawater, if be attached on net cage, not only block cage netting, easily cause the environment of cultivation to be deteriorated, and increase net cage clothing resistance, the scratch of spring tide fish in flood season body easily occurs; If be attached to hull bottom, increase ship resistance, the speed of a ship or plane is reduced; If be attached on buoy, buoyancy can be reduced.And antifouling paint effective at present can cause must affect Marine ecosystems, therefore, can try to carry out there be preventing and kill off of target from the larva setting stage of its growth and development stage for barnacle.And want to carry out when barnacle adheres in early days testing its adhesive rate, molecule marking method becomes first-selected technique means, avoid the interference of biomorph textural difference, directly by detecting the genetic stability information that in organism, macromole comprises, quantitative description, analysis adhesion condition, improve accuracy and the efficiency of the main marine fouling organism species of Forepart identification.
The adhesion detection of barnacle at least following several in extremely important: (1) fast more a collection of antifouling coating to suppress the difference of kentrogon adhesive ability at marime fouling early stage (before the visible barnacle growth of naked eyes); (2) certain coating difference of larva adhesive rate in larva adhesive rate and actual seawater in indoor barnacle pure culture system is compared.(3) high-quality coating suppresses the Mechanism Study of marime fouling: the apposition growth sometimes needing to be determined at certain specific coatingsurface barnacle is suppressed is because larva attachment is suppressed or after attachment, growth is suppressed.
Chinese patent (CN101654704A) reports the Subspecies Taxonomy that one group of primer is used for distinguishing different barnacle, can distinguish white ridge barnacle and line barnacle.The larva that the primer that this patent relates to can be used for measuring more barnacle subspecies adheres to.
Summary of the invention
The object of the present invention is to provide a kind of in marime fouling the mensuration to stained early stage kentrogon attachment.The specificity measured is ensured by barnacle Auele Specific Primer.Design based on the 18SrDNA of barnacle during this Auele Specific Primer, its sequence is AmpR1:5 '-ATGCTTTCGCAGTAGTTCGTTG-3 '.The specificity can guaranteeing barnacle gene amplification is used together with its universal primer F566 with ribosomal gene 18SrDNA (5 '-GGCATCACAGACCTGTTATTGC-3 ').Amplification can realize quantitative assay by the gel image scanning after qPCR or general PCR terminate.
The present invention is achieved through the following technical solutions:
A kind of based on molecular marking technique, in main marime fouling eukaryote, detect the method for kentrogon degree of adhesion, the method comprises the following steps:
(1) the design of barnacle Auele Specific Primer;
(2) the collection of stained early stage (naked eyes adhere to barnacle as seen before) stained sample of seawater and genome extract;
(3) utilize barnacle Auele Specific Primer to carry out pcr amplification to measure the attachment of kentrogon to said gene group sample.
And barnacle specific primer design method is as follows: main marime fouling Eukaryotic 18S ribosomal gene sequence, find out specificity SNP (single nucleotide polymorphism) site of barnacle.This site uses as 3 ' least significant end position of primer.The base principle of design of 3 ' penultimate of primer is, does not have mispairing or form a mispairing (weak mispairing: A-C, T-G between it and target sequence; Or the mispairing of medium tenacity: A-A, C-C, G-G), also form a mispairing (strong mispairing: T-T, T-C, A-G with non-target sequences; Or the mispairing of medium tenacity: A-A, C-C, G-G).So, primer and target sequence match completely or only have the mispairing of a 3 ' penultimate, generally do not affect expanding effect, and have continuous two mispairing of two 3 ' least significant ends with non-target sequences, generally cause increasing unsuccessfully.So can obtain barnacle Auele Specific Primer.
And one of barnacle Auele Specific Primer of aforesaid method design is AmpR1:5 '-ATGCTTTCGCAGTAGTTCGTTG-3 '.This primer uses and can complete the mensuration that in early stage stained sample, whether kentrogon exists and judge together with universal primer F566:5 '-GGCATCACAGACCTGTTATTGC-3 '.
(1) design of primers
By amplification (amplimer is 5 '-AACCTGGTTGATCCTGCCAGT-3 ' and 5 '-GATCCTTCTGCAGGTTCACCTAC-3 ') and the cloning and sequencing of 18SrDNA in the stained sample of certain seawater, obtain a collection of effective clone, carry out BLAST bioinformatic analysis afterwards, confirm the respective 18S sequence of a collection of stained eukaryote (containing barnacle).A lot of 18S sequence also can be downloaded arrangement by database and obtain.By to the Multiple Sequence Alignment of above-mentioned 18S sequence with repeatedly compare, several characteristic SNP (single nucleotide polymorphism) site finding out barnacle is multiple.Design primer is attempted in 3 ' the least significant end position using these SNP site as primer.The base principle of design of 3 ' penultimate of primer is, forms a mispairing, also form a mispairing with non-target sequences between it and target sequence.So, primer and target sequence only have the mispairing of a 3 ' penultimate, and have continuous two mispairing of two 3 ' least significant ends with non-target sequences.Amplification length is in 100-500 base so that for qPCR (also may be used for general PCR).These primer pairs carry out PCR with clone's pure plasmid of barnacle 18S sequence for template and verify specific amplification after synthesis, and qPCR checking (requires only amplification out special object band, the analysis of Tm value only has master tape (not containing primer dimer), determines the specificity of each pair of primer.Select best specific amplification primer (to).Auele Specific Primer also can match with universal primer and use.More than a pair best primer can be designed in principle.Because the subspecies of barnacle itself are a lot, the Auele Specific Primer of certain or some specific subspecies can be designed according to aforesaid method, also can design the Auele Specific Primer that certain class balanoid zone, balanus zone divides other species specific (as coil pipe worm).One of best primer pair is AmpR1:5 '-ATGCTTTCGCAGTAGTTCGTTG-3 ' and universal primer F566:5 '-GGCATCACAGACCTGTTATTGC-3 '.This primer pair amplifies product out (about 400 base pair) is used to prove to be all barnacle 18S sequence through DNA sequencing.By analysis, this best primer pair is applicable to the mensuration (form 1) of following barnacle kind:
The barnacle kind that form 1 Auele Specific Primer is suitable for
(2) the collection of stained early stage (naked eyes adhere to barnacle as seen before) stained sample of seawater and (grand) genome extract
Be applicable to the stained sample sampling in marine on-site sampling or indoor pure culture system.The collection in worksite of marine early stage stained sample and grand genome extract and can adopt hot phenol chloroform extraction method, its concrete steps are: 1) by stained sample at sea (scene) scrape off about 200 microlitres, include the kentrogon (just only having kentrogon in pure culture system) of various marine organisms and attachment, add the saturated phenol of 600 microlitre (pH8), concuss one minute.Take back laboratory.2) after 75 degree of water-baths shake 10 minutes, room temperature leaves standstill 3min, the centrifugal 10min of 10000r/min; 3) supernatant liquid is drawn to adding equal-volume phenol, the centrifugal 10min of 10000r/min after concuss 1min in new EP pipe; 4) get its supernatant liquid and add equal-volume phenol chloroform (1:1) again, the centrifugal 15min of 10000r/min after mixing 1min; 5) get supernatant liquid and add isopyknic chloroform again, the centrifugal 15min of 10000r/min after mixing 1min; Get (grand) genome that supernatant is sample.
(3) utilize barnacle Auele Specific Primer to carry out general pcr amplification to measure the attachment of kentrogon to above-mentioned (grand) genome sample
The cumulative volume of pcr amplification reaction system is 12 μ L, and reaction system is as follows:
The composition (NPK62, NPK64 test kit for GREDBIO) of form 2PCR system
PCR loop parameter is: 94 DEG C of 4min-(94 DEG C of 30sec-62 DEG C 50sec-72 DEG C 50sec) × 38cycles; After pcr amplification terminates, by 12 μ L products, the agarose gel electrophoresis with 1.2% detects, ethidium bromide staining, and observes and saving result with gel imaging system; Result judges: only have band to expand and band length at about 400bp, be then barnacle.
(4) utilize barnacle Auele Specific Primer to carry out to above-mentioned (grand) genome sample the attachment that qPCR amplification measures kentrogon
General PCR system in above table 2 is carried out in the mode of qPCR after also can adding suitable fluorescence dye, the qPCR carried out like this is the quantivative approach of the total copy number for the 18S ribosomal gene adhering to barnacle in sample, but not the detailed number of the barnacle cell comprised.Such quantitative assay also has its certain practical value.The reference material quantitatively used can be the cloned plasmids of the 18S ribosomal gene (fragment) of any one barnacle in form 1.The concentration of Accurate Determining cloned plasmids stoste sample, and be diluted to 10 by 10 times
-10the secondary standard samples quantitative as qPCR.Because the sized molecules amount of cloned plasmids is unique, mass concentration and volumetric molar concentration can Accurate Measurements, and for single qPCR product, functional quality concentration and volumetric molar concentration can; And in qPCR reaction in this technology, what standard model increased is single product, and sample amplification is the length mix products closely of many kinds of barnacle, so the concentration of standard model must use volumetric molar concentration (or Molecules), the result that sample measures out is also volumetric molar concentration (or Molecules, often liter of how many molecules).Specific amplification ensures that the melt curve analysis of qPCR product only has one group of peak of a unimodal or melting temp closely (difference is generally no more than 5 degree); The specificity of amplified production and the configuration of barnacle standard model can ensure the validity that qPCR is quantitative.
Advantage of the present invention and beneficial effect:
1. provide a kind of kentrogon adhesion detection method not relying on pure culture system;
2. method provided by the invention had both been applicable to the kentrogon adhesion detection of the on-the-spot stained sample of seawater, was also applicable to the kentrogon adhesion detection on different coating surface in indoor pure culture system;
3. the present invention is applicable to the wider barnacle subspecies (see form 1) of scope in classification aspect;
4. the consumptive material involved by enforcement of the present invention is all common molecular biology reagents, is conveniently easy to get, with low cost.Described qPCR system configurations can use any common test kit.For the NPK62 test kit of Shandong large just (GREDBIO), the composition of qPCR system is shown in form 2.
Accompanying drawing explanation
Fig. 1 is primer specificity checking.
Embodiment:
The present invention is described in further detail by following examples by reference to the accompanying drawings, but is not limited to following embodiment.
This primer pair of embodiment 1 can make a distinction barnacle from several frequently seen main fouling organism
Use primers F 566, primer AmpR1 increase to common several main fouling organism, checking primer specificity, wherein 1-10 template is respectively handle knurl Ascidian, the little barnacle in east (Chthamaluschallengeri), line barnacle (Amphibalanusamphitrite), white ridge barnacle (Fistulobalanusalbicostatus), careless tongue worm, beautiful tongue worm, Shi Shi golden star, oyster A, oyster B, Mytilus edulis.
Embodiment 2 measures the attachment of barnacle from seawater link plate sample
At gravelstone island code head marine site, Weihai link plate, measure nano material and mix the coatingsurface of rear formation to the impact of kentrogon degree of adhesion here in different coating matrix (being iron oxide red and polydimethylsiloxane two kinds of matrix).During link plate, seawater temperature is about 15 degree, and the surface of steel plate stained sample collection time is 1. the 6th day, 2. the 14th day and 3. the 22nd day.Extract the grand genome of stained sample, use the barnacle Auele Specific Primer in the present invention and form 2 experiment condition amplification target 400bp fragment.Result is as form 3.
Form 3 four kinds of nano materials are in the test of the stained early stage kentrogon degree of adhesion of iron oxide red and PDMS two kinds of matrix coatings surface
Note :+number to represent the relative intensity of 400bp target product sweep signal in PCR glue figure, can tentatively reflect attachment kentrogon number.
Embodiment 3 measures the relative extent of different nano coating surface barnacle attachment
At gravelstone island code head marine site, Weihai link plate, measure different nano material mix in polydimethylsiloxane matrix after formed coatingsurface on the impact of kentrogon degree of adhesion.During link plate, seawater temperature is about 13 degree, and the surface of steel plate stained sample collection time is 1. the 6th day, 2. the 14th day and 3. the 22nd day.Extract the grand genome of stained sample, use the barnacle Auele Specific Primer in the present invention and form 2 experiment condition amplification target 400bp fragment.Result is as form 4.
Form 40 eight kinds of nano materials are in the test of the stained early stage kentrogon degree of adhesion in PDMS matrix coating surface
Note :+number to represent the relative intensity of 400bp target product sweep signal in PCR glue figure, can tentatively reflect attachment kentrogon number.
Claims (3)
1. detect a method for the early stage degree of adhesion of kentrogon, it is characterized in that: the method comprises the following steps:
(1) the design of barnacle Auele Specific Primer;
(2) in early days stained, before naked eyes adhere to barnacle as seen, the collection of the stained sample of seawater and genome extract;
(3) utilize barnacle Auele Specific Primer to carry out pcr amplification to measure the attachment of kentrogon to said gene group sample.
2. the method described in the claims 1, is characterized in that: barnacle specific primer design method is as follows: main marime fouling Eukaryotic 18S ribosomal gene sequence, finds out the specificity mononucleotide polymorphism site of barnacle.This site uses as 3 ' least significant end position of primer.The base principle of design of 3 ' penultimate of primer is, does not have mispairing or form a mispairing (weak mispairing: A-C, T-G between it and target sequence; Or the mispairing of medium tenacity: A-A, C-C, G-G), also form a mispairing (strong mispairing: T-T, T-C, A-G with non-target sequences; Or the mispairing of medium tenacity: A-A, C-C, G-G).So, primer and target sequence match completely or only have the mispairing of a 3 ' penultimate, generally do not affect expanding effect, and have continuous two mispairing of two 3 ' least significant ends with non-target sequences, generally cause increasing unsuccessfully.So can obtain barnacle Auele Specific Primer.
3. according to the described method described in the claims 1, it is characterized in that one of barnacle Auele Specific Primer is AmpR1:5 '-ATGCTTTCGCAGTAGTTCGTTG-3 ', this primer uses and can complete the mensuration that in early stage stained sample, whether kentrogon exists and judge together with universal primer F566:5 '-GGCATCACAGACCTGTTATTGC-3 '.
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| CN108893527A (en) * | 2018-06-13 | 2018-11-27 | 哈尔滨工业大学（威海） | A method of detection bryozoan is being stained early stage degree of adhesion |
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| CN108893527A (en) * | 2018-06-13 | 2018-11-27 | 哈尔滨工业大学（威海） | A method of detection bryozoan is being stained early stage degree of adhesion |
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Application publication date: 20160420 |
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