KR101934955B1 - Primer, DNA probe for detecting Bugula neritina and method for detcting Bugula neritina using the same - Google Patents

Abstract

The present invention provides a primer and a DNA probe for detecting, identifying, or determining Bugula neritina in a molecular biological method, and a method for detecting Bugula neritina in a qualitative and quantitative method using the same. The primer and the DNA probe of the present invention are gene markers specific to Bugula neritina species, and thus can be used for detecting, identifying and determining Bugula neritina in the qualitative and quantitative method. In addition, as the method for detecting Bugula neritina of the present invention is a simple, molecular biological method, distribution and spread of Bugula neritina, which is harmful invasive species against the domestic marine ecosystem, can be easily identified by using the same. The present invention is also useful in establishing a management strategy for Bugula neritina.

Description

TECHNICAL FIELD The present invention relates to a primer, a DNA probe, and a method for detecting a large-sized moss bug using the same.

The present invention relates to primers, DNA probes and the like which are used for detecting, identifying or discriminating large-leaf moss worms and a method for qualitatively or quantitatively detecting large-leaf moss worms using the same.

Bugula neritina is a typical offensive animal in Korea and is attached to artificial structures such as a buoy of a farm, a bottom of a ship, a fishing net, an anchor and all other fishing gears and a port wall. Bugula neritina ( Bugula neritina ) is a typical exotic species spreading all over the world, frequently found in shipyards , harbors and farm facilities. The large bundle moss bugs have been reported to adhere to the structures of farms and fishing boats, causing economic damage as well as causing the ship to slow down and overfuel.

The origin of the large bivalve moss bugs is known as the Mediterranean Sea, but now it is spreading worldwide and is spreading throughout the world, including China, Japan, the Pacific (South China Sea, Philippines, Vancouver Island - California coast, Hawaii, Galapagos Islands, Chile, Australia, New Zealand) It is reported to have invaded into the Atlantic Ocean (East coast of North America, Caribbean - Brazil, Mediterranean coast, European waters, Southwest Africa) and the Arctic sea of Pakistan, Sumatra, Red Sea, Suez Canal and Mauritius.

Various techniques have been developed to prevent the deterioration of the performance of vessels, offshore structures and the like due to the large-scale moss worms. For example, Korean Patent Laid-Open Publication No. 10-2017-0052601 discloses a method for preventing adhesion of adhered organisms to a structure in water, and a method of irradiating light having a wavelength of 409 to 412 nm to the structure Lt; / RTI > Korean Patent Laid-Open Publication No. 10-2015-0082501 discloses (A) a diorganopolysiloxane having two or more silanol groups in a molecule, (B) an organosilane having at least two hydrolysable groups in one molecule, and / or A partial hydrolyzed condensate thereof, and (C) a pyrithione metal salt.

On the other hand, in order to prevent the destruction of the marine ecosystem by the large-bore moss bugs, it is necessary to precede to grasp the distribution status of large moss bugs. Conventional large-scale moss bug identification method relies on morphological identification or DNA barcode method. However, morphological identification requires only taxonomic expertise because taxonomic expertise is required. The DNA barcode method uses a universal primer to amplify the 5'-terminal region of the COI gene and amplify the nucleotide sequence of the amplified gene And compared with the gene of the large bundle moss beetle COI (Cytochrome C Oxidase Subunit I), which was time-consuming and costly and impossible to quantitatively measure.

SUMMARY OF THE INVENTION The present invention has been made under the background of the prior art, and an object of the present invention is to provide a primer and a DNA probe used for detecting, identifying, or identifying a large-bore moss worm by a molecular biological method, Or quantitatively detecting the presence or absence of the antigen.

In order to solve the above-mentioned object, the present invention provides a primer set for detection of Bugula neritina comprising a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2.

In order to solve the above object, the present invention provides a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 4 and having a form wherein a part of the nucleotide sequence is deleted from the nucleotide sequence of SEQ ID NO: 3 or a polynucleotide having a complementary base sequence It provides a large bundle moss worm (Bugula neritina) detected for the DNA probe.

In order to solve the above object, the present invention provides a bacterium having a nucleotide sequence of SEQ ID NO: 4 or a bacterium belonging to the genus Bugula neritina ). < / RTI >

(A1) A DNA sample to be detected is used as a template and a large bivalve mugwort PCR (polymerase chain reaction) using a neritina species-specific primer set; And (a2) adding to the polymerase chain reaction (PCR) product a large bivalve mugwort neritina) species provides a large bundle moss worm (Bugula neritina) detection method comprising a step to determine whether a specific DNA markers exist.

In order to solve the above-mentioned object, the present invention provides (b1) a DNA sample to be detected is used as a template, and a large bivalve mugwort performing PCR (polymerase chain reaction) using neritina species-specific primer set and Bugula neritina species-specific DNA marker detection means; And (b2) measuring fluorescence intensity during or after the PCR is performed. The method of detecting Bugula neritina comprises the steps of:

In order to solve the above-mentioned object, the present invention relates to a bugula neritina ) Species specific primer set and large bug moss ( Bugula neritina) species provides a specific DNA marker detecting large bundle moss worm (Bugula neritina) detection kit including means.

Since the primers and DNA probes of the present invention are gene markers specific to large-bark moss species, they can be used to qualitatively or quantitatively detect, identify, or discriminate large-bark moss bugs. In addition, since the method of detecting a large-sized moss worms of the present invention is a simple molecular biological method, it can easily confirm the distribution and spread of a large-sized moss worm, which is a harmful invasion species of domestic marine ecosystem, . ≪ / RTI >

FIG. 1 is a schematic diagram showing a part of the nucleotide sequence of COI (Cytochrome C Oxidase Subunit I) of 3 large moss moths collected in Korea corresponding to a large moss moss species-specific primer and a large- Of FIG.
FIG. 2 shows the results of PCR using BuNe_SF1 and BuNe_SR1, which are genomic DNAs of three large moss moths collected in Korea and 19 other marine mammals collected in Korea as templates and specific primers of large moss moss species, The amplified product was analyzed by electrophoresis.
FIG. 3 is a standard curve that can be used for the quantitative detection of large-leaf moss worms by real-time PCR.
Figure 4 shows the result of analyzing the distribution of large moss mosses in Korea through real-time PCR and real-time polymerase chain reaction (B) to be.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a primer set that can be used for detecting, identifying, or determining whether a large-sized moss worm exists in a predetermined sample by PCR (polymerase chain reaction). A primer set for detecting Bugula neritina according to an embodiment of the present invention comprises a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2. In accordance with one embodiment of the present invention, Bugula neritina) detection primer set for the case of performing PCR using a large bundle of moss insect species-specific since the configuration of the nucleotide sequence, the mold (template) to a large bundle moss worm (Bugula neritina) genomic DNA larger bundle moss worm (Bugula neritina ) species-specific DNA marker. The large bundle of moss worm (Bugula neritina) species-specific DNA marker is a polynucleotide fragment consisting of the nucleotide sequence of SEQ ID NO: 3. The polynucleotide fragment consisting of the nucleotide sequence of SEQ ID NO: 3 or its complementary base sequence is called a Bugula neritina ), it can be used as a DNA marker to distinguish Bugula neritina from other organisms. On the other hand, a large bundle of moss worm (Bugula neritina) detection primer set according to an embodiment of the present invention, when performing PCR using the genomic DNA of other organisms other than the mold (template) to a large bundle moss worm (Bugula neritina) No separate amplification is generated.

One aspect of the present invention relates to a method for detecting, identifying, or identifying whether a large-sized moss worm is present in a predetermined sample by PCR (polymerase chain reaction) or measuring the amount of a large moss worm contained in a predetermined sample The present invention relates to a DNA probe which can be used as a DNA probe. In accordance with one embodiment of the present invention, Bugula The DNA probe for detecting neritina comprises a polynucleotide having a form comprising the nucleotide sequence of SEQ ID NO: 4 and having a form in which part of the nucleotide sequence is deleted from the nucleotide sequence of SEQ ID NO: 3 or a polynucleotide having a complementary base sequence thereof. In addition, a large bundle of moss worm (Bugula neritina) detecting DNA probes in accordance with one embodiment of the present invention preferably comprises a nucleotide sequence or a complementary base sequence of SEQ ID NO: 4. In accordance with one embodiment of the present invention, Bugula neritina) detecting DNA probe is the above-described large bundle moss worm (Bugula neritina ) detection primer set, which is a product amplified by a large moss worm ( Bugula neritina ) species-specific DNA markers, it is possible to add a large number of mugwort ( Bugula) neritina ) can be used to determine whether a species-specific DNA marker is present or to measure the amount present. According to one embodiment of the invention, a large bivalve moss bug neritina ) detecting DNA probe is preferably labeled at the terminal with a fluorescent substance. When the end of a DNA probe for detecting Bugula neritina according to an embodiment of the present invention is labeled with a fluorescent substance, a target sample (for example, a real-time polymerase chain reaction, also referred to as qPCR) , It is possible not only to detect, identify, or discriminate whether or not a large bundle moss worm exists, but also to measure the amount of the large bundle moss worm contained in the sample to be tested. Specifically, a DNA probe for detecting a large bugula neritina according to an embodiment of the present invention is a DNA probe for detecting a Bugula neritina , wherein one end (for example, the 5 'end) is labeled with a reporter dye and the other end ) May be TaqMan probes labeled with quencher dye. The reporter dye at the 5 'terminus is selected from the group consisting of 6-carboxyfluorescein (FAM), 2', 4 ', 5', 7 ', - tetrachloro-4,7-dichlorofluorane 4'-dimethoxy-4 ', 5', 7 ', -tetrachloro-4-7-dichlorofluorescein, TET and 2', 7'-dimethoxy- ', 5'-6-carboxyrhodamine, JOE) and VIC ™. The quencher dye at the 3'-end may be any of those developed to date including tetramethylrhodamine (TAMRA), BHQ-1, BHQ-2 and a minor groove binder with a non-fluorescent quencher (NFQ) It may be used. According to one embodiment of the invention, a large bivalve moss bug When neritina) DNA probes for detecting the taekmaen probes (TaqMan probes) large bundle moss worm (Bugula neritina) by PCR case having the form of species-specific DNA marker is amplified large bundle moss worm (Bugula neritina) for detecting DNA probe When the DNA polymerase that binds to a part of the amplified product and is polymerizing the DNA at the 5 'side meets with the reporter dye labeled at the 5' end of the probe, the fluorescence is generated. Thus, large bugula nematina ( Bugula neritina ) can be detected qualitatively or quantitatively through the inherent fluorescence emitted by the reporter dye. On the other hand, when the PCR was carried out, a large bivalve mugwort ( Bugula neritina ) If a species-specific DNA marker is not amplified or a polynucleotide fragment having a different base sequence is amplified, the fluorescence of the reporter dye labeled at the 5'-end of the probe is absorbed by the labeled light-absorbing dye at the 3'- No.

One aspect of the present invention relates to a method of detecting a large bug mugwort ( Bugula neritina ) through PCR (polymerase chain reaction). Large bundle in accordance with one embodiment of the present invention moss worm (Bugula neritina) detection method (a1) with a DNA sample to be detected as a template and a large bundle moss worm (Bugula PCR (polymerase chain reaction) using a neritina species-specific primer set; And (a2) adding to the polymerase chain reaction (PCR) product a large bivalve mugwort neritina ) species-specific DNA marker is present. In accordance with one embodiment of the present invention, Bugula neritina ) detection method in which the large bivalve mugwort ( Bugula The neritina species-specific primer set consists of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2. In addition, the large bundle moss worm (Bugula in large bundles moss worm (Bugula neritina) detection method according to an embodiment of the present invention. neritina ) species-specific DNA marker comprises the nucleotide sequence of SEQ ID NO: 3 or a complementary base sequence thereof. In addition, large bundle in accordance with one embodiment of the present invention moss worm (Bugula In the neritinase detection method, step (a2) is preferably performed by electrophoresis of a polymerase chain reaction (PCR) product using agarose gel or polyacrylamide gel, RTI ID = 0.0 > DNA < / RTI > When the size of the DNA band as a result of the electrophoresis in the step (a2) is 185 bp (base pair), the large bivalve mugwort neritina ) species-specific DNA marker.

Another aspect of the present invention relates to a method for detecting large Bugula neritina through real-time polymerase chain reaction (PCR). Large bundle in accordance with another embodiment of the present invention moss worm (Bugula (b1) using a DNA sample to be detected as a template and using a large bivalve mugwort ( Bugula) performing PCR (polymerase chain reaction) using neritina species-specific primer set and Bugula neritina species-specific DNA marker detection means; And (b2) measuring fluorescence intensity during or after the PCR. Large bundle in accordance with another embodiment of the present invention moss worm (Bugula neritina) the large bundle moss worm (Bugula neritina) species-specific primers in the detection method is composed of a reverse primer consisting of the nucleotide sequence of the forward primer and SEQ ID NO: 2 consisting of the nucleotide sequence of SEQ ID NO: 1. In addition, large bundle in accordance with another embodiment of the present invention moss worm (Bugula neritina ) detection method in which the large bivalve mugwort ( Bugula neritina ) species-specific DNA marker comprises the nucleotide sequence of SEQ ID NO: 3 or a complementary base sequence thereof. In addition, large bundle in accordance with another embodiment of the present invention moss worm (Bugula neritina) is the large bundle moss worm (Bugula neritina) species-specific DNA marker detection means is larger bundle moss worm (Bugula neritina) detecting DNA probe described above in the detection method. The bugula of the present invention neritina) when the detection method of detecting a large bundle moss worm (Bugula neritina) with real-time PCR (Real-time polymerase chain reaction) larger bundle by PCR amplification of water detecting means moss worm (Bugula neritina) species-specific DNA marker detection means Instead of excluding the use of nonspecific detection means capable of binding double-stranded DNS (ds DNA), such as SYBR Green, the non-specific detection means are not associated with large bugula nematina , such as primer dimer It can also interfere with precise monitoring of large bug worms ( Bugula neritina ) in combination with products.

The bugula of the present invention described above The DNA samples to be detected in the neritina detection method include genomic DNA. In the present invention, various known methods can be used for extracting the genomic DNA from animal tissue or a sample containing the same, and the genomic DNA can be extracted using a commercial genomic DNA extraction kit.

One aspect of the present invention relates to a method for producing Bugula neritina detection kit. In one example of the present invention, other large moss worms ( Bugula neritina) detection kit comprises a large bundle moss worm (Bugula neritina) species-specific primer sets and the large bundle moss worm (Bugula neritina) species-specific DNA marker detection means. Large bundles moss worm (Bugula neritina) the large bundle moss worms in the detection kit (Bugula neritina) species-specific primer sets in accordance with an embodiment of the present invention, the nucleotide sequence of the second forward primer and SEQ ID NO: consisting of the nucleotide sequence of SEQ ID NO: 1 Lt; / RTI > primer. In addition, large bundle in accordance with one embodiment of the present invention moss worm (Bugula neritina) in the detection kit large bundle moss worm (Bugula neritina) species-specific DNA marker comprises a nucleotide sequence or its complementary nucleotide sequence of SEQ ID NO: 3. In addition, the large bundle moss worm (Bugula in large bundles moss worm (Bugula neritina) detection kit in accordance with one embodiment of the present invention. neritina) species-specific DNA marker detection means is above a large bundle moss worm (Bugula neritina ). In addition, large bundle in accordance with one embodiment of the present invention moss worm (Bugula neritina detection kit may further include amplification means capable of amplifying the DNA sample in addition to the primer set and the DNA marker detection means. The amplification means may be a conventional means for performing PCR (PCR), for example, a polymerase chain reaction

And can be a reaction mixture in which the polymerase chain reaction can be performed. The reaction mixture may contain a reaction buffer, a Taq DNA polymerase, dNTP, and MgCl _{2. When the} PCR reaction is a real-time PCR, it may further comprise means for measuring the degree of fluorescence Lt; / RTI > The amplification means may further comprise a polymerase chain reaction reactor in addition to the reaction mixture. The polymerase chain reaction may be a conventional polymerase chain reaction, and may be, for example, a real-time PCR, a thermal block PCR, or a micro-PCR , But is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

One. Large bundle moss bug  Species-specific primer lt; / RTI > Large bundle moss bug  Fabrication of species-specific probes

The nucleotide sequence of the COI (Cytochrome C Oxidase Subunit I) in the closely related species of the giant animal, the echinoderms living in the marine environment, the zoospore, and the chordate animal are retrieved from the GenBank of the National Center for Biotechnology Information (NCBI) And then compared with the nucleotide sequence of COI (Cytochrome C Oxidase Subunit I) of three large moss moths collected in Korea. Based on this, a large - mongrel species - specific primer was constructed. Taxonomic groups compared with 3 large moss mosses collected in Korea include 13 species of 7 species of Phylum Bryozoa; 56 species of 24 species of seagrass (Class Ascidiacea) of Phylum Chordata; 30 species of Class Hydrozoa of Phylum Cnidaria, 27 species of 23 species of Class Anthzoa and 11 species of 6 species of jellyfish (Class Scyphozoa); Phylum Echinodermata Spider 13 species of Class Ophiuroidea 13 species, Class Asteroidea 6 species 10 species, Class Echinoidea species 16 species 19 species, Sea Holocyte species 13 species 14 And Crinozoa (Class Crinozoa) 4 species and 7 individuals. A total of 128 species were 200 individuals. Specific species and individual names are as follows.

* 3 large moss worms collected in Korea

Bugula neritina (anomokhang), Bugula neritina (Donghae Port), Bugula neritina ( ayazin )

* Phylum Bryozoa 7 species 13 species

Flustrellidra hispida (DQ157889), Flustrellidra hispida (DQ157889), Bacillus niger (AY690838), Bugula neritina (NC010197), Celleporella hyalina (JQ839275), Celleporella hyalina (JQ839276), Celleporella hyalina (NC018344), Flustra foliacea (NC008192), Membranipora grandicella (NC018355), Tubulipora flabellaris (EU563937), Watersipora subtorquata (EU365892), Watersipora subtorquata (NC011820)

* Phylum Chordata Class Ascidiacea (24 species, 56 individuals)

Botrylloides nigrum (HF548559), Botrylloides nigrum (HF548559), Botrylloides leachii (HF548553), Botrylloides leachii (HG931921), Botrylloides leachii (NC024103), Botrylloides nigrum (HF548559), Botrylloides nigrum (HF548559), Aspergillus niger Botryllus schlosseri (HF548550), Botryllus schlosseri (HF548551), Botrylloides violaceus (NC021467), Botrylloides pizoni (HF548554), Botrylloides pizoni (HG931922), Botrylloides pizoni (NC024104), Botrylloides violaceus (HF548552) ), Ciona savignyi (NC004570), Ciona intestinalis (NC004470), Ciona intestinalis (NC004447), Ciona intestinalis (NC017929), Ciona savignyi (NC045709), Botrylus schlosseri (HG931923) Clavelina lepadiformis (AM292603), Clavelina lepadiformis (FJ839918), Clavelina lepadiformis (NC012887), Clavelina phlegraea (AM292604), Clavelina phlegraea (NC024105), Didemnum and xillum (KM259616) , Halocynthia spinosus (NC021466), Herdmania momus (NC021466), Diplomas listerianum (FN313539), Diplosomes listerianum (NC013556), Halocynthia roretzi (AB024528), Halocynthia roretzi Phallusia mammilata (NC009833), Polycarpa mytiligera (HF548556), Phallusia mytiligera (HF548556), Phallusia fumigata (AM292602), Phallusia fumigata (NC009834) , Styela plicata (NC013565), Styela plicata (NC013565), Polycarpa mytiligera (NC021464), Pyura gangelion (HF548557), Pyura gangelion (NC021465), Rhodosoma turcicum HF548560, Rhodosoma turcicum NC021468, Styela clava HG931920,

* Phylum Cnidaria Class Hydrozoa (22 species, 30 individuals)

, Cubaia aphrodite (NC016467), Ectopleura Aplysina (NC016467), Amphimedon compressa (EU237474), Chondrilla aff (EU237478), Clava multicornis (JN700935), Clava multicornis (NC016465), Craspedacusta sowerbyi (JN593332), Craspedacusta sowerbyi Hydra oligacytis (EU237491), Hydra oligacytis (EU237491), Ephydatia muelleri (EU237481), Ephydatia muelleri (NC010202), Halisarca dujardini (EU237483), Hippospongia lachne (NC010214), Hydra sinensis (JX089978), Hydra sinensis (NC021406), Hydra vulgaris (HM369414), Igernella notabilis (EU237485), Iotrochota birotulata (EU237486), Laomedea flexuosa (NC016463), Plakinastrella cf. Oncodes (EU237487), Topsentia ophiraphidites (EU237482), Turritopsis dohrnii (KT020766), Turritopsis dohrnii (KT899097), Turritopsis dohrnii (NC031213), Vaceletia sp. (EU237489), Xestospongia muta (EU237490)

* Phylum Echinodermata Class Ophiuroidea 7 species 13 species

, Ophiopholus aculeata (AF314589), Amphipholis squamata (FN562578), Amphipholis squamata (NC013876), Astrospartus mediterraneus (FN562580), Astrospartus mediterraneus (NC013878), Ophiacantha linea (NC023254), Ophiocomina nigra (NC005334), Ophiura albida (AM404180), Ophiura albida (NC010691), Ophiura lutkeni (AY184223), Ophiura lutkeni (NC005930)

* Echinodermata Class Asteroidea (Echinodermata Class 6) 10 species

Astropasia polyacanthus (AB183560), Astropecten polyacanthus (AB183560), Acanthaster brevispinus (AB231476), Acanthaster brevispinus (NC007789), Acanthaster planner (AB231475), Acanthaster planner (NC007788), Aphelasterias japonica (NC025766), Asterias amurensis (NC006666), Luidia quinalia (AB183558)

* Phylum Echinodermata Class Echinoidea (16 species, 19 individuals)

Nocospatangus alta (NC023255), Paracentrotus lividus (NC023255), Mesocentrotus nudus (NC020771), Nocospatangus alta (NC023255), Aristocene spp. Strongylocentrotus pallidus (NC009941), Strongylocentrotus intermedius (NC023772), Strongylocentrotus pallidus (NC009941), Strongylocentrotus droperidus (NC09940), Strongylocentrotus intermedius (NC023772), Pseudocentrotus depressus (NC023773), Sterechinus neumayeri (NC027063), Strongylocentrotus droebachiensis (EU054306) ), Strongylocentrotus purpuratus (NC001453), Temnopleurus hardwickii (NC026200)

* Phylum Echinodermata Class Holothuroidea (13 species, 14 individuals)

, Parrotichopus californicus (NC013874), Amphipholis squamata (NC013876), Apostichopus japonicus (EU294194), Astrospartus mediterraneus (NC013878), Balanoglossus clavigerus (NC013877), Cucumaria miniata (AY182376), Holothuria forskali (NC013884), Holothuria scabra (NC026727), Parastichopus nigripunctatus (NC013432), Parastichopus parvimensis (NC029699), Peniagone sp. (KF915304), Stichopus horrens (HQ000092), Stichopus horrens (NC014454)

* Phylum Cnidaria Class Anthozoa (23 species, 27 individuals)

Dendronephthya pestia (HQ694726), Dendronephthya pateria (HQ694726), Acanella eburnea (EF672731), Acanella eburnea (NC011016), Alicia sansibarensis (NC027610), Bolocera tuediae (NC022470), Dendronephthya castanea Euplexaura crassa (NC020458), Heliopora coerulea (NC020375), Junceella fragilis (NC024181), Dendronephthya suensoni (NC022809), Echinogorgi complexa (NC020457), Euplexaura crassa (H0694528). (DQ825686), Scleronephthya gracillimum (DQ825686), Pseudopterogorgia bipinnata (NC008157), Pseudopterogorgia bipinnata (NC008157), Renilla muelleri (NC018378), Madagis mirabilis (EU400212), Muricea crassa (NC029697), Muricea purpurea (NC029698), Narella hawaiinensis (KM015351) (NC023344), Sinularia peculiaris (NC018379), Stylophora pistillata (EU400214), Urticina eques (NC022469)

* Jellyfish Jellyfish (Phylum Cnidaria Class Scyphozoa) 6 species 11 individuals

Aurelia aurita (DQ787873), Aurelia aurita (HQ694729), Aurelia aurita (NC008446), Aurelia sp. (LC005413), Aurelia sp. Haliclystus antarcticus (NC030337), Chrysaora quinquecirrha (NC020459), Haliclystus antarcticus (KU947038), Haliclystus antarcticus (NC030337), Cassiopea frondosa (NC016466)

* Phylum Echinodermata Class Crinozoa 4 species, 7 individuals

(NC007690), Phytophthora gracilis (NC007690), Phytophthora gracilis (NC007690), Antilon mediterranea (AM404181), Antedon mediterranea (NC010692), Florometra serratissima (NC001878), Neogymnocrinus richeri (DQ068951), Neogymnocrinus richeri

A conserved region with few mutations in 3 large moss mosses and different from other comparative species was selected and primers were constructed. In particular, when the 3 'terminal region is not identical, the site of the 3' terminal region (1-5 bp) that is different from the other species is selected using the characteristics of the polymerase chain reaction (PCR) Respectively. Table 1 below shows primers that amplify large-moss moth species-specific gene markers by PCR (polymerase chain reaction).

Name and Function SEQ ID NO: The base sequence (5 '-> 3') BuNe_SF1: Forward primer One 5'-GGT ACA TTA TAC TTT TTA TTT GGA C-3 ' BuNe_SR1: Reverse primer 2 5'-CCC CCA ATT ATA ACT GGT ATG -3 '

When PCR was performed using the genomic DNA extracted from the large-bore moss bug as a template and the primer described in Table 1, the nucleotide sequence of SEQ ID NO: 3 (SEQ ID NO: 3) corresponding to a part of the entire nucleotide sequence of the large Biceps moth, COI (Cytochrome C Oxidase Subunit I) And a complementary nucleotide sequence capable of hybridization to the amplified DNA marker can be used as a probe for detecting a large-sized moss worm.

In addition, the present inventors selected a region having no mutation in 3 large moss mosses among the internal base sequences of the 185 bp long large moss moss species-specific DNA marker having the nucleotide sequence of SEQ ID NO: 3 and using this as a final probe Respectively. In order to use the final probe for real-time polymerase chain reaction (qPCR), a reporter dye (FAM) was labeled at the 5 'end, and a fluorescent dye quencher dye, BHQ-1. Table 2 below shows the final probe for the detection of a large bundle moss worm produced by the present inventor.

designation SEQ ID NO: The base sequence (5 '-> 3') BuNe probe 4 5'-Fam-CCA GGC AGC TTA TTA GGT AAC GACCA-BHQ-1 -3 '

FIG. 1 is a schematic diagram showing a part of the nucleotide sequence of COI (Cytochrome C Oxidase Subunit I) of 3 large moss moths collected in Korea corresponding to a large moss moss species-specific primer and a large- Of FIG.

2. Large bundle moss bug  Species-specific The primer  Used Large bundle of moss worms  Qualitative detection experiment

(1) Experimental method

We extracted genomic DNA from 3 domestic large moss mosses, 5 mosses from large moss moss worms, 2 disturbed harmful benthos and 12 marine benthos. The specific information of the animal to be analyzed is as follows.

* 3 large moss worms collected in Korea

Bugula neritina (anomokhang), Bugula neritina (Donghae Port), Bugula neritina ( ayazin )

5 relatives of large bivalve moss bugs

Tricellaria occidentalis , Watersipora subtorquata , Schizoporella, unicornis ), California moss bug ( Bugulina californica ), Membranipora tuberculata

* 2 disturbed benthic creatures

Asturias amurensis ), Starfish ( Patiria pectinifera )

* 12 species of marine macaroni

Herdmania monus ), tube hydra ( Ectopleura crocea , Ascidiella aspersa , Bougainvillia muscus ), thorny sparrow ( Antedon serrata ), long beard spider ( Heliometra glaciali ), snake spider starfish ( Ophiactis savignyi ), horn spider starfish ( Ophiopholis mirabilis), Hardwick branched urchins (Temnopleurus hardwickii), thin gwangeuk urchins (Prionocidaris japonica), pentagonal gwangsam (Eupentacta chronhjelmi), visible anchor sea cucumber (Protankyra bidentata)

The polymerase chain reaction (PCR) was performed using the extracted genomic DNA as a template and using the large-mosquito insect species-specific primer shown in Table 1 above. Thereafter, the products amplified by PCR (polymerase chain reaction) were electrophoresed on an agarose gel, and were separated and analyzed by size.

(2) Genomic DNA extraction method of animal to be analyzed

The genomic DNA was extracted with DNeasy blood and tissue DNA isolation kit (QIAGEN, Hilden, Germany). The extraction procedure followed the procedure provided by the kit manufacturer.

(3) PCR (polymerase chain reaction) conditions

* Primers used: large strand moss insect species specific primers BuNe_SF1 and BuNe_SR1

* PCR reaction solution composition: The total volume of the PCR reaction solution is 25 μl, and the contents of the PCR reaction solution are as follows.

- 10 x Ex Taq Buffer with 25 mM MgCl ₂ (Clontech, USA)

 - 2.5 mM dNTPs (Clontech, USA)

 - 10 pmol primer

 - 1 μl template genomic DNA

 - 5 U / Ta Taq polymerase (Clontech, USA)

* PCR process conditions: The PCR process consists of Denaturation, Anealing and Extension. The specific conditions are as follows.

 - Denaturation: 94 ° C, 5 min.

 - Anealing: 94 ° C, 30 sec .; 65 ° C, 90 sec .; 72 ° C, 90 sec .; 30 cycles

 Extension: 72 캜, 7 min.

(4) Results of electrophoresis analysis

FIG. 2 shows the results of PCR using BuNe_SF1 and BuNe_SR1, which are genomic DNAs of three large moss moths collected in Korea and 19 other marine mammals collected in Korea as templates and specific primers of large moss moss species, The amplified product was analyzed by electrophoresis. The description of each lane in FIG. 2 is as follows.

lane 1: 100 bp DNA ladder marker; lane 2, 3, and 4: genomic DNA of three large moss moths collected in Korea as a template; * lane 5: using the genomic DNA of the California moss worm ( Bugulina californica ) as a template; * lane 6: using the genomic DNA of Watersipora subtorquata as a template; * lane 7: Tricellaria occidentalis ) as a template; * lane 8: Schizoporelli unicornis ) as a template; * lane 9: Membranipora tuberculata ) as a template; * lane 10: using the genomic DNA of the tube hydra ( Ectopleura crocea ) as a template; * lane 11: Bougainvillia muscus genomic DNA as a template; * lane 12: Ascidiella aspersa ) as a template; * lane 13 and 14: 100 bp DNA ladder marker; * lane 15: using the genomic DNA of Herdmania momus as a template; * lane 16: Using the genetic DNA of anthony serrata as a template; * lane 17: using the genomic DNA of Heliometra glacialis as a template; * lane 18: using the genomic DNA of the starfish ( Patiria pectinifera ) as a template; * lane 19: using the genomic DNA of the starfish ( Asterias amurensis ) as a template; * lane 20: uses the genomic DNA of the starfish ( Ophiactis savignyi ) as a template; * lane 21: using the genomic DNA of the horned starfish ( Ophiopholis mirabilis ) as a template; * lane 22: Hardwing basin ( Temnopleurus hardwickii ) as a template; * lane 23: using the genomic DNA of Prionocidaris japonica as a template; * lane 24: Eupentacta chronhjelmi ) as a template; * lane 25: Protankyra bidentata ) as a template; lane 26: 100 bp DNA ladder marker

3. Real time PCR (Real-time polymerase chain reaction) Large bundle of moss worms  Analysis of domestic distribution status

(1) Experimental method

In order to confirm the presence and detection amount of large moss mosses distributed in the whole country including Jeju Island, BuNe_SF1 and BuNe_SR1, which are large moss moss species specific primers, and final probes for large moss moss species - specific DNA markers Real-time polymerase chain reaction (QPCR) analysis was performed using a BuNe probe. Specifically, in the west coast, the south coast, and the east coast, there are 22 areas (Daejin Port, Gijin Port, Gonghyeon Port, Zhangjiagang, Zizhong Port, Sichuan Port, Mukho Port, Donghae Port, , Jeju Island (Jeju Port, Jocheon Port, Gwacheon Port), Busan Port, Gwangyang Port, Tongyoung Port, Mokpo Port, (September 16th), winter (February 17th), spring (16th) in Bukchon Port, Gimnyeong Port, Jindal Port, Seongsan Port Pohang, Seokseong Port, Wuhmi Port, Seogwipo Port, Mosul Port, Hanlim Port, March), the presence of large bivalve moss bugs was visually inspected. In addition, after collecting seawater from 35 areas, genomic DNA of various organisms contained in seawater was extracted, and real - time PCR analysis was performed using it as a template and the distribution region of large moss bugs was confirmed.

(2) Genomic DNA extraction method of various organisms contained in seawater

8 L of seawater was collected from domestic shore and then filtered using a membrane filter (pore size: 0.2 μm; Merck Millipore, Darmstadt, Germany). Then, the membrane filter was frozen in a 1.5-ml tube, and genomic DNA of various organisms contained in seawater was extracted using a genomic DNA extraction kit (Bioneer, Korea) in the laboratory. The extraction procedure followed the procedure provided by the kit manufacturer.

(3) Real-time PCR conditions

* Primers and probes used: BuNe_SF1 and BuNe_SR1, which are large-moss moss specimen-specific primers, and BuNe probe, which is the final probe for the large-moss moss species-specific DNA marker

* PCR Reaction Solution Composition: The total volume of the PCR reaction solution is 20 μl.

 - DEPC treated water

 - 5 μl template genomic DNA (extracted from seawater samples)

 - 5 pmol TaqMan probe (metabion, Germany)

 - 10 pmol primer

 - PCR Probe Mix Hi-ROX (PCR BIOSYSTEMS, UK)

* PCR process conditions: The PCR process consists of denaturation and binding. Specific conditions are as follows.

 - Denaturation: 94 ° C, 3 min.

 - Anealing: 75 캜, 5 sec .; 60 C, 30 sec .; 45 cycles

(4) Real-time PCR standard curve for quantitative detection of large moss moss bugs

A standard curve was constructed to identify the distribution area of large bivalve moth bugs and to determine the amount of detection by absolute quantitative analysis of real-time PCR. FIG. 3 is a standard curve that can be used for the quantitative detection of large-leaf moss worms by real-time PCR. In the standard curve of FIG. 3, the X axis indicates the amount of DNA of the large bivalve moss worm contained in the standard sample, and the Y axis indicates the amount of the PCR amplification product when a real-time PCR is performed using a standard sample having a predetermined large- Represents the number of PCR cycles when the reference fluorescence value is detectable. Measuring the number of PCR cycles when the reference fluorescence value is detectable from the seawater sample and substituting it into the C _T value corresponding to the Y axis of the real-time PCR standard curve, the amount of DNA of the large moss bugs contained in the seawater is calculated can do.

(5) Real-time polymerase chain reaction (PCR)

Figure 4 shows the result of analyzing the distribution of large moss mosses in Korea through real-time PCR and real-time polymerase chain reaction (B) to be.

The real-time PCR analysis showed that the population of Bugula neritina was collected in 27 areas, but the distribution was observed in all 35 areas. In the 8 sites where no individuals were found in the field survey, It was found that moss worms were inhabited. On the west coast, southern coast, and east coast region (22 areas), distribution was confirmed in 20 regions through field survey. However, real-time PCR analysis showed that DNA of large moss bugs was detected in 22 regions and 2 of mokpo Further distribution was confirmed in the area. In Jeju Island area (13 areas), the distribution was confirmed in 7 areas through field survey, but 13 areas were confirmed through real-time PCR analysis. In addition, 6 areas (Jeju port, Jocheon port, Bukchon port, , And the distribution of the distribution was confirmed. The distribution pattern of Bugula neritina was widely distributed throughout the country including the eastern coast, south coast, and west coast of Korea including the unspread areas. PCR amplification was performed using a real-time PCR analysis.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the scope of protection of the present invention is not limited to the specific embodiments but should be construed as including all embodiments belonging to the claims attached hereto.

<110> SAHMYOOK UNIVERSITY INDUSTRY-ACADEMIC COOPERATION <120> Primer, DNA probe for detecting Bugula neritina and method for          detcting Bugula neritina using the same <130> DP-17-335 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BuNe_SF1 (Forward primer specific to Bugula neritina) <400> 1 ggtacattat actttttatt tggac 25 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BuNe_SR1 (Reverse primer specific to Bugula neritina) <400> 2 cccccaatta taactggtat g 21 <210> 3 <211> 185 <212> DNA <213> Artificial Sequence <220> <223> DNA marker specific to Bugula neritina <400> 3 ttgagatat cttattcgag tagaattaag acaaccaggc agcttattag gtaacgacca actttataat 120 gtaattgtca cagctcatgc atttttaata atttttttta tagtcatacc agttataatt 180 ggggg 185 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> BuNe probe (DNA probe for detecting Bugula neritina) <400> 4 ccaggcagct tattaggtaa cgacca 26

Claims (13)

A reverse primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2 ( Bugula neritina ) detection primer set.
delete delete delete delete (a1) performing PCR (polymerase chain reaction) using the DNA sample to be detected as a template and using the primer set of (1); And
(a2) a large bundle moss worm (Bugula neritina) detection method comprising a step to determine whether a large bundle moss worm (Bugula neritina) species-specific DNA marker present in the PCR (polymerase chain reaction) product.
7. The method according to claim 6, wherein the bugula neritina) species-specific DNA markers are large bundles moss worm (Bugula neritina) detecting method, characterized in that consisting of the nucleotide sequence or its complementary nucleotide sequence of SEQ ID NO: 3.
The method of claim 6, wherein (a2) step PCR (polymerase chain reaction) product electrophoresis a later, larger bundle being configured to determine the size of the DNA band resulting of the electrophoretic moss worm (Bugula neritina ) detection method.
delete delete The primer set of claim 1 and a large group of moss worms ( Bugula &lt; RTI ID = 0.0 &gt; neritina ) Large-scale moss worms including species-specific DNA marker detection means ( Bugula neritina ) detection kit.
12. The method according to claim 11, wherein the bugula neritina) species-specific DNA markers are large bundles moss worm (Bugula neritina) detection kit, characterized in that consisting of the nucleotide sequence or its complementary nucleotide sequence of SEQ ID NO: 3.
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