KR20240109319A - Primer and dna probe for detecting ascidiella aspersa, and method for detecting ascidiella aspersa using the same - Google Patents
Primer and dna probe for detecting ascidiella aspersa, and method for detecting ascidiella aspersa using the same Download PDFInfo
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- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
본 발명은 서열번호 1의 염기서열로 이루어진 정방향 프라이머 (Forward Primer) 및 서열번호 2의 염기서열로 이루어진 역방향 프라이머 (Reverse Primer)를 포함하는 거친대추멍게 검출용 프라이머 세트 및 서열번호 3의 염기서열 또는 이의 상보적 염기서열로 이루어진 프로브, 및 상기 프라이머 세트 및 프로브를 이용하여 거친대추멍게를 검출하는 방법에 관한 것으로, 본 발명에 따르면 본 발명에 따른 프라이머 세트 및 프로브는 거친대추멍게를 정성적 또는 정량적으로 검출 가능할 뿐만 아니라 거친대추멍게의 분포 및 확산 여부를 확인할 수 있다.The present invention provides a primer set for detecting rough jujube sea squirt, including a forward primer consisting of the base sequence of SEQ ID NO: 1 and a reverse primer consisting of the base sequence of SEQ ID NO: 2, and the base sequence of SEQ ID NO: 3 or It relates to a probe consisting of a complementary base sequence, and a method for detecting rough jujube sea squirt using the primer set and probe. According to the present invention, the primer set and probe according to the present invention are used to detect rough jujube sea squirt qualitatively or quantitatively. Not only can it be detected, but the distribution and spread of rough jujube sea squirts can also be confirmed.
Description
본 발명은 거친대추멍게 검출용 프라이머 및 프로브, 및 이를 이용한 거친대추멍게 검출 방법에 관한 것으로, 더욱 구체적으로 서열번호 1의 염기서열로 이루어진 정방향 프라이머 (Forward Primer) 및 서열번호 2의 염기서열로 이루어진 역방향 프라이머 (Reverse Primer)를 포함하는 거친대추멍게 검출용 프라이머 세트, 및 서열번호 3의 염기서열 또는 이의 상보적 염기서열로 이루어진 프로브, 및 상기 프라이머 세트 및 프로브를 이용하여 거친대추멍게를 검출하는 방법에 관한 것이다.The present invention relates to primers and probes for detecting rough jujube sea squirts and a method for detecting rough jujube sea squirts using the same, and more specifically, to a forward primer consisting of the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2. A primer set for detecting rough jujube sea squirts including a reverse primer, a probe consisting of the base sequence of SEQ ID NO: 3 or its complementary base sequence, and a method for detecting rough jujube sea squirts using the primer set and probe. It's about.
거친대추멍게 (Ascidiella aspersa)는 해양 고착성 저서동물로서 척삭동물문 해초강 편새해초목 대추멍게과에 속하며, ‘유럽산 멍게 (European sea squirt)’로 알려져 있다(Inglis et al. 2008; Mackenzie 2011). 해양 외래종인 거친대추멍게는 여름철에 유생이 부착하여 성장을 시작하고, 수온이 낮아지는 겨울철에는 성장을 멈추는 것으로 알려져 있다(Millar 1952). 이러한 거친대추멍게는 자연 암반, 항구의 항벽, 부두 말뚝, 밧줄 등과 같이 거칠고 단단한 표면에 부착하여 서식하며(Curtis 2005; Inglis et al. 2008), 빠른 성장률과 높은 밀도로 군락을 형성하여 침입한 지역의 해양저서동물 군집의 생물다양성을 감소시킬 뿐만 아니라(Curtis 2005; Mackenzie 2011; Picton and Morrow 2016), 먹이 경쟁이나 포식의 위험이 적은 환경인 양식장 등에서 굴, 가리비 등의 양식종의 성장을 저해하여 양식 산업에 경제적 피해를 주는 (Carman et al. 2010; Park et al. 2017) 유해해양생물이다. 종래에 거친대추멍게와 같은 유해해양생물이 양식장의 어망, 부표 등 인공구조물에 부착하여 인공구조물의 성능이 저하되는 것을 막음과 동시에 양식종의 성장을 향상시키기 위한 기술이 개발된 바 있다. 구체적으로, 한국등록특허 제0885934호는 어망용 방오도료에 관한 것으로, 바인더 성분으로서 로진 및 아크릴수지를 포함하고, 방오제 성분으로서 바셀린을 포함함으로써 표면인장력을 감소시키고 해수 중에서 강도 및 유연성을 향상시켜 도료가 어망에 견고하고 유연하게 도포되도록 함으로써 어망의 사용 시 어망의 굴곡과 비틀림에도 도료가 잘 적응하여 탈리가 발생하지 않도록 하여 한 번의 도포로 어망을 장시간 사용할 수 있도록 하고, 어망을 사용하여도 도료가 부착된 상태에서 미세한 홈 등이 발생하지 않도록 함으로써 어망에 오손생물 및 이물질이 부착되는 것을 방지하도록 한 기술이다.Rough jujube sea squirt ( Ascidiella) aspersa ) is a marine sessile benthic animal belonging to the Jujube sea squirt of the Chordata phylum Seaweed class Seaweed order Jujube sea squirt and is known as the ‘European sea squirt’ (Inglis et al. 2008; Mackenzie 2011). Rough jujube sea squirt, a marine invasive species, is known to begin growing by attaching larvae in the summer and to stop growing in the winter when the water temperature drops (Millar 1952). These rough jujube sea squirts live by attaching themselves to rough and hard surfaces such as natural rocks, harbor walls, pier piles, ropes, etc. (Curtis 2005; Inglis et al. 2008), and form colonies with rapid growth rates and high densities, invading areas. It not only reduces the biodiversity of marine benthic fauna communities (Curtis 2005; Mackenzie 2011; Picton and Morrow 2016), but also inhibits the growth of farmed species such as oysters and scallops in fish farms where there is little risk of food competition or predation. It is a harmful marine organism that causes economic damage to the aquaculture industry (Carman et al. 2010; Park et al. 2017). In the past, technologies have been developed to prevent harmful marine organisms such as rough jujube sea squirts from attaching to artificial structures such as fishing nets and buoys in fish farms, thereby preventing deterioration of the performance of artificial structures and at the same time improving the growth of farmed species. Specifically, Korean Patent No. 0885934 relates to an antifouling paint for fishing nets, which contains rosin and acrylic resin as a binder component and vaseline as an antifouling component to reduce surface tension and improve strength and flexibility in seawater. By ensuring that the paint is applied firmly and flexibly to the fishing net, the paint adapts well to the bends and twists of the fishing net and prevents detachment from occurring. This allows the fishing net to be used for a long time with a single application, and the paint remains intact even when the fishing net is used. This is a technology that prevents fouling organisms and foreign substances from attaching to the fishing net by preventing fine grooves, etc. from forming in the attached state.
상기와 같은 종래의 기술은 해양유해생물이 해양의 인공구조물에 부착되는 것을 방지할 수는 있으나, 해양 생태계를 교란시키는 것을 방지하기 위한 방법이 될 수 없으므로, 해양 생태계의 교란 및 파괴를 방지하기 위한 해양유해생물의 검출을 통한 분포 현황을 파악하는 것이 선행되어야 한다. 종래의 거친대추멍게 확인 방법은 형태적 동정에 의지하거나 DNA 바코드(Barcode) 방법을 이용하였다. 그러나 형태적 동정은 분류학적인 전문 지식이 필요하여 분류 전문가만이 가능하며, DNA 바코드(Barcode) 방법은 유니버셜 프라이머(universal primer)를 이용하여 COI(Cytochrome C Oxidase Subunit I) 유전자의 5′ 말단 부위를 증폭하고 증폭된 유전자의 염기서열을 분석하고 거친대추멍게 COI(Cytochrome C Oxidase Subunit I) 유전자와 비교하는 방법으로서 시간과 비용이 많이 들고 정량적인 측정이 불가능하였다. Although the above-mentioned conventional technology can prevent marine harmful organisms from attaching to artificial structures in the ocean, it cannot be used as a method to prevent disturbance of the marine ecosystem, so it is necessary to prevent disturbance and destruction of the marine ecosystem. Identifying the distribution status through detection of marine harmful organisms should be a priority. Conventional methods for identifying rough jujube sea squirts relied on morphological identification or used DNA barcoding. However, morphological identification requires taxonomic expertise and can only be performed by classification experts, and the DNA barcode method uses a universal primer to identify the 5′ end region of the COI (Cytochrome C Oxidase Subunit I) gene. This method was amplified, analyzed the base sequence of the amplified gene, and compared it with the rough jujube sea squirt COI (Cytochrome C Oxidase Subunit I) gene. It was time-consuming, expensive, and quantitative measurement was impossible.
이에, 본 발명자들은 상기 종래기술들의 문제점들을 극복하기 위하여 예의 연구노력한 결과, 서열번호 1의 염기서열로 이루어진 정방향 프라이머 (Forward Primer) 및 서열번호 2의 염기서열로 이루어진 역방향 프라이머 (Reverse Primer)를 포함하는 거친대추멍게 검출용 프라이머 세트와 서열번호 3의 염기서열 또는 이의 상보적 염기서열로 이루어진 프로브를 이용할 경우, 거친대추멍게를 정성적 또는 정량적으로 검출 가능할 뿐만 아니라 거친대추멍게의 분포 및 확산 여부를 확인할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the present inventors have made extensive research efforts to overcome the problems of the prior technologies, and as a result, a forward primer consisting of the base sequence of SEQ ID NO: 1 and a reverse primer consisting of the base sequence of SEQ ID NO: 2 are included. When using a primer set for detecting rough jujube sea squirts and a probe consisting of the base sequence of SEQ ID NO. After confirming that it could be confirmed, the present invention was completed.
따라서, 본 발명의 주된 목적은 거친대추멍게를 정성적 또는 정량적으로 검출 가능할 뿐만 아니라 거친대추멍게의 분포 및 확산 여부를 확인할 수 있는 거친대추멍게 검출용 프라이머 및 프로브를 제공하는 데 있다.Therefore, the main purpose of the present invention is to provide primers and probes for detecting rough jujube sea squirts, which can not only detect rough jujube sea squirts qualitatively or quantitatively, but also confirm the distribution and spread of rough jujube sea squirts.
본 발명의 다른 목적은 상기 거친대추멍게 검출용 프라이머 및 프로브를 이용한 거친대추멍게의 검출방법 및 해양 생태계 내 거친대추멍게 분포 조사 방법을 제공하는 데 있다.Another object of the present invention is to provide a method for detecting rough jujube sea squirts using the primers and probes for detecting rough jujube sea squirts and a method for investigating the distribution of rough jujube sea squirts in the marine ecosystem.
본 발명의 한 양태에 따르면, 본 발명은 서열번호 1의 염기서열로 이루어진 정방향 프라이머 (Forward Primer) 및 서열번호 2의 염기서열로 이루어진 역방향 프라이머 (Reverse Primer)를 포함하는 거친대추멍게 검출용 프라이머 세트를 제공한다. According to one aspect of the present invention, the present invention is a primer set for detecting rough jujube sea squirt, comprising a forward primer consisting of the base sequence of SEQ ID NO: 1 and a reverse primer consisting of the base sequence of SEQ ID NO: 2. provides.
거친대추멍게는 유해해양생물로서 해양의 인공구조물에 부착하여 양식종의 성장을 저해함에 따라 경제적 피해를 주고 있으므로, 해양 생태계를 교란시키는 거친대추멍게의 검출을 통한 분포 현황을 파악하는 것이 필요하다. 이에, 본 발명자들은 거친대추멍게를 정성적 또는 정량적으로 검출 가능할 뿐만 아니라 거친대추멍게의 분포 및 확산 여부를 확인하기 위한 프라이머 세트 및 프로브를 발명하게 되었다.Rough jujube sea squirts are harmful marine organisms that attach to artificial structures in the ocean and hinder the growth of farmed species, causing economic damage. Therefore, it is necessary to determine the distribution status through detection of rough jujube sea squirts that disturb the marine ecosystem. Accordingly, the present inventors have invented a primer set and probe that can not only detect rough jujube sea squirts qualitatively or quantitatively, but also confirm the distribution and spread of rough jujube sea squirts.
상기 본 발명에 따른 거친대추멍게 검출용 프라이머 세트는 거친대추멍게 종 특이적 염기서열로 구성되어 있으며, 상기 프라이머 세트를 이용하여 거친대추멍게 DNA를 주형으로 하여 중합효소 연쇄반응을 실시할 경우 거친대추멍게 종 특이적 DNA 마커를 증폭시키므로, 상기 종 특이적 DNA 마커에 대한 프로브를 이용하여 거친대추멍게만을 특이적으로 검출할 수 있다.The primer set for detecting rough jujube sea squirt according to the present invention consists of a rough jujube sea squirt species-specific base sequence, and when polymerase chain reaction is performed using the rough jujube sea squirt DNA as a template using the primer set, rough jujube sea squirt DNA is used as a template. Since the sea squirt species-specific DNA marker is amplified, only the rough jujube sea squirt can be specifically detected using a probe for the species-specific DNA marker.
상기 본 발명에 따른 프라이머는 포스포르아미다이트 고체 지지체 방법, 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 프라이머는 거친대추멍게를 검출할 수 있는 효과를 나타내는 한, 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 예로는 메틸화, 캡화, 천연 뉴클레오티드 하나 이상의 동족체로의 치환, 및 뉴클레오티드 간의 변형, 예를 들면, 하전되지 않은 연결체(예를 들어, 메틸 포스포네이트, 포스포트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체(예를 들어, 포스포로 티오에이트, 포스포로디티오에이트 등)로의 변형을 포함할 수 있다. 핵산은 하나 이상의 부가적인 공유 결합된 잔기, 예를 들면, 단백질(예를 들어, 뉴클레아제, 독소, 항체, 시그널 펩타이드, 폴리-L-리신 등), 삽입제(예를 들어, 아크리딘, 프로랄렌 등), 킬레이트화제(예를 들어, 금속, 방사성 금속, 철, 산화성 금속 등) 및 알킬화제를 함유할 수 있다. 또한, 본 발명의 프라이머는 필요한 경우, 분광학적, 광화학적, 생화학적, 면역화학적 또는 화학적 수단에 의해 직접적으로 또는 간접적으로 검출 가능한 표지를 포함할 수 있다. 표지의 예로는, 효소(예를 들어, 호스래디쉬 퍼옥시다제, 알칼린 포스파타아제), 방사성 동위원소(예를 들어, 32P), 형광성 분자 및 화학그룹(예를 들어, 바이오틴) 등이 있다.The primer according to the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These primers can be modified using many means known in the art as long as they are effective in detecting rough jujube sea squirts. Examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologues, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoroamidate). , carbamates, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). Nucleic acids may contain one or more additional covalently linked residues, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), insertions (e.g., acridine, etc.) , propalene, etc.), chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. Additionally, the primer of the present invention may, if necessary, contain a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (e.g., horseradish peroxidase, alkaline phosphatase), radioisotopes (e.g., 32P), fluorescent molecules and chemical groups (e.g., biotin), etc. there is.
본 발명의 다른 한 양태에 따르면, 본 발명은 서열번호 1의 염기서열로 이루어진 정방향 프라이머 및 서열번호 2의 염기서열로 이루어진 역방향 프라이머로 이루어진 거친대추멍게 검출용 프라이머 세트를 포함하는 거친대추멍게 검출용 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a primer set for detecting rough jujube sea squirt, comprising a forward primer consisting of the base sequence of SEQ ID NO: 1 and a reverse primer consisting of the base sequence of SEQ ID NO: 2. A composition is provided.
본 발명의 거친대추멍게 검출용 조성물에 있어서, 상기 조성물은 서열번호 3의 염기서열 또는 이의 상보적 염기서열로 이루어진 프로브를 더 포함한다. In the composition for detecting rough jujube sea squirt of the present invention, the composition further includes a probe consisting of the base sequence of SEQ ID NO: 3 or a complementary base sequence thereof.
본 발명에서의 상기 용어 ‘프로브’는 DNA 또는 RNA와 특이적으로 결합할 수 있는 수개 내지 수백 개의 염기에 해당하는 핵산 단편을 의미하며, 올리고뉴클레오티드(oligonucleotide) 프로브, 단쇄 DNA(single stranded DNA) 프로브, 이중쇄 DNA(double stranded DNA) 프로브 또는 RNA 프로브 등의 형태로 제작될 수 있다.In the present invention, the term 'probe' refers to a nucleic acid fragment of several to hundreds of bases that can specifically bind to DNA or RNA, including an oligonucleotide probe and a single stranded DNA probe. , can be produced in the form of a double stranded DNA probe or RNA probe.
상기 프로브는 포스포르아미다이트 고체 지지체 방법, 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 프로브는 거친대추멍게를 검출할 수 있는 효과를 나타내는 한, 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 예로는 메틸화, 캡화, 천연 뉴클레오티드 하나 이상의 동족체로의 치환, 및 뉴클레오티드 간의 변형, 예를 들면, 하전되지 않은 연결체(예를 들어, 메틸 포스포네이트, 포스포트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체(예를 들어, 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형을 포함할 수 있다. 핵산은 하나 이상의 부가적인 공유 결합된 잔기, 예를 들면, 단백질(예를 들어, 뉴클레아제, 독소, 항체, 시그널 펩타이드, 폴리-L-리신 등), 삽입제(예를들어, 아크리딘, 프로랄렌 등), 킬레이트화제(예를 들어, 금속, 방사성 금속, 철, 산화성 금속 등) 및 알킬화제를 함유할 수 있다.The probe can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. These probes can be modified using many means known in the art as long as they are effective in detecting rough jujube sea squirts. Examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologues, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoroamidate). , carbamate, etc.) or a charged linker (e.g., phosphorothioate, phosphorodithioate, etc.). Nucleic acids may contain one or more additional covalently linked residues, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), insertions (e.g., acridine, etc.) , propalene, etc.), chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents.
본 발명의 거친대추멍게 검출용 조성물에 있어서, 상기 프로브는 이의 5′말단에 리포터가 추가로 더 접합될 수 있으며, 상기 리포터는 당업계에서 종래에 리포터로 사용되는 어떠한 물질도 사용가능하나, 바람직하게는 상기 프로브는 5′ 말단에 FAM(6-carboxyfluorescein), 텍사스 레드(texas red), 플루오레신(fluorescein), HEX(2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE(2',7'-dimethoxy-4',5'-6-carboxyrhodamine), 플루오레신 클로로트리아지닐(fluorescein chlorotriazinyl), 로다민 그린(rhodamine green), 로다민 레드(rhodamine red), 테트라메틸로다민(tetramethylrhodamine), FITC(fluorescein isothiocyanate), 오레곤 그린(oregon green), 알렉사 플루오로(alexa fluor), ROX(6-Carboxyl-X-Rhodamine), TET(Tetrachloro-Fluorescein), TRITC(tertramethylrodamine isothiocyanate), TAMRA(6-carboxytetramethyl-rhodamine), NED(N-(1-Naphthyl) ethylenediamine) 및 씨아디카르보시아닌(thiadicarbocyanine)으로 구성된 군으로부터 선택되는 하나 이상일 수 있으며, 본 발명의 실시예에서는 FAM(6-carboxyfluorescein)를 이용하였으나, 이에 제한되지 않는다.In the composition for detecting rough jujube sea squirts of the present invention, a reporter may be additionally conjugated to the 5' end of the probe, and the reporter may be any material conventionally used as a reporter in the art, but is preferred. Specifically, the probe contains FAM (6-carboxyfluorescein), Texas Red, fluorescein, and HEX (2',4',5',7'-tetrachloro-6-carboxy-) at the 5' end. 4,7-dichlorofluorescein), JOE (2',7'-dimethoxy-4',5'-6-carboxyrhodamine), fluorescein chlorotriazinyl, rhodamine green, rhodamine red (rhodamine red), tetramethylrhodamine, FITC (fluorescein isothiocyanate), Oregon green, Alexa fluor, ROX (6-Carboxyl-X-Rhodamine), TET (Tetrachloro-Fluorescein) ), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), and thiadicarbocyanine, and may be one or more selected from the group consisting of the present invention. In the example, FAM (6-carboxyfluorescein) was used, but the method is not limited thereto.
본 발명의 거친대추멍게 검출용 조성물에 있어서, 상기 프로브는 이의 3′말단에 소광자가 추가로 더 접합될 수 있으며, 상기 소광자는 당업계에서 종래에 리포터로 사용되는 어떠한 물질도 사용가능하나, 바람직하게는 BHQ1(black hole quencher 1), BHQ2(black hole quencher 2), BHQ3(black hole quencher 3), AMRA(6-carboxytetramethyl-rhodamine), NFQ(nonfluorescent quencher), 답실(dabcyl), DDQ(Deep Dark Quencher), 블랙베리 퀸처(Blackberry Quencher) 및 아이오와 블랙(Iowa black)로 구성된 군으로부터 선택되는 하나 이상일 수 있으며, 본 발명의 실시예에서는 BHQ1(black hole quencher 1)을 이용하였으나, 이에 제한되지 않는다.In the composition for detecting rough jujube sea squirts of the present invention, the probe may be further conjugated with a quencher at its 3' end, and the quencher may be any material conventionally used as a reporter in the art, but is preferred. BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), AMRA (6-carboxytetramethyl-rhodamine), NFQ (nonfluorescent quencher), dabcyl, DDQ (Deep Dark) Quencher), blackberry quencher (Blackberry Quencher), and Iowa black (Iowa black). In the embodiment of the present invention, BHQ1 (black hole quencher 1) was used, but is not limited thereto.
상기 조성물에는 상기 프라이머 세트 및 프로브 이외에 역전사 중합효소, DNA 중합효소, Mg2 +와 같은 조인자, dATP, dCTP, dGTP 및 dTTP가 포함될 수 있다. 역전사된 cDNA를 증폭하기 위하여 다양한 DNA 중합효소가 본 발명의 증폭 단계에 이용될 수 있으며, DNA 중합효소의 예로 E. coli DNA 중합효소 I의 클레나우 단편, 열안정성 DNA 중합효소 또는 박테리오파지 T7 DNA 중합효소가 있다. 중합효소는 박테리아 그 자체로부터 분리하거나 상업적으로 구입하거나 중합효소를 암호화하는 클로닝 유전자의 높은 레벨을 발현하는 세포로부터 수득할 수 있다.In addition to the primer set and probe , the composition may include reverse transcription polymerase, DNA polymerase, cofactors such as Mg 2+ , dATP, dCTP, dGTP, and dTTP. To amplify the reverse transcribed cDNA, various DNA polymerases can be used in the amplification step of the present invention. Examples of DNA polymerases include the Klenow fragment of E. coli DNA polymerase I, thermostable DNA polymerase, or bacteriophage T7 DNA polymerase. There are enzymes. Polymerase can be isolated from the bacteria themselves, purchased commercially, or obtained from cells expressing high levels of the cloned gene encoding the polymerase.
본 발명의 일 실험예에 따르면, 본 발명에 따른 서열번호 1의 염기서열로 이루어진 정방향 프라이머 및 서열번호 2의 염기서열로 이루어진 역방향 프라이머로 이루어진 거친대추멍게 검출용 프라이머 세트를 포함하는 거친대추멍게 검출용 조성물를 이용하여 PCR을 통해 거친대추멍게의 정성적 검출을 확인한 결과, 본 발명에 따른 프라이머 세트 또는 이를 포함하는 조성물은 거친대추멍게의 근연종, 교란유해 저서동물 및 해양 저서동물은 검출하지 못하였으나, 거친대추멍게 개체만을 특이적으로 검출하는 것을 확인하였다(실험예 1, 도 2 참조). 또한, 본 발명에 따른 서열번호 1의 염기서열로 이루어진 정방향 프라이머 및 서열번호 2의 염기서열로 이루어진 역방향 프라이머, 및 서열번호 3의 염기서열 또는 이의 상보적 염기서열로 이루어진 프로브를 포함하는 거친대추멍게 검출용 조성물를 이용하여 실시한 PCR을 통해 거친대추멍게의 정성적 검출을 확인한 결과, 현장 조사를 통해 발견하지 못했던 거친대추멍게가 서식 지역을 확인할 수 있었다(실험예 2, 도 4 참조). 이러한 결과는, 본 발명에 따른 프라이머 세트 및 프로브, 및 이를 포함하는 조성물은 거친대추멍게의 근연종 등과의 교차반응이 없어 거친대추멍게 종만을 특이적으로 검출할 수 있으며, 이러한 특성에 따라 거친대추멍게의 분포 현황을 정확히 파악할 수 있으므로, 거친대추멍게를 검출하고, 분포 현황을 파악하는 데 유용하게 사용될 수 있음을 보여준다.According to an experimental example of the present invention, detection of rough jujube sea squirts comprising a primer set for detecting rough jujube sea squirts consisting of a forward primer consisting of a base sequence of SEQ ID NO: 1 and a reverse primer consisting of a base sequence of SEQ ID NO: 2 according to the present invention. As a result of confirming the qualitative detection of rough jujube sea squirt through PCR using the composition, the primer set according to the present invention or the composition containing the same did not detect related species of rough jujube sea squirt, harmful benthic animals, and marine benthic animals. , it was confirmed that only rough jujube sea squirt individuals were specifically detected (Experimental Example 1, see Figure 2). In addition, a rough jujube sea squirt comprising a forward primer consisting of the base sequence of SEQ ID NO: 1 according to the present invention, a reverse primer consisting of the base sequence of SEQ ID NO: 2, and a probe consisting of the base sequence of SEQ ID NO: 3 or a complementary base sequence thereof. As a result of confirming the qualitative detection of the rough jujube sea squirt through PCR conducted using the detection composition, the habitat area of the rough jujube sea squirt, which was not found through field investigation, was confirmed (Experimental Example 2, see Figure 4). These results show that the primer set and probe according to the present invention, and the composition containing the same, do not have cross-reactivity with related species of the rough jujube sea squirt, so they can specifically detect only the rough jujube sea squirt species, and according to these characteristics, the rough jujube sea squirt species can be specifically detected. Since the distribution status of sea squirts can be accurately determined, it shows that it can be usefully used to detect rough jujube sea squirts and determine their distribution status.
본 발명의 다른 한 양태에 따르면, 서열번호 1의 염기서열로 이루어진 정방향 프라이머 및 서열번호 2의 염기서열로 이루어진 역방향 프라이머로 이루어진 거친대추멍게 검출용 프라이머 세트를 포함하거나 서열번호 3의 염기서열 또는 이의 상보적 염기서열로 이루어진 프로브를 더 포함하는 조성물을 포함하는 거친대추멍게 검출용 키트를 제공한다.According to another aspect of the present invention, it includes a primer set for detecting rough jujube sea squirt consisting of a forward primer consisting of the base sequence of SEQ ID NO: 1 and a reverse primer consisting of the base sequence of SEQ ID NO: 2, or the base sequence of SEQ ID NO: 3 or thereof. Provided is a kit for detecting rough jujube sea squirts, including a composition further comprising a probe consisting of a complementary base sequence.
상기 키트는 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성 성분 조성물, 용액 또는 장치를 더 포함할 수 있다. 상기 키트는 프라이머 세트, 프로브 이외에도 튜브 또는 다른 적절한 컨테이너, 반응 완충액(pH 및 마그네슘 농도는 다양), 데옥시뉴클레오티드(dNTPs), Taq-폴리머라제 및 역전사 효소와 같은 효소, DNase 억제제, RNase 억제제, DEPC-수(DEPC-water) 및 멸균수 등을 포함할 수 있다. 한편, 키트에 포함되는 성분들은 액상 형태로 제조될 수도 있고, 포함 성분들의 자유도를 낮추어 제품의 안정성을 제고하기 위해 건조된 형태로 제조될 수도 있다. 이러한 건조된 형태로의 제조를 위해서는 건조 단계의 적용이 필요하고, 이때 가온건조, 자연건조, 감압건조, 동결건조 또는 이들의 복합 공정이 사용될 수 있다.The kit may further include one or more other component compositions, solutions, or devices suitable for the analysis method. In addition to primer sets and probes, the kits also contain tubes or other suitable containers, reaction buffers (pH and magnesium concentration vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase inhibitors, RNase inhibitors, DEPC. -Can include water (DEPC-water) and sterilized water. Meanwhile, the ingredients included in the kit may be manufactured in liquid form or in dried form to improve the stability of the product by lowering the degree of freedom of the included ingredients. To manufacture this dried form, a drying step is required, and in this case, heated drying, natural drying, reduced pressure drying, freeze-drying, or a combination process thereof may be used.
본 발명의 다른 한 양태에 따르면, 본 발명은 분리된 시료로부터 DNA를 분리하는 제1 단계, 상기 제1 단계의 DNA를 주형으로 제1항에 따른 프라이머 세트를 이용하여 중합효소 연쇄반응을 수행하여 표적 서열을 증폭하는 제2 단계, 및 상기 제2 단계의 증폭 산물에 거친대추멍게 종 특이적 DNA 마커의 존재 여부를 확인하는 제3 단계를 포함하는 거친대추멍게의 검출방법을 제공한다.According to another aspect of the present invention, the present invention involves the first step of isolating DNA from a separated sample, performing a polymerase chain reaction using the DNA of the first step as a template and using the primer set according to claim 1. A method for detecting rough jujube sea squirts is provided, comprising a second step of amplifying a target sequence, and a third step of confirming the presence of a rough jujube sea squirt species-specific DNA marker in the amplification product of the second step.
상기 시료는 임상시료 또는 환경시료로부터 수득되는 것일 수 있으며, 예를 들어 해양으로부터 수득되는 해수일 수 있다.The sample may be obtained from a clinical sample or an environmental sample, for example, may be seawater obtained from the ocean.
본 발명의 거친대추멍게 검출방법에 있어서, 상기 제3 단계는 서열번호 3의 염기서열 또는 이의 상보적 염기서열로 이루어진 프로브를 이용하여 수행한다.In the method for detecting rough jujube sea squirt of the present invention, the third step is performed using a probe consisting of the base sequence of SEQ ID NO: 3 or its complementary base sequence.
본 발명의 거친대추멍게의 검출방법에서 상기 제2 단계에서의 증폭은, 바람직하게 90~95℃, 1~10분을 한 세트로 1 사이클(cycle)의 조건으로 반응을 진행하여 변성을 수행하고, 순차적으로 90~95℃에서 5~60초, 65~75℃에서 60~120초를 한 세트로 30 내지 40 사이클(cycle)의 조건으로 반응을 진행하여 신장을 수행하는 것이 좋다. 상기 변성을 수행하는 과정에서 1분 미만의 반응시간에서는 변성이 원활하게 이루어지지 않으며, 10분이 초과하면 과도한 변성이 일어나 다음 단계의 증폭 반응을 수행하는데 부적합하게 된다. 상기 신장을 수행하는 과정에서 90~95℃에서 5~60초간 반응시킨 다음, 반응 온도가 60℃ 미만이라면 충분히 원하는 서열이 증폭되지 않아 시료 내 거친대추멍게의 존재를 검출할 수 없게 되며, 70℃를 초과하면 과도한 증폭이 일어날 수 있다. 따라서, 상기 조건은 본 발명의 프라이머 세트를 이용하여 혼재된 DNA 속에서 거친대추멍게를 증폭하여 진단, 검출하기 위한 최적의 조건인 것이다. 상기 수치 범위를 벗어나는 경우, 시료 내 거친대추멍게의 증폭이 원활하게 이루어지지 않아, 시료 내 존재하는 거친대추멍게의 존재를 진단하는데 어려움이 있을 수 있다.In the method for detecting rough jujube sea squirts of the present invention, the amplification in the second step is preferably performed at 90-95°C, 1-10 minutes, and denaturation is carried out under the conditions of 1 cycle as a set. , it is recommended to carry out the elongation by sequentially performing the reaction under conditions of 30 to 40 cycles of 5 to 60 seconds at 90 to 95°C and 60 to 120 seconds at 65 to 75°C. In the process of performing the denaturation, denaturation does not occur smoothly if the reaction time is less than 1 minute, and if it exceeds 10 minutes, excessive denaturation occurs, making it unsuitable for performing the amplification reaction in the next step. In the process of performing the elongation, the reaction is performed at 90-95°C for 5-60 seconds, and if the reaction temperature is less than 60°C, the desired sequence is not sufficiently amplified, making it impossible to detect the presence of rough jujube sea squirts in the sample. If it exceeds , excessive amplification may occur. Therefore, the above conditions are the optimal conditions for diagnosis and detection by amplifying the rough jujube sea squirt in mixed DNA using the primer set of the present invention. If the value is outside the above range, the amplification of the rough jujube sea squirt in the sample may not occur smoothly, and it may be difficult to diagnose the presence of the rough jujube sea squirt present in the sample.
본 발명의 다른 한 양태에 따르면, 본 발명은 분리된 시료로부터 DNA를 분리하는 제1 단계, 제1항에 따른 프라이머 세트 및 서열번호 3의 염기서열 또는 이의 상보적 염기서열로 이루어진 프로브를 이용하여 실시간 중합효소 연쇄반응을 실시하는 제2 단계, 및 상기 제2 단계의 실시간 중합효소 연쇄반응을 통해 확인된 실시간 PCR 표준 곡선을 이용하여 거친대추멍게의 DNA 양을 산출하는 단계를 포함하는 해양 생태계 내 거친대추멍게 분포 조사 방법을 제공한다. According to another aspect of the present invention, the first step of isolating DNA from a separated sample, using a primer set according to claim 1 and a probe consisting of the base sequence of SEQ ID NO: 3 or a complementary base sequence thereof, A second step of performing a real-time polymerase chain reaction, and a step of calculating the amount of DNA of the rough jujube sea squirt using the real-time PCR standard curve confirmed through the real-time polymerase chain reaction of the second step. Provides a method for investigating the distribution of rough jujube sea squirts.
본 발명의 해양 생태계 내 거친대추멍게 분포 조사 방법에서 상기 제2 단계에서의 연쇄반응은, 바람직하게 90~95℃, 1~5분을 한 세트로 1 사이클(cycle)의 조건으로 반응을 진행하여 변성을 수행하고, 순차적으로 70~80℃에서 1~10초, 55~65℃에서 20~40초를 한 세트로 40 내지 50 사이클(cycle)의 조건으로 반응을 진행하여 신장을 수행하는 것이 좋다. 상기 변성을 수행하는 과정에서 1분 미만의 반응시간에서는 변성이 원활하게 이루어지지 않으며, 5분이 초과하면 과도한 변성이 일어나 다음 단계의 증폭 반응을 수행하는데 부적합하게 된다. 상기 신장을 수행하는 과정에서 70~85℃에서 1~10초간 반응시킨 다음, 반응 온도가 60℃ 미만이라면 충분히 원하는 서열이 증폭되지 않아 시료 내 거친대추멍게의 존재를 검출할 수 없게 되며, 70℃를 초과하면 과도한 증폭이 일어날 수 있다. 따라서, 상기 조건은 본 발명의 프라이머 세트를 이용하여 혼재된 DNA 속에서 거친대추멍게를 증폭하여 진단, 검출하기 위한 최적의 조건인 것이다. 상기 수치 범위를 벗어나는 경우, 시료 내 거친대추멍게의 증폭이 원활하게 이루어지지 않아, 시료 내 존재하는 거친대추멍게의 존재를 진단하는데 어려움이 있을 수 있다.In the method for investigating the distribution of rough jujube sea squirts in the marine ecosystem of the present invention, the chain reaction in the second step is preferably carried out under the conditions of 1 cycle at 90 to 95 ° C. for 1 to 5 minutes as a set. It is recommended to carry out denaturation and carry out extension by sequentially performing the reaction under conditions of 40 to 50 cycles of 1 to 10 seconds at 70 to 80°C and 20 to 40 seconds at 55 to 65°C. . In the process of performing the denaturation, denaturation does not occur smoothly if the reaction time is less than 1 minute, and if it exceeds 5 minutes, excessive denaturation occurs, making it unsuitable for performing the amplification reaction in the next step. In the process of performing the elongation, the reaction is performed at 70-85°C for 1-10 seconds, and if the reaction temperature is less than 60°C, the desired sequence is not sufficiently amplified, making it impossible to detect the presence of rough jujube sea squirts in the sample. If it exceeds , excessive amplification may occur. Therefore, the above conditions are the optimal conditions for diagnosis and detection by amplifying the rough jujube sea squirt in mixed DNA using the primer set of the present invention. If the value is outside the above range, the amplification of the rough jujube sea squirt in the sample may not occur smoothly, and it may be difficult to diagnose the presence of the rough jujube sea squirt present in the sample.
전술한 바와 같이, 본 발명에 따른 프라이머 세트 및 프로브는 거친대추멍게를 정성적 또는 정량적으로 검출 가능할 뿐만 아니라 거친대추멍게의 분포 및 확산 여부를 확인할 수 있다.As described above, the primer set and probe according to the present invention can not only detect rough jujube sea squirts qualitatively or quantitatively, but also confirm the distribution and spread of rough jujube sea squirts.
도 1은 국내에서 채집된 거친대추멍게 3개체의 COI(Cytochrome C Oxidase Subunit I) 유전자 염기서열에서 거친대추멍게 종 특이적 프라이머(primer)에 해당하는 부분 및 거친대추멍게 검출용 최종 프로브에 해당하는 부분을 나타낸 것이다.
도 2는 실험예 1에 따른 국내에서 채집된 거친대추멍게 3개체 및 다른 해양 동물 총 19개체의 유전체 DNA를 주형(template)으로 사용하고 거친대추멍게 종 특이적 프라이머인 AsAs_F와 AsAs_R을 사용하여 PCR을 수행하고 증폭된 산물을 전기영동(electrophoresis) 방법으로 분석한 결과이다(lane 1, 2 및 3: 국내에서 채집된 거친대추멍게 3개체의 유전체 DNA를 주형으로 사용함; lane 4: 유령멍게(Ciona robusta)의 유전체 DNA를 주형으로 사용함; lane 5: 노랑꼭지유령멍게(Ciona savignyi)의 유전체 DNA를 주형으로 사용함; lane 6: 보라판멍게(Botrylloides violaceus)의 유전체 DNA를 주형으로 사용함; lane 7: 미더덕(Styela clava)의 유전체 DNA를 주형으로 사용함; lane 8: 주름미더덕(Styela plicata)의 유전체 DNA를 주형으로 사용함; lane 9: 관히드라(Ectopleura crocea)의 유전체 DNA를 주형으로 사용함; lane 10: 분지보우갠빌히드라(Bougainvillia muscus)의 유전체 DNA를 주형으로 사용함; lane 11: 자주빛이끼벌레(Watersipora subtorquata)의 유전체 DNA를 주형으로 사용함; lane 12: 분홍멍게(Herdmania momus)의 유전체 DNA를 주형으로 사용함; lane 13: 가시예쁜갯고사리(Antedon serrata)의 유전체 DNA를 주형으로 사용함; lane 14: 긴수염갯고사리(Heliometra glacialis)의 유전체 DNA를 주형으로 사용함; lane 15: 별불가사리(Patiria pectinifera)의 유전체 DNA를 주형으로 사용함; lane 16: 아무르불가사리(Asterias amurensis)의 유전체 DNA를 주형으로 사용함; lane 17: 뱀거미불가사리(Ophiactis savignyi)의 유전체 DNA를 주형으로 사용함; lane 18: 뿔거미불가사리(Ophiopholis mirabilis)의 유전체 DNA를 주형으로 사용함; lane 19: 하드윅분지성게(Temnopleurus hardwickii)의 유전체 DNA를 주형으로 사용함; lane 20: 가는관극성게(Prionocidaris japonica)의 유전체 DNA를 주형으로 사용함; lane 21: 오각광삼(Eupentacta chronhjelmi)의 유전체 DNA를 주형으로 사용함; lane 22: 가시닻해삼(Protankyra bidentata)의 유전체 DNA를 주형으로 사용함; ladder marker: 100bp DNA ladder marker 사용함).
도 3은 실시간 PCR에 의한 거친대추멍게의 정량적 검출시 사용될 수 있는 표준 곡선이다.
도 4는 현장 조사를 통해 거친대추멍게의 국내 분포 현황을 분석한 결과(A) 및 실시간 PCR(Real-time polymerase chain reaction)을 통해 거친대추멍게의 국내 분포 현황을 분석한 결과(B)이다.Figure 1 shows the part corresponding to the rough jujube sea squirt species-specific primer and the final probe for the detection of the rough jujube sea squirt in the COI (Cytochrome C Oxidase Subunit I) gene base sequence of three rough jujube sea squirts collected in Korea. It shows the part.
Figure 2 shows PCR using the genomic DNA of 3 specimens of rough jujube sea squirt and a total of 19 specimens of other marine animals collected in Korea according to Experimental Example 1 as a template and using the species-specific primers AsAs_F and AsAs_R. This is the result of performing and analyzing the amplified product using electrophoresis (lanes 1, 2, and 3: genomic DNA from three rough jujube sea squirts collected in Korea was used as a template; lane 4: ghost sea squirt (Ciona robusta) as a template; lane 5: the genomic DNA of the yellow-topped ghost sea squirt (Ciona savignyi) as a template; lane 6: the genomic DNA of the purple sea squirt (Botrylloides violaceus) as a template; Lane 8: using the genomic DNA of Styela clava as a template; lane 9: using the genomic DNA of Ectopleura crocea as a template; Lane 11: genomic DNA of Bougainvillia muscus was used as a template; lane 12: genomic DNA of pink sea squirt (Herdmania momus) was used as a template; Lane 13: Used the genomic DNA of Antedon serrata as a template; Lane 15: Used the genomic DNA of Heliometra glacialis as a template; Using genomic DNA as a template; lane 16: using the genomic DNA of Asterias amurensis as a template; lane 18: using the genomic DNA of a spider starfish (Ophiactis savignyi) as a template; mirabilis) genomic DNA was used as a template; lane 19: Genomic DNA of Hardwick's Basin sea urchin (Temnopleurus hardwickii) was used as a template; lane 20: Genomic DNA of Prionocidaris japonica was used as a template; lane 21: genomic DNA of Eupentacta chronhjelmi was used as a template; lane 22: genomic DNA of anchor sea cucumber (Protankyra bidentata) was used as a template; ladder marker: 100bp DNA ladder marker was used).
Figure 3 is a standard curve that can be used for quantitative detection of rough jujube sea squirt by real-time PCR.
Figure 4 shows the results of analyzing the domestic distribution status of rough jujube sea squirts through field surveys (A) and the results of analyzing the domestic distribution status of rough jujube sea squirts through real-time PCR (Real-time polymerase chain reaction) (B).
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. Since these examples are merely for illustrating the present invention, the scope of the present invention is not to be construed as limited by these examples.
실시예Example : : 거친대추멍게Rough jujube sea squirt 종 특이적 species specific 프라이머primer (primer) 및 (primer) and 거친대추멍게Rough jujube sea squirt 종특이적species specific 프로브probe (probe) 제작(probe) production
거친대추멍게가 속하는 척삭동물의 근연종, 해양에 서식하는 태형동물, 극피동물, 자포동물 등의 COI(Cytochrome C Oxidase Subunit I) 유전자 염기서열을 NCBI(National Center for Biotechnology Information)의 Genbank에서 검색한 후 국내에서 채집된 거친대추멍게 3개체의 COI(Cytochrome C Oxidase Subunit I) 유전자 염기서열과 비교하고, 이를 바탕으로 거친대추멍게 종 특이적 프라이머(primer)를 제작하였다. 국내에서 채집된 거친대추멍게 3개체와 비교된 분류군으로는 척삭동물문(Phylum Chordata)의 해초강(Class Ascidiacea)의 24종 56개체; 태형동물문(Phylum Bryozoa)의 7종 13개체; 자포동물문(Phylum Cnidaria)의 히드라충강(Class Hydrozoa) 22종 30개체, 산호충강(Class Anthzoa) 23종 27개체 그리고 해파리강 (Class Scyphozoa) 6종 11개체; 극피동물문(Phylum Echinodermata) 거미불가사리강((Class Ophiuroidea) 7종 13개체, 불가사리강(Class Asteroidea) 6종 10개체, 성게강(Class Echinoidea) 16종 19개체, 해삼강(Class Holothuroidea) 13종 14개체 그리고 바다나리강(Class Crinozoa) 4종 7개체 등 총 128종 200개체이었으며, 구체적인 종 및 개체명은 다음과 같다.The COI (Cytochrome C Oxidase Subunit I) gene sequences of marine sea squirts, related species of chordates, marine bryozoans, echinoderms, and cnidarians were retrieved from Genbank of the National Center for Biotechnology Information (NCBI). Afterwards, the base sequences of the COI (Cytochrome C Oxidase Subunit I) genes of three specimens of the rough jujube sea squirt collected in Korea were compared, and based on this, a species-specific primer for the rough jujube sea squirt was produced. The taxa compared with the three rough jujube sea squirts collected in Korea include 56 individuals of 24 species from Class Ascidiacea of the Phylum Chordata; 13 specimens from 7 species of Phylum Bryozoa; Of the Phylum Cnidaria, 30 specimens of 22 species of Class Hydrozoa, 27 specimens of 23 species of Class Anthzoa, and 11 specimens of 6 species of Class Scyphozoa; Phylum Echinodermata, Class Ophiuroidea, 7 species, 13 specimens, Starfish (Class Asteroidea), 6 species, 10 specimens, Sea Urchin Class (Class Echinoidea, 16 species, 19 specimens, Sea Cucumber Class (Class Holothuroidea), 13 species, 14 There were a total of 200 individuals of 128 species, including 7 individuals of 4 species of Class Crinozoa, and the specific species and individual names are as follows.
<국내에서 채집된 거친대추멍게 3개체><Three rough jujube sea squirts collected in Korea>
Ascidiella aspersa (비응항), Ascidiella aspersa (통영항), Ascidiella aspersa (양포항) Ascidiella aspersa (Bieunghang), Ascidiella aspersa (Tongyeong Port), Ascidiella aspersa (Yangpo Port)
<척삭동물문 해초강(Phylum Chordata Class Ascidiacea) 24종 56개체><24 species, 56 individuals of Phylum Chordata Class Ascidiacea>
Aplidium conicum (FN313538), Aplidium conicum (NC013584), Ascidiella zara (KY235396), Ascidiella zara (KY235398), Botrylloides leachii (HF548553), Botrylloides leachii (HG931921), Botrylloides leachii (NC024103), Botrylloides nigrum (HF548559), Botrylloides nigrum (NC021467), Botrylloides pizoni (HF548554), Botrylloides pizoni (HG931922), Botrylloides pizoni (NC024104), Botrylloides violaceus (HF548552), Botrylloides violaceus (NC024256), Botryllus schlosseri (FM177702), Botryllus schlosseri (HF548550), Botryllus schlosseri (HF548551), Botryllus schlosseri (HG931923), Botryllus schlosseri (NC021463), Ciona intestinalis (AJ517314), Ciona intestinalis (AM292218), Ciona intestinalis (NC004447), Ciona intestinalis (NC017929), Ciona savignyi (AB079784), Ciona savignyi (NC004570), Clavelina lepadiformis (AM292603), Clavelina lepadiformis (FJ839918), Clavelina lepadiformis (NC012887), Clavelina phlegraea (AM292604), Clavelina phlegraea (NC024105), Didemnum vexillum (KM259616), Didemnum vexillum (KM259617), Didemnum vexillum (NC026107), Diplosoma listerianum (FN313539), Diplosoma listerianum (NC013556), Halocynthia roretzi (AB024528), Halocynthia roretzi (NC002177), Halocynthia spinosa (HF548558), Halocynthia spinosa (NC021466), Herdmania momus (FN296153), Herdmania momus (NC013561), Microcosmus sulcatus (AM292321), Microcosmus sulcatus (NC013752), Phallusia fumigata (AM292602), Phallusia fumigata (NC009834), Phallusia mammillata (AM292320), Phallusia mammillata (NC009833), Polycarpa mytiligera (HF548556), Polycarpa mytiligera (NC021464), Pyura gangelion (HF548557), Pyura gangelion (NC021465), Rhodosoma turcicum (HF548560), Rhodosoma turcicum (NC021468), Styela clava (HG931920), Styela plicata (AM292601), Styela plicata (NC013565) Aplidium conicum (FN313538), Aplidium conicum (NC013584), Ascidiella zara(KY235396), Ascidiella zara (KY235398), Botrilloides leachii (HF548553), Botrilloides leachii (HG931921), Botrilloides leachii (NC024103), Botrilloides nigrum (HF548559), Botrilloides nigrum (NC021467), Botrilloides pizoni(HF548554), Botrilloides pizoni (HG931922), Botrilloides pizoni (NC024104), Botrilloides violaceus (HF548552), Botrilloides violaceus (NC024256), Botryllus schlosseri (FM177702), Botryllus schlosseri (HF548550), Botryllus schlosseri (HF548551), Botryllus schlosseri (HG931923), Botryllus schlosseri(NC021463), Ciona intestinalis (AJ517314), Ciona intestinalis (AM292218), Ciona intestinalis (NC004447), Ciona intestinalis (NC017929), Ciona savignyi (AB079784), Ciona savignyi (NC004570), Clavelina lepadiformis (AM292603), Clavelina lepadiformis (FJ839918), Clavelina lepadiformis (NC012887), Clavelina phlegraea (AM292604), Clavelina phlegraea (NC024105), Didemnum vexillum (KM259616), Didemnum vexillum (KM259617), Didemnum vexillum (NC026107), Diplosoma listerianum (FN313539), Diplosoma listerianum (NC013556), Halocynthia roretz (AB024528), Halocynthia roretz (NC002177), Halocynthia spinosa (HF548558), Halocynthia spinosa (NC021466), Herdmania momus(FN296153), Herdmania momus (NC013561), Microcosmus sulcatus (AM292321), Microcosmus sulcatus (NC013752), Phallusia fumigata (AM292602), Phallusia fumigata (NC009834), Phallusia mammillata (AM292320), Phallusia mammillata (NC009833), Polycarpa mytiligera (HF548556), Polycarpa mytiligera (NC021464), Pyura gangelion (HF548557), Pyura gangelion (NC021465), Rhodosoma turcicum(HF548560), Rhodosoma turcicum (NC021468), Stylela clava (HG931920), Stylela plicata(AM292601), Stylela plicata(NC013565)
<태형동물문(Phylum Bryozoa) 7종 13개체><13 specimens of 7 species of Phylum Bryozoa>
Bugula neritina (AY690838), Bugula neritina (NC010197), Celleporella hyalina (JQ839275), Celleporella yalina (JQ839276), Celleporella hyalina (NC018344), Flustra foliacea (JQ061319), Flustra foliacea (NC016722), Flustrellidra hispida (DQ157889), Flustrellidra hispida (NC008192), Membranipora grandicella (NC018355), Tubulipora flabellaris (EU563937), Watersipora subtorquata (EU365892), Watersipora subtorquata (NC011820) Bugula neritina (AY690838), Bugula neritina (NC010197), Celleporella hyalina (JQ839275), Celleporella yalina (JQ839276), Celleporella Hyalina (NC018344), Flustra foliacea (JQ061319), Flustra foliacea (NC016722), Flustrellidra hispida (DQ157889), Flustrellidra hispida (NC008192), Membranipora grandicella (NC018355), Tubulipora flabellaris (EU563937), Watersipora subtorquata (EU365892), Watersipora subtorquata (NC011820)
<자포동물문 히드라충강(Phylum Cnidaria Class Hydrozoa) 22종 30개체><22 species, 30 individuals of Phylum Cnidaria Class Hydrozoa>
Amphimedon compressa (EU237474), Chondrilla aff . nucula (EU237478), Clava multicornis (JN700935), Clava multicornis (NC016465), Craspedacusta sowerbyi (JN593332), Craspedacusta sowerbyi (NC018537), Cubaia aphrodite (JN700942), Cubaia aphrodite (NC016467), Ectopleura Aplysina fulva (EU237476), Ectopleura Callyspongia plicifera (EU237477), Ephydatia muelleri (EU237481), Ephydatia muelleri (NC010202), Halisarca dujardini (EU237483), Hippospongia lachne (EU237484), Hydra magnipapillata (NC011221), Hydra oligactis (EU237491), Hydra oligactis (NC010214), Hydra sinensis (JX089978), Hydra sinensis (NC021406), Hydra vulgaris (HM369414), Igernella notabilis (EU237485), Iotrochota birotulata (EU237486), Laomedea flexuosa (NC016463), Plakinastrella cf. onkodes (EU237487), Topsentia ophiraphidites (EU237482), Turritopsis dohrnii (KT020766), Turritopsis dohrnii (KT899097), Turritopsis dohrnii (NC031213), Vaceletia sp. (EU237489), Xestospongia muta (EU237490) Amphimedon compressa (EU237474), Chondrilla aff . nucula (EU237478), Clava multicornis (JN700935), Clava multicornis (NC016465), Craspedacusta sowerbyi (JN593332), Craspedacusta sowerbyi (NC018537), Cubaia aphrodite (JN700942), Cubaia aphrodite (NC016467), Ectopleura Aplysina fulva (EU237476), Ectopleura Callyspongia plicifera (EU237477), Ephydatia muelleri (EU237481), Ephydatia muelleri (NC010202), Halisarca dujardini (EU237483), Hippospongia lachne (EU237484), Hydra magnipapillata (NC011221), Hydra oligactis (EU237491), Hydra oligactis (NC010214), Hydra sinensis (JX089978), Hydra sinensis (NC021406), Hydra vulgaris (HM369414), Igernella notabilis (EU237485), Iotrochota birotulata (EU237486), Laomedea flexuosa (NC016463), Plakinastrella cf. onkodes (EU237487), Topsentia ophiraphidites (EU237482), Turritopsis dohrnii (KT020766), Turritopsis dohrnii (KT899097), Turritopsis dohrnii (NC031213), Vaceletia sp. (EU237489), Xestospongia muta (EU237490)
<극피동물문 <Echinoderm phylum 거미불가사리강Spider Starfish River (Phylum (Phylum EchinodermataEchinodermata Class Class OphiuroideaOphiuroidea ) 7종 13개체>) 7 species, 13 individuals>
Amphipholis squamata (FN562578), Amphipholis squamata (NC013876), Astrospartus mediterraneus FN562580), Astrospartus mediterraneus (NC013878), Ophiacantha linea (NC023254), Ophiocomina nigra (FN562577), Ophiocomina nigra (NC013874), Ophiopholis aculeata (AF314589), Ophiopholis aculeata (NC005334), Ophiura albida (AM404180), Ophiura albida (NC010691), Ophiura lutkeni (AY184223), Ophiura lutkeni (NC005930) Amphipholis squamata (FN562578), Amphipholis squamata (NC013876), Astrospartus mediterraneus FN562580), Astrospartus mediterraneus (NC013878), Ophiacantha linea (NC023254), Ophiocomina nigra (FN562577), Ophiocomina nigra (NC013874), Ophiopholis aculeata (AF314589), Ophiopholis aculeata (NC005334), Ophiura albida (AM404180), Ophiura albida (NC010691), Ophiura lutkeni (AY184223), Ophiura lutkeni (NC005930)
<극피동물문 <Echinoderm phylum 불가사리강starfish river (Phylum (Phylum EchinodermataEchinodermata Class Class AsteroideaAsteroidea ) 6종 10개체>) 6 types, 10 individuals>
Acanthaster brevispinus (AB231476), Acanthaster brevispinus (NC007789), Acanthaster planci (AB231475), Acanthaster planci (NC007788), Aphelasterias japonica (NC025766), Asterias amurensis (AB183559), Asterias amurensis (NC006665), Astropecten polyacanthus (AB183560), Astropecten polyacanthus (NC006666), Luidia quinalia (AB183558) Acanthaster brevispinus (AB231476), Acanthaster brevispinus (NC007789), Acanthaster planci (AB231475), Acanthaster planci (NC007788), Aphelasterias japonica (NC025766), Asterias amurensis (AB183559), Asterias amurensis (NC006665), Astropecten polyacanthus (AB183560), Astropecten polyacanthus (NC006666), Luidia quinalia (AB183558)
<극피동물문 성게강(Phylum Echinodermata Class Echinoidea) 16종 19개체><19 individuals of 16 species of Phylum Echinodermata Class Echinoidea>
Arbacia lixula (NC001770), Echinocardium cordatum (NC013881), Heliocidaris crassispina (NC023774), Hemicentrotus pulcherrimus (NC023771), Loxechinus albus (JX888466), Mesocentrotus franciscanus (NC024177), Mesocentrotus nudus (NC020771), Nacospatangus alta (NC023255), Paracentrotus lividus (J04815), Pseudocentrotus depressus (KC490913), Pseudocentrotus depressus (NC023773), Sterechinus neumayeri (NC027063), Strongylocentrotus droebachiensis (EU054306), Strongylocentrotus droebachiensis (NC009940), Strongylocentrotus intermedius (KC490912), Strongylocentrotus intermedius (NC023772), Strongylocentrotus pallidus (NC009941), Strongylocentrotus purpuratus (NC001453), Temnopleurus hardwickii (NC026200) Arbacia lixula (NC001770), Echinocardium cordatum (NC013881), Heliocidaris crassispina (NC023774), Hemicentrotus pulcherrimus (NC023771), Loxechinus albus (JX888466), Mesocentrotus franciscanus (NC024177), Mesocentrotus nudus (NC020771), Nacospatangus alt a (NC023255), Paracentrotus lividus (J04815), Pseudocentrotus depressus (KC490913), Pseudocentrotus depressus (NC023773), Sterechinus neumayeri (NC027063), Strongylocentrotus droebachiensis (EU054306), Strongylocentrotus droebachiensis (NC009940), Strongylocentrotus intermedius (KC490912), Strongylocentrotus intermedius (NC023772), Strongylocentrotus pallidus (NC009941), Strongylocentrotus purpuratus (NC001453), Temnopleurus hardwickii (NC026200)
<극피동물문 <Echinoderm phylum 해삼강sea cucumber river (Phylum (Phylum EchinodermataEchinodermata Class Class HolothuroideaHolothuroidea ) 13종 14개체>) 13 species, 14 individuals>
Amphipholis squamata (NC013876), Apostichopus japonicus (EU294194), Astrospartus mediterraneus (NC013878), Balanoglossus clavigerus (NC013877), Cucumaria miniata (AY182376), Holothuria forskali (NC013884), Holothuria scabra (NC027086), Ophiocomina nigra (NC013874), Parastichopus californicus (NC026727), Parastichopus nigripunctatus (NC013432), Parastichopus parvimensis (NC029699), Peniagone sp. (KF915304), Stichopus horrens (HQ000092), Stichopus horrens (NC014454) Amphipholis squamata (NC013876), Apostichopus japonicus (EU294194), Astrospartus mediterraneus (NC013878), Balanoglossus clavigerus (NC013877), Cucumaria miniata (AY182376), Holothuria forskali (NC013884), Holothuria scabra (NC027086), Ophiocomina nigra (NC013874), Parastichopus californicus (NC026727), Parastichopus nigripunctatus (NC013432), Parastichopus parvimensis (NC029699), Peniagone sp. (KF915304), Stichopus horrors (HQ000092), Stichopus horrens (NC014454)
<극피동물문 바다나리강(Phylum Echinodermata Class Crinozoa) 4종 7개체><4 species, 7 individuals of Phylum Echinodermata Class Crinozoa>
Antedon mediterranea (AM404181), Antedon mediterranea (NC010692), Florometra serratissima (NC001878), Neogymnocrinus richeri (DQ068951), Neogymnocrinus richeri (NC007689), Phanogenia gracilis (DQ068952), Phanogenia gracilis (NC007690) Antedon mediterranea (AM404181), Antedon mediterranea (NC010692), Florometra serratissima (NC001878), Neogymnocrinus richeri (DQ068951), Neogymnocrinus richeri (NC007689), Phanogenia gracilis (DQ068952), Phanogenia gracilis (NC007690)
<자포동물문 산호충강(Phylum Cnidaria Class Anthozoa) 23종 27개체><27 individuals of 23 species of Phylum Cnidaria Class Anthozoa>
Acanella eburnea (EF672731), Acanella eburnea (NC011016), Alicia sansibarensis NC027610), Bolocera uediae (NC022470), Dendronephthya castanea (NC023343), Dendronephthya gigantea (FJ372991), Dendronephthya mollis (NC020456), Dendronephthya putteri (HQ694726), Dendronephthya putteri (JQ886185), Dendronephthya suensoni (NC022809), Echinogorgia complexa (NC020457), Euplexaura crassa (HQ694728), Euplexaura crassa (NC020458), Heliopora coerulea (NC020375), Junceella fragilis (NC024181). Madracis mirabilis (EU400212), Muricea crassa (NC029697), Muricea purpurea (NC029698), Narella hawaiinensis (KM015351), Pseudopterogorgia bipinnata (DQ640646), Pseudopterogorgia bipinnata (NC008157), Renilla muelleri (NC018378), Savalia savaglia (DQ825686), Scleronephthya gracillimum (NC023344), Sinularia peculiaris (NC018379), Stylophora pistillata (EU400214), Urticina eques (NC022469) Acanella eburnea (EF672731), Acanella eburnea (NC011016), Alicia sansibarensis NC027610), Bolocera uediae (NC022470), Dendronephthya castanea (NC023343), Dendronephthya gigantea (FJ372991), Dendronephthya mollis (NC020456), Dendronephthya putteri (HQ694726), Dendronephthya putteri (JQ886185), Dendronephthya suensoni (NC022809), Echinogorgia complexa (NC020457), Euplexaura crassa (HQ694728), Euplexaura crassa (NC020458), Heliopora coerulea (NC020375), Juncella fragilis (NC024181). Madracis mirabilis (EU400212), Muricea crassa (NC029697), Muricea purpurea (NC029698), Narella hawaiinensis (KM015351), Pseudopterogorgia bipinnata (DQ640646), Pseudopterogorgia bipinnata (NC008157), Renilla muelleri (NC018378), Savalia savaglia (DQ825686), Scleronephthya gracillimum (NC023344), Sinularia peculiaris (NC018379), Stylophora pistillata (EU400214), Urticina eques (NC022469)
<자포동물문 해파리강(Phylum Cnidaria Class Scyphozoa) 6종 11개체><11 specimens from 6 species of Phylum Cnidaria Class Scyphozoa>
Aurelia aurita (DQ787873), Aurelia aurita (HQ694729), Aurelia aurita (NC008446), Aurelia sp. (LC005413), Aurelia sp. (LC005414), Cassiopea frondosa (JN700936), Cassiopea frondosa (NC016466), Chrysaora quinquecirrha (HQ694730), Chrysaora quinquecirrha (NC020459), Haliclystus antarcticus (KU947038), Haliclystus antarcticus (NC030337)Aurelia aurita (DQ787873), Aurelia aurita (HQ694729), Aurelia aurita (NC008446), Aurelia sp. (LC005413), Aurelia sp. (LC005414), Cassiopea frondosa (JN700936), Cassiopea frondosa (NC016466), Chrysaora quinquecirrha (HQ694730), Chrysaora quinquecirrha (NC020459), Haliclystus antarcticus (KU947038), Haliclystus antarcticus (NC030337)
거친대추멍게 3개체에서 변이가 적고 다른 비교 종들과는 차이를 보이는 보존적 부위를 선정하여 프라이머 (primer)를 제작하였다. 특히, 프라이머는 3′ 말단 부위가 일치하지 않는 경우 증폭이 잘 일어나지 않는 PCR(polymerase chain reaction)의 특성을 이용하여 3′ 말단 부위(1-5bp)에서 다른 종과 차이를 보이는 부위를 선정하여 제작하였다. 하기 표 1에 PCR(polymerase chain reaction)을 통해 거친대추멍게 종 특이적 유전자 마커를 증폭하는 프라이머를 나타내었다.Primers were produced by selecting conservative regions in three rough jujube sea squirts that showed little variation and were different from other comparative species. In particular, primers were created by selecting a region that differs from other species in the 3' end region (1-5bp) by using the characteristics of PCR (polymerase chain reaction), which does not amplify easily when the 3' end region does not match. did. Table 1 below shows primers that amplify species-specific genetic markers through PCR (polymerase chain reaction).
거친대추멍게로부터 추출된 유전체 DNA를 주형으로 하고 상기 표 1에 기재된 프라이머를 이용하여 PCR을 진행하면 거친대추멍게 COI(Cytochrome C Oxidase Subunit I) 유전자전체 염기서열 중 일부에 해당하는 서열번호 3의 염기서열을 갖는 160 bp 길이의 거친대추멍게 종 특이적 DNA 마커가 증폭되며, 이에 대해 혼성화가 가능한 상보적인 염기서열 등을 거친대추멍게 검출용 프로브로 사용할 수 있다.When PCR is performed using the genomic DNA extracted from the rough jujube sea squirt as a template and the primers listed in Table 1 above, the base of SEQ ID No. 3, which is part of the entire nucleotide sequence of the rough jujube sea squirt COI (Cytochrome C Oxidase Subunit I) gene, is obtained. A 160 bp long rough jujube sea squirt species-specific DNA marker with a sequence is amplified, and a complementary base sequence capable of hybridizing to this can be used as a probe for detecting the rough jujube sea squirt.
또한, 본 발명자는 서열번호 3의 염기서열을 갖는 160 bp 길이의 거친대추멍게 종 특이적 DNA 마커의 내부 염기서열 중 거친대추멍게 3개체에서 변이가 없는 부위를 선정하고 이를 최종 프로브로 사용하였다. 아울러, 최종 프로브를 실시간 PCR(Real-time polymerase chain reaction; quantitative polymerase chain reaction, qPCR이라고도 함)에 이용하기 위해 서열번호 3의 염기서열 5′ 말단에 리포터 염료(reporter dye)인 FAM을 표지하고 3′ 말단에 흡광 염료(quencher dye)인 BHQ1를 표지하였다. 하기 표 2는 본 발명에 따른 거친대추멍게 검출용 최종 프로브 서열이다.In addition, the present inventors selected a region without mutation in three specimens of the rough jujube sea squirt among the internal base sequences of the 160 bp long rough jujube sea squirt species-specific DNA marker with the base sequence of SEQ ID NO: 3 and used this as the final probe. In addition, in order to use the final probe in real-time PCR (Real-time polymerase chain reaction; also known as quantitative polymerase chain reaction, qPCR), the 5′ end of the base sequence of SEQ ID NO: 3 was labeled with FAM, a reporter dye, and 3 ′ The terminal was labeled with BHQ1, a quencher dye. Table 2 below shows the final probe sequence for detecting rough jujube sea squirt according to the present invention.
실험예Experiment example 1: One: 거친대추멍게Rough jujube sea squirt 종 특이적 species specific 프라이머(primer)를primer 이용한 used 거친대추멍게의Rough jujube sea squirt 정성적 검출 실험 Qualitative detection experiment
국내에서 채집된 거친대추멍게 3개체, 거친대추멍게의 근연종 5개체, 교란유해 저서동물 2개체 및 해양 저서동물 12개체 각각의 유전체 DNA를 추출하였다. 분석 대상 동물의 구체적인 정보는 다음과 같다.Genomic DNA was extracted from three specimens of rough jujube sea squirt, five related species of rough jujube sea squirt, two specimens of disturbed harmful benthic fauna, and 12 specimens of marine benthic fauna collected in Korea. Specific information on the animals subject to analysis is as follows.
<국내에서 채집된 거친대추멍게 3개체><Three rough jujube sea squirts collected in Korea>
Ascidiella aspersa(비응항), Ascidiella aspersa(통영항), Ascidiella aspersa(양포항) Ascidiella aspersa , Ascidiella aspersa (Tongyeong Port), Ascidiella aspersa (Yangpo Port)
<거친대추멍게의 근연종 5개체><Five closely related species of rough jujube sea squirt>
유령멍게(Ciona robusta), 노랑꼭지유령멍게(Ciona savignyi ), 보라판멍게(Botrylloides violaceus), 미더덕(Styela clava), 주름미더덕(Styela plicata) Ghost sea squirt ( Ciona robusta ), yellow-tipped ghost sea squirt ( Ciona savignyi ) , Botrylloides violaceus , Styela clava , Styela plicata
<교란유해 저서동물 2개체><Two disturbed harmful benthic animals>
아므르불가사리(Asterias amurensis), 별불가사리(Patiria pectinifera)Amure starfish ( Asterias amurensis ), Starfish ( Patiria pectinifera )
<해양 저서동물 12개체><12 marine benthic animals>
관히드라(Ectopleura crocea), 분지보우갠빌히드라(Bougainvillia muscus), 자주빛이끼벌레(Watersipora subtorquata), 가시예쁜갯고사리(Antedon serrata), 긴수염갯고사리(Heliometra glaciali), 뱀거미불가사리(Ophiactis savignyi), 뿔거미불가사리(Ophiopholis mirabilis), 하드윅분지성게(Temnopleurus hardwickii), 가는관극성게(Prionocidaris japonica), 오각광삼(Eupentacta chronhjelmi), 가시닻해삼 (Protankyra bidentata), 분홍멍게(Herdmania monus) Ectopleura crocea ), Bougainvillia hydra ( Bougainvillia) muscus ), purple moss bug ( Watersipora subtorquata ), thorny sea fern ( Antedon serrata ), long-haired mantis fern ( Heliometra glaciali ), and spider starfish ( Ophiactis savignyi ), horned spider starfish ( Ophiopholis mirabilis ), Hardwick's Basin sea urchin ( Temnopleurus hardwickii ), thin-crowned sea urchin ( Prionocidaris japonica ), and Eupentacta chronhjelmi ), anchor sea cucumber ( Protankyra ) bidentata ), pink sea squirt ( Herdmania monus )
상기와 같은 분석 대상 동물을 에탄올에 보관 후, DNeasy blood and tissue DNA isolation kit(QIAGEN, Hilden, Germany)를 이용하여 유전체 DNA(genomic DNA)를 kit 제조사에서 제공하는 과정에 따라 분석 대상 동물의 유전체 DNA를 추출하였다. 상기 추출된 유전체 DNA를 주형(template)으로 사용하고, 상기 표 1에 기재된 거친대추멍게 종 특이적 프라이머(primer)를 사용하여 하기 표 3에 기재된 조건으로 PCR(polymerase chain reaction) 과정을 수행하였다. 이후, PCR(polymerase chain reaction) 과정에 의해 증폭된 산물을 아가로스 겔(agarose gel)에 전기영동하여 크기별로 분리 및 분석하였으며, 그 결과를 도 2에 나타내었다.After storing the animal subject to analysis in ethanol as described above, genomic DNA of the animal subject to analysis was extracted using the DNeasy blood and tissue DNA isolation kit (QIAGEN, Hilden, Germany) according to the process provided by the kit manufacturer. was extracted. The extracted genomic DNA was used as a template, and a polymerase chain reaction (PCR) process was performed under the conditions shown in Table 3 below using the rough jujube sea squirt species-specific primers shown in Table 1. Afterwards, the product amplified by the PCR (polymerase chain reaction) process was electrophoresed on an agarose gel to be separated and analyzed by size, and the results are shown in Figure 2.
- 10× Ex Taq Buffer with 25 mM MgCl2(clontech, USA)
- 2.5 mM dNTPs(clontech, USA)
- 10 pmol primer
- 2㎕ template genomic DNA
- 5U/㎕ Taq polymerase(clontech, USA)The total volume of the PCR reaction solution is 25㎕, and its contents are as follows.
- 10× Ex Taq Buffer with 25 mM MgCl2 (clontech, USA)
- 2.5mM dNTPs (clontech, USA)
- 10 pmol primer
- 2㎕ template genomic DNA
- 5U/㎕ Taq polymerase (clontech, USA)
조건process
condition
그 결과 도 2와 같이, 본 발명에 따른 프라이머는 거친대추멍게 3개체만을 특이적으로 검출하는 것을 확인하였다.As a result, as shown in Figure 2, it was confirmed that the primer according to the present invention specifically detected only three rough jujube sea squirts.
실험예Experiment example 2: 실시간 2: Real time PCRPCR (Real-time polymerase chain reaction)을 이용한 (Real-time polymerase chain reaction) 거친대추멍게의Rough jujube sea squirt 국내 분포 현황 분석 Domestic distribution status analysis
(1) 실험방법(1) Experimental method
제주도 지역을 포함한 전국 지역에 분포하는 거친대추멍게의 유무와 검출량을 확인하기 위해서 거친대추멍게 종 특이적 프라이머인 AsAs_F와 AsAs_R 그리고 거친대추멍게 종 특이적 DNA 마커에 대한 최종 프로브(probe)인 AsAs_probe를 이용하여 실시간 PCR(Real-time polymerase chain reaction; quantitative polymerase chain reaction, qPCR이라고도 함) 분석을 수행하였다. 구체적으로, 서해안, 남해안, 동해안에서는 13개 지역(인천항, 당진항, 비응항, 목포항, 완도항, 광양항, 여수항, 통영항, 부산항, 울산항, 양포항, 동해항, 속초항)에서 여름(21년 8월), 가을(21년 10월), 겨울(21년 1월)에 그리고 제주도는 5개 지역(제주항, 성산포항, 서귀포항, 한림항, 도두항)에서 가을(21년 10월), 겨울(21년 1월), 봄(21년 4월)에 현장 조사를 통해 거친대추멍게 개체의 존재 여부를 육안으로 검사하였다. 또한, 총 18개 지역에서 해수를 채취한 후, 해수에 함유된 다양한 생물의 유전체 DNA를 추출하고 이를 주형(template)으로 사용하여 실시간 PCR 분석을 수행하고 거친대추멍게 국내 분포 지역을 확인하였다. 해수에 함유된 다양한 생물의 유전체 DNA 추출은 국내 해안에서 8L의 해수를 채취한 후, 현장에서 멤브레인 필터(pore size : 0.2㎛; Merck Millipore, Darmstadt, Germany)를 이용하여 여과하였다. 이후, 멤브레인 필터를 1.5㎖ 튜브에 냉동 보관 후, 실험실에서 Genomic DNA extraction kit(Bioneer, Korea)를 이용하여 해수에 함유된 다양한 생물의 유전체 DNA를 추출하였다. 추출 방법은 kit 제조사에서 제공하는 과정에 따랐다. 또한, 실시간 PCR 분석은 하기 표 4에 기재된 조건으로 진행하였다.In order to confirm the presence and detection amount of rough jujube sea squirts distributed throughout the country, including the Jeju Island region, the rough jujube sea squirt species-specific primers AsAs_F and AsAs_R and the final probe for the rough jujube sea squirt species-specific DNA marker, AsAs_probe, were used. Real-time PCR (Real-time polymerase chain reaction; also known as quantitative polymerase chain reaction, qPCR) analysis was performed. Specifically, on the west coast, south coast, and east coast, summer (August 21) in 13 regions (Incheon Port, Dangjin Port, Bieung Port, Mokpo Port, Wando Port, Gwangyang Port, Yeosu Port, Tongyeong Port, Busan Port, Ulsan Port, Yangpo Port, Donghae Port, and Sokcho Port), In fall (October 21) and winter (January 21), and in Jeju Island in 5 regions (Jeju Port, Seongsanpo Port, Seogwipo Port, Hallim Port, Dodu Port) in fall (October 21) and winter (January 21) ), the presence of rough jujube sea squirt individuals was visually inspected through a field survey in spring (April 21). In addition, after collecting seawater from a total of 18 regions, the genomic DNA of various organisms contained in the seawater was extracted, real-time PCR analysis was performed using this as a template, and the domestic distribution area of the jujube sea squirt was confirmed. To extract the genomic DNA of various organisms contained in seawater, 8L of seawater was collected from the coast of Korea and filtered on site using a membrane filter (pore size: 0.2㎛; Merck Millipore, Darmstadt, Germany). Afterwards, the membrane filter was stored frozen in a 1.5 ml tube, and the genomic DNA of various organisms contained in seawater was extracted in the laboratory using a Genomic DNA extraction kit (Bioneer, Korea). The extraction method followed the process provided by the kit manufacturer. In addition, real-time PCR analysis was conducted under the conditions shown in Table 4 below.
- DEPC treated water
- 5㎕ template genomic DNA(해수 시료에서 추출함)
- 5 pmol TaqMan probe(metabion, Germany)
- 10 pmol primer
- PCR Probe Mix Hi-ROX(PCR BIOSYSTEMS, UK)The total volume of the PCR reaction solution is 20㎕, and its contents are as follows.
- DEPC treated water
- 5㎕ template genomic DNA (extracted from seawater sample)
- 5 pmol TaqMan probe (metabion, Germany)
- 10 pmol primer
- PCR Probe Mix Hi-ROX (PCR BIOSYSTEMS, UK)
조건process
condition
(2) 거친대추멍게의 정량적 검출을 위한 실시간 PCR 표준 곡선(2) Real-time PCR standard curve for quantitative detection of rough jujube sea squirt
실시간 PCR의 절대 정량 분석법을 통해 거친대추멍게의 분포 지역을 확인함과 동시에 검출양을 판단하기 위하여 표준 곡선(standard curve)을 제작하였다. 도 3은 실시간 PCR에 의한 거친대추멍게의 정량적 검출시 사용될 수 있는 표준 곡선이다. 도 3의 표준 곡선에서 X축은 표준 시료에 함유된 거친대추멍게의 DNA 양을 나타내고 Y축은 소정의 거친대추멍게 DNA 양을 가진 표준 시료를 주형으로 사용하여 실시간 PCR을 수행할 때 PCR 증폭 산물이 검출할 수 있는 기준 형광 값을 가질 때의 PCR Cycle 수를 나타낸다. 해수 시료로부터 검출할 수 있는 기준 형광 값을 가질 때의 PCR Cycle 수를 측정하고, 이를 실시간 PCR 표준 곡선의 Y축에 해당하는 CT 값에 대입하면 해수에 들어있는 거친대추멍게의 DNA의 양을 산출할 수 있다.Through the absolute quantitative analysis of real-time PCR, a standard curve was created to confirm the distribution area of the rough jujube sea squirt and at the same time determine the amount of detection. Figure 3 is a standard curve that can be used for quantitative detection of rough jujube sea squirt by real-time PCR. In the standard curve of Figure 3, the Indicates the number of PCR cycles when a standard fluorescence value is available. By measuring the number of PCR cycles when a standard fluorescence value detectable from a seawater sample is obtained and substituting this into the CT value corresponding to the Y-axis of the real-time PCR standard curve, the amount of DNA of rough jujube sea squirt in seawater is calculated. can do.
상기 실시간 PCR 및 실시간 PCR 표준 곡선을 이용하여 분석한 거친대추멍게의 국내 분포 현황 결과를 도 4에 나타내었다.The results of the domestic distribution status of rough jujube sea squirt analyzed using the real-time PCR and real-time PCR standard curve are shown in Figure 4.
도 4에서 확인할 수 있듯이, 거친대추멍게(Ascidiella aspersa)의 개체가 채집된 지역은 14개 지역이었으나, 실시간 PCR 분석을 통해서는 전체 18개 지역에서 분포를 확인하였으며, 현장 조사에서 개체를 발견하지 못한 8개 지역에서도 거친대추멍게가 서식하고 있음을 알 수 있었다. 서해안, 남해안, 동해안 지역(14개 지역)은 현장 조사를 통하여 10개 지역에서 분포를 확인하였으나 실시간 PCR 분석을 통해서는 1개 지역에서 거친대추멍게의 DNA가 검출되어 인천, 당진, 광양, 부산 4개 지역에서 추가로 분포를 확인하였다. 제주도 지역(5개 지역)은 현장 조사를 통하여 전지역에서 분포 확인이 안되었으나, 실시간 PCR 분석을 통해서는 전 지역(제주항, 성산포항, 서귀포항, 한림항, 도두항)에서 분포를 확인하였다. 거친대추멍게(Ascidiella aspersa)의 분포 지역이 전국적인 분포를 보이는 가운데, 확산하지 않은 지역까지 포함하여 국내 연안의 동해안, 남해안, 서해안을 포함한 제주도 지역까지 널리 확대되고 있는 양상을 확인하였으며, 현장 조사를 통해 발견하지 못했던 지역을 실시간 PCR 분석을 통해 확인할 수 있었다.As can be seen in Figure 4, rough jujube sea squirt ( Ascidiella Aspersa ) individuals were collected in 14 regions, but their distribution was confirmed in a total of 18 regions through real-time PCR analysis, and it was confirmed that rough jujube sea squirts also inhabit 8 regions where no individuals were found in the field survey. Could know. In the west coast, south coast, and east coast regions (14 regions), distribution was confirmed in 10 regions through field surveys, but through real-time PCR analysis, DNA of rough jujube sea squirt was detected in 1 region, and 4 regions were found in Incheon, Dangjin, Gwangyang, and Busan. Distribution was additionally confirmed in 4 regions. In the Jeju Island region (5 regions), distribution could not be confirmed throughout the entire region through field surveys, but distribution was confirmed throughout the entire region (Jeju Port, Seongsanpo Port, Seogwipo Port, Hallim Port, and Dodu Port) through real-time PCR analysis. While the distribution area of the rough jujube sea squirt ( Ascidiella aspersa ) is distributed nationwide, it was confirmed that it is expanding widely to Jeju Island, including the east coast, south coast, and west coast of the country, including areas where it has not spread, and field surveys were conducted. Areas that could not be discovered through real-time PCR analysis were confirmed.
서열목록 전자파일 첨부Sequence list electronic file attached
Claims (9)
A primer set for detecting rough jujube sea squirts, including a forward primer consisting of the base sequence of SEQ ID NO: 1 and a reverse primer consisting of the base sequence of SEQ ID NO: 2.
A composition for detecting rough jujube sea squirts comprising the primer set according to claim 1.
상기 조성물은 서열번호 3의 염기서열 또는 이의 상보적 염기서열로 이루어진 프로브를 더 포함하는 것을 특징으로 하는 거친대추멍게 검출용 조성물.
According to paragraph 2,
A composition for detecting rough jujube sea squirt, characterized in that the composition further comprises a probe consisting of the base sequence of SEQ ID NO: 3 or a complementary base sequence thereof.
상기 프로브는 5′ 말단에 FAM(6-carboxyfluorescein), 텍사스 레드(texas red), 플루오레신(fluorescein), HEX(2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE(2',7'-dimethoxy-4',5'-6-carboxyrhodamine), 플루오레신 클로로트리아지닐(fluorescein chlorotriazinyl), 로다민 그린(rhodamine green), 로다민 레드(rhodamine red), 테트라메틸로다민(tetramethylrhodamine), FITC(fluorescein isothiocyanate), 오레곤 그린(oregon green), 알렉사 플루오로(alexa fluor), ROX(6-Carboxyl-X-Rhodamine), TET(Tetrachloro-Fluorescein), TRITC(tertramethylrodamine isothiocyanate), TAMRA(6-carboxytetramethyl-rhodamine), NED(N-(1-Naphthyl) ethylenediamine) 및 씨아디카르보시아닌(thiadicarbocyanine)으로 구성된 군으로부터 선택되는 하나 이상의 리포터가 추가로 접합된 것을 특징으로 하는 거친대추멍게 검출용 조성물.
According to paragraph 3,
The probe contains FAM (6-carboxyfluorescein), Texas Red, fluorescein, HEX (2',4',5',7'-tetrachloro-6-carboxy-4, 7-dichlorofluorescein, JOE (2',7'-dimethoxy-4',5'-6-carboxyrhodamine), fluorescein chlorotriazinyl, rhodamine green, rhodamine red red), tetramethylrhodamine, FITC (fluorescein isothiocyanate), Oregon green, Alexa fluor, ROX (6-Carboxyl-X-Rhodamine), TET (Tetrachloro-Fluorescein), One or more reporters selected from the group consisting of TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), and thiadicarbocyanine are additionally conjugated. A composition for detecting rough jujube sea squirts.
상기 프로브는 3′ 말단에 BHQ1(black hole quencher 1), BHQ2(black hole quencher 2), BHQ3(black hole quencher 3), AMRA(6-carboxytetramethyl-rhodamine), NFQ(nonfluorescent quencher), 답실(dabcyl), DDQ(Deep Dark Quencher), 블랙베리 퀸처(Blackberry Quencher) 및 아이오와 블랙(Iowa black)로 구성된 군으로부터 선택되는 하나 이상의 소광자가 추가로 접합된 것을 특징으로 하는 거친대추멍게 검출용 조성물.
According to paragraph 3,
The probe contains BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), AMRA (6-carboxytetramethyl-rhodamine), NFQ (nonfluorescent quencher), and dabcyl at the 3′ end. A composition for detecting rough jujube sea squirts, characterized in that one or more quenchers selected from the group consisting of DDQ (Deep Dark Quencher), Blackberry Quencher, and Iowa black are additionally conjugated.
A kit for detecting rough jujube sea squirts comprising the composition according to claim 2.
상기 제1 단계의 DNA를 주형으로 제1항에 따른 프라이머 세트를 이용하여 중합효소 연쇄반응을 수행하여 표적 서열을 증폭하는 제2 단계; 및
상기 제2 단계의 증폭 산물에 거친대추멍게 종 특이적 DNA 마커의 존재 여부를 확인하는 제3 단계를 포함하는 거친대추멍게의 검출방법.
A first step of isolating DNA from the separated sample;
A second step of amplifying the target sequence by performing a polymerase chain reaction using the DNA of the first step as a template and the primer set according to claim 1; and
A method for detecting rough jujube sea squirts comprising a third step of confirming the presence of a rough jujube sea squirt species-specific DNA marker in the amplification product of the second step.
상기 제3 단계는 서열번호 3의 염기서열 또는 이의 상보적 염기서열로 이루어진 프로브를 이용하여 수행하는 것을 특징으로 하는 거친대추멍게의 검출방법.
In clause 7,
The third step is a method of detecting rough jujube sea squirt, characterized in that it is performed using a probe consisting of the base sequence of SEQ ID NO: 3 or its complementary base sequence.
제1항에 따른 프라이머 세트 및 서열번호 3의 염기서열 또는 이의 상보적 염기서열로 이루어진 프로브를 이용하여 실시간 중합효소 연쇄반응을 실시하는 제2 단계; 및
상기 제2 단계의 실시간 중합효소 연쇄반응을 통해 확인된 실시간 PCR 표준 곡선을 이용하여 거친대추멍게의 DNA 양을 산출하는 단계를 포함하는 해양 생태계 내 거친대추멍게 분포 조사 방법.A first step of isolating DNA from the separated sample;
A second step of performing a real-time polymerase chain reaction using the primer set according to claim 1 and a probe consisting of the base sequence of SEQ ID NO: 3 or its complementary base sequence; and
A method for investigating the distribution of rough jujube sea squirts in a marine ecosystem, comprising the step of calculating the amount of DNA of the rough jujube sea squirts using the real-time PCR standard curve confirmed through the real-time polymerase chain reaction in the second step.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20240109319A true KR20240109319A (en) | 2024-07-11 |
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