[논문]PCR 기법을 이용한 바지락포자충 Perkinsus 진단 기술개발

박경일; 박영미; 이제희; 최광식

PCR 기법을 이용한 바지락포자충 Perkinsus 진단 기술개발
Development of a PCR Assay for Detection of the Protozoan Parasite Perkinsus 원문보기

환경생물 = Korean journal of environmental biology, v.20 no.2, 2002년, pp.109 - 117

박경일 (제주대학교 해양과학대학 해양생산과학부) , 박영미 (제주대학교 해양과학대학 해양생산과학부) , 이제희 (제주대학교 해양과학대학 해양생산과학부) , 최광식 (제주대학교 해양과학대학 해양생산과학부)

초록
AI-Helper

이 연구에서는 해산 연체동물의 폐사를 유발하는 기생성 원생동물인 Perkinsus를 신속하고 특이적으로 검출하기 위하여 PCR 진단법을 개발하였다. 이를 위해 Perkinsus 속 4종을 공통적으로 증폭하거나 P. atlanticus만을 특이적으로 증폭할 수 있는 두 가지의 primer를 제작하였다. PCR분석은 기존 Perkinsus 진단법인 fluid thioglycollate medium (FTM) 방법과 2 M NaOH 기법을 병행하여 실시하였다. 실험구로서 전라남도 완도산, 제주도 김녕산, 제주도 서귀포산, 제주도 성산산 바지락과 in vitro 배양된 바지락포자충이 이용되었으며, 대조구로서 전라남도 강진에서 채집된 꼬막, T. granosa을 사용하였다. 실험 결과 Perkinsus특이성 DNA band가 전남 완도산과 제주도 성산산 바지락, in vitro 배양된 바지락 포자충에서 확인되었으나, 대조구였던 꼬막과 제주도 서귀포산, 제주도 김녕산 바지락에서는 나타나지 않았다. 이같은 진단 결과는 FTM과 2M NaOH 진단 결과와 일치하였다. 한편 P. atlanticus에 특이적인 primer에 의해 증폭된 band가 확인됨에 따라 국내산 바지락에서 검출되는 바지락포자충은 P. atlanticus와 동일 종임이 확인되었다. 결론적으로, 본 연구를 통하여 개발된 PCR을 이용한 진단법은 해산 연체동물내 Perkinsus 속 기생충과 P. atlanticus의 감염을 신속하고도 종 특이적으로 진단할 수 있어 수ㆍ출입 수산물의 검역과 바지락 포자충의 생태학적 특성을 규명하는데 효과적으로 이용될 수 있을 것으로 기대된다.

Abstract ▼ AI-Helper

Detection of protozoan parasites Perkinsus sp. and P. atlanticus was developed in this study using a specific polymerase chain reaction (PCR) to diagnose the presence of those organisms that causes extensive mortalities of marine shellfishes. The PCR was conducted together with fluid thioglycollate medium (FTM) method and 2 M NaOH lysis method. For the test, Manila clams, Ruditapes philippinarum, were collected from four coastal locations in Korea including Wando Island, Gimnyeong, Sungsan and Sogwipo in Jeju. In addition, trophozites of Perkinsus sp. cultivated in vitro and the granular ark clam, Tegillarca granosa, taken from Gangjin on the south coast of Korea, were used as positive and negative controls, respectively. Expected DNA bands were detected in the samples from Wando Island, Sungsan and the in vitro cultured Perkinsus sp. when the probes specific for the genus Perkinsus and P. atlanticus were used. The samples were also positively diagnosed by the FTM and 2 M NaOH methods. In contrast, the Manila clams from Gimnyeong and Sogwipo, and the granular arks clams from Gangjin showed no detectable signs of infection with the PCR, the FTM method and the 2 M NaOH lysis method. On the other hand, being amplified by p. atlanticus specific primer, it is suggested that the protozoan parasite Perkinsus sp. found in the Korean Manila clam is P. atlanticus. Finally the PCR- based assay developed in the present study can be used in detection of Perkinsus infection and discrimination of Peykinsus species in quarantine stations or laboratories due to the high sensitivity and specificity as well as its rapid detection.

주제어

참고문헌 (46)

Ahn K-J and K-H Kim. 2001. Effect of temperature and salinity on in vitro zoosporulation of Perkinsus sp. in Manila clams Ruditapes phiIippinarum. Dis. Aquat. Org. 48:43-46

상세보기
Almeida M, F Berthe, A Thebault and MT Dinis. 1999. Whole clam culture as a quantitative diagnostic Proce-dure of Perkinsus atlanticus (Apicomplexa, Perkinsea) in clams Ruditapes decussatus. Aquaculture. 177:325-332

상세보기
Andrews JD. 1996. History of Perkinsus marinus, a Path-ogen of oysters in the Chesapeake Bay 1950-1984. J. Shellfish Res. 15:13-16
Auzoux-Bordenave S, AM Vigario, F Ruano, I Domart-Coulon and D Doumenc. 1995. In vitro sporulation of the clam pathogen Perkinsus atlanticus (Apicomplexa, Perkinsea) under various environmental conditions. J. Shellfish Res. 14:469-475
Azevedo C. 1989. Fine structure of Perkinsus atlanticus n. sp. (Apicomplexa, Perkinsea) Parasite of the clam Rudi-tapes decussatus from Portugal. J. Parasitol. 75:627-635

상세보기
Blackbourn J, SM Bower and GR Meyer. 1998. Perkinsus qugwadi sp. nov. (incertae sedis), a pathogenic Proto-zoan parasite of Japanese scallops, Patinopecten yes-soensis, cultured in British Columbia, Canada. Can. J. Zool. 76:942-953

상세보기
Burreson EM and L Ragone Calvo. 1996. Epizootiology of Perkinsus marinas disease of oysters in Chesapeake Bay, with emphasis on data since 1985. J. Shellfish Res. 15:17-34
Bushek D, SE Ford and SK Allen Jr. 1994. Evaluation of methods using Ray's thioglycollate medium for diagno-sis of Perkinsus marinum infection in the eastern Oys-ter, Crassostrea virginica. Ann. Rev. Fish Dis. 4:201-217

상세보기
Cavalier-Smith T. 1999. Zooflagellate phylogeny and the systematics of protozoa. Biol. Bull. 196:393-396

상세보기
Chagot D, M Comps, V Boulo, F Ruano and H Grizel. 1987. Histological study of a cellular reaction in Ruditapes decussatus infected by a protozoan. Aquaculture. 67:260-261

상세보기
Choi D-L, J-N Kwon and S-W Park. 1998. Infection andrapid detection of Perkinsus sp. in cultured babyneck clam, Ruditapes philippinarum from western coast of Korea. J. Fish Pathol. 11:69-76
Choi K-S and K-I Park. 1997. Report on occurrence of Perhinsus sp. in the Manila clam Ruditapes PhiliP-pinarum in Korea. Korean J. Aquacult. 10:227-237
Choi K-S, DH Lewis, EN Powell, PF Frelier and SM Ray. 1991. A polyclonal antibody developed from Perkinsus marinas hypnospores fails to cross-react with other life stages of P. marinus in oyster (Crassostrea Virgini-co) tissues. J. Shellfish Res. 10:411-415
Choi K-S, EA Wilson, DH Lewis, EN Powell and SM Ray. 1989. The energetic cost of Perkinsus marinus Para-sitism in oysters: quantification of the thioglycollate method. J. Shellfish Res. 8:125-131
Choi K-S and K-I Park. 2001. Infection intensity, Pre-valence and histopathology of Perkinsus sp. in the Manila clam, Ruditapes phitippinarum, in West Ariake Sound, Japan. J. Shellfish Res. (in press)
Coss CA, JA Robledo, GM Ruiz and GR Vasta. 2001. Description of Perkinsus andrewsi n. sp. isolated from the Baltic clam (Macoma balthica) by characterization of the ribosomal RNA locus, and development of a Spe-cies-specific PCR-based diagnostic assay, J. Eukaryo-tic Microbiol. 48:52-61

상세보기
Crosby MP and CF Roberts. 1990. Seasonal infection inten-sity cycle of the parasite Perkinsus marinus (and an ab-sence of HapIosporidium spp.) on oysters from a South Carolina salt marsh. Dis. Aquat. Org. 9:149-155

상세보기
Dungan CF and BS Roberson. 1993. Binding specificities of mono-and polyclonal antibodies to the protozoan oyster pathogen Perkinsus marinus. Dis. Aquat. Org. 15:9-22

상세보기
Figueras A, G Lorenzo, MC Ordas, M Gouy and B Novoa. 2000. Sequence of the small subunit ribosomal RNA gene of Perkinsus atlanticus-Iike isolated from Carpet shell clam in Galicia, Spain. Mar. Biotechnol. 2:419-428
Fisher WS and LM Oliver. 1996. A whole-oyster procedure for diagnosis of Perkinsus marinus disease using Ray's fluid thioglycollate culture medium. J. Shellfish. Res. 15:109-117
Hamaguchi M, N Suzuki, H Usuki and H Ishioka. 1998. Perkinsus protozoan infection in Short-necked clam Tapes (Ruditapes) Phitippinarum in Japan. Fish Pathol. 33: 473-480

상세보기
Kinne O. 1983. Diseases of marine animals. Biologische Anstal Helgoland, Hamburg, Germany
Korea Fisheries Association. 2000. Korean Fisheries Yearbook. Korea Fisheries Association, Seoul, Korea
Krantz GE and SJ Jordan. 1996. Management alternatives for protecting Crassostrea virginica fisheries in Per-kinsus marinas enzootic and epizootic areas. J. Shell-fish Res. 15:167-176
Levine ND. 1978 Perkinsus gen. n. and other new taxa in the protozoan Phylum Apicomplexa. J. Parasitol. 64:549

상세보기
Lee M-K, B-Y Cho, S-J Lee, J-Y Kang, H-D Jeong, S-H Huh and M-D Huh. 2001. Histopathological lesions of Manila clam, Tapes philippinarum, from Hadong and Namhae coastal areas of Korea. Aquaculture. 201:199-209

상세보기
Liang YB, XC Zhang, LI Wang, B Yang, Y Zhang and CL Cai. 2001. Prevalence of Perkinsus sp. in the Manila clam Ruditapes philippmarum along northern coast of Yellow Sea in China. Oceanol. Limnol. Siniga. 32:502-511
Mackin JG. 1962. Oyster disease caused by Dermocysti-dium marinum and other microorganisms in Louisi-ana. Publ. Inst. Mar. Sci. Univ. Texas 7:132-229
Mackin JG and JL Boswell. 1956. The life cycle and rela-tionships of Dermocystidium marinum. Proc. Natl. Shellfish Assoc. 46:112-115
Noren F, $\phi$ Moestrup, A-S Rehnstam-Holm. 1999. Par-vilucifera infectans Noren et Moestrup gen. et sp. nov. (Perkinsozoa phylum nov.): a parasitic flagellate Capa-ble of killing toxic microalgae. Eur. J. Protistol, 35: 233-254

상세보기
Ordas MC and A Figueras. 1998. In vitro culture of Per-kinsus atlanticus, a parasite of the carpet shell clam Ruditapes decussatus. Dis. Aquat. Org. 33:129-136

상세보기
Ordas MC, A Ordas, C Beloso and A Figueras. 2000. Imm-une parameters in carpet shell clams naturally infected with Perkinsus atlanticus. Fish Shellfish Immunol. 10:597-609

상세보기
Park K-I. 1999. Occurrence of Perkinsus sp. in the Manila clam, Ruditapes philippinarum and development of diagnostic methods. M.Sc. thesis, Cheju National University
Park K-I and K-S Choi. 2001. Spatial distribution and infection intensity of the protozoan parasite Perkinsus sp. in the Manila clam Ruditapes philippinarum in Korea. Aquaculture. 203:9-22

상세보기
Park K-I, Choi K-S and Choi J-W. 1999. Epizootiology of Perkinsus sp. found in the Manila clam, Ruditapes Phi-tippinarum in Komsoe Bay, Korea. J. Korean Fish. Soc. 32:303-309
Perkins FO. 1996. The structure of Perkinsus marinas (Mackin, Owen and Collier, 1950) Levine, 1978 with comments on the taxonomy and phylogeny of Perkinsus spp. J. Shellfish Res. 15:67-87
Ray SM. 1952. A culture technique for the diagnosis of infection with Dermocystidium marinum Mackin, OW-en and Collier in Oyster. Science. 116:360-361

상세보기
Ray SM and JG Mackin. 1954. Studies of pathogenesis of Dermocystidium marinum. Proc. Natl. Shellfish Assoc. 45:164-167
Robledo JAF, JD Gauthier, CA Coss, AC Wright and GR Vasta. 1998. Species-specificity and sensitivity of a PCR-based assay for Perkinsus marinus in the eastern oyster, Crassostrea virginica: a comparison with the fl-uid thioglycollate assay. J. Parasitol. 84:1237-1244

상세보기
Rodriguez F and JI Navas. 1995. A comparison of gill and hemolymph assays for the thioglycollate diagnosis of Perkinsus atlanticus (Apicomplexa, Perkinsea) in Cla-ms, Ruditapes decussatus (L.) and Ruditapes Philippin-arum(Adams et. Reeve). Aquaculture. 132:145-152

상세보기
Romestand B, J Torreilles and P Roch. 2001. Production of monoclonal antibodies against the Protozoa, Perkinsus marinus: estimation of parasite multiplication in vitro. Aquat. Living Resour. 14:351-357

상세보기
Ryu P-Y, Chung J and Oh J-S. 1995. Polymerase chain reaction of mycobacterial DNA by FTC-2000 capillary PCR machine. J. Korean Soc. Microbiol. 30:415-424
Siddall ME, KS Reece, JE Graves and EM Burreson. 1997. 'Total evidence' refutes the inclusion of Perkinsus Spe-cies in the phylum Apicomplexa. Parasitology. 115:165-176

상세보기
Stein JE, JG Mackin. 1957. An evaluation of the culture method used in determining the in tensity of Dermocys-tidium marinum infection in the oyster Crassostrea virginica. Texas A & M Res. Found. Project 23, Tech. Rept. 22:1-5
Sunila I, RM Hamilton and CF Dungan. 2001. UItra-structural characteristics of the in vitro cell cycle of the protozoan pathogen of oysters, Perkinsus marinas. J. Eukaryot. Microbiol. 48:348-361

상세보기
Wilson EA, EN Powell, M Alison Craig, TL Wade and JM Brooks. 1990. The distribution of Perkinsus marinus in Gulf coast oysters: Its relationship with temperature, reproduction and pollutant body burden, Int. Revue ges. Hydrobiol. 75:533-550

상세보기

저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증