对虾一种无包涵体杆状病毒病原的PCR检测
Detection of a nonoccluded baculovirus in penaeid shrimp by PCR
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摘要: 为建立对虾无包涵体杆状病毒的快速诊断技术,应用PCR技术分别对暴发性流行病发生前、流行中和发病后的对虾样品进行了检测.根据已克隆的对虾杆状病毒部分基因组序列而设计的引物能特异性地扩增出靶DNA片段,最低可以检测1pg的病素DNA.由典型发病症状对虾的胃、腮、肝胰腺、中肠、游泳足、肌内和心脏等器官和组织中成功地检测到病毒.不同组织和器官经扩增得到的信号强弱不同:病虾的胃扩增得到的信号最强,中肠、肌肉、心脏和游泳足次之,腮和肝胰腺信号最弱,说明病毒在不同组织和器官中数量及感染程度不同.在对虾发病前及发病后,用二次PCR还能检测表面不发病呈隐性感染状态的对虾携带的病毒;而对野生健康虾的检测结果为阴性.研究表明,PCR是检测对虾暴发性流行病快速、灵敏而准确的方法,对病毒病的早期诊断、防治和高健康对虾品种的选育具有指导意义.Abstract: Specimen of Penaeus chznenszs were collected from hatchery ponds at Jimo, Qingdao before, during and after explosive epidemic disease respectively in July to September 1997.In order to establish rapid diagnosis techniques for shrimp explosive epidemic virus, diseased samples are detected by polymerase chain reaction(PCR).The primers which are synthesized according to the genome sequence of partially cloned shrimp baculovirus can specifically amplify the target baculoviral DNA, the viral DNA can be detected as low in quantity as 1 pg.The virus can be detected from the stomach, gill, hapatopancrease, min-gut, pereopod, muscle and heart of the penaeid shrimps with typical symptom of explosive epidemic disease.The positive signals vary strength from the strongest in stomach,the min-gut, muscle, heart and pereopod in the middle, to the weadest in gill and hapatopancrease.This result demon-strates the different numbers and affection extent of virus in different tissues or organs.Virus can also be detected by nested PCR during latent and convalescent periods from shrimps showing no obvious sympton of acute disease, whereas the results on healthy wild P.Chinensis are negative.With its sensitivity, accuracy and specificity, PCR is significant for the early diagnosis of viral disease and the high healthy stock brding of penaeid shrimp.
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Key words:
- Penaeid shrimp /
- non-occluded baculovirus /
- PCR /
- pathogen detection
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Lightner D V, Poulos B T, Bruce. L, et al. New developments in penaeid virology:application of biotechnology in research and disease diagnosis for shrimp virus of concern in the American. In:Fulks W, Main K L, ed. Diseases of Cultured Penaeid Shrimp in Asia and the United States. Honolulu. Hawaii, 1992. 233～253 黄健,宋晓玲,于佳,等.杆状病毒性的皮下及造血组织球死对虾暴发性流行病的病原和病理学.海洋水产研究,1995,16(1):1～10 Chang P S, Lo C F, Kou G H, et al. Purfication and amplification of DNS from Penaeus monodon-type baculovirus(MBV) Journal of invertebrate Pathology, 1993,(62):116～120 Kimura T, Yamano K, Nakano H, et al. Detection of penaeid rod-shaped DNA virus(PRDV) by PCR. Fish Pathology, 1996, 31(2):93～98 孔杰,石拓,刘萍,等,中国对虾一种C型杆状病毒的纯化技术及形态特征研究.海洋与湖沼,1997,28(3):233～237 Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning, a Laboratory Manual. 2nd ed. Cold Spring Harbor, New York:Cold Spring Harbor Laboratory Press, 1989 张立人,张建红,陈棣华,等,东方对虾杆状病毒在宿主细胞内的装配.电子显微学报,1994,13(5):354 彭宝珍,任家鸣,沈菊英,等.急性致死性对虾病的杆状病毒病原研究.病毒学报,1995,11(2):151～157 Overstreet R M, Stuck K C, Krol R A, et al. Experimental infections with baculovirus penaei in the white shrimp Penaeus vannamei(Crustaces:Decaplda) as a bioassay. Journal of the World Aquaculture Society, 1989, 19(4):175～187 Bruce L D, Redman R M, Lightner D V, et al. Application of gene probe to determine target organs of penaeid shrimp baculovirus in situ hybridization. Aquaculture, 1994, 120:45～53 Lu C C, Tang K F J, Kou G H, et al. Detection of Penaeus monodon-type baculovirus(MBV) infection in Penaeus monodon Fabricius by in situ hybridization. Journal of Fist Disease, 1995, 18:337～345 包振民,胡景杰,姜明,等.杆状病毒感染越冬亲虾的研究越冬亲虾感染及其垂直传播的可能性.青岛海洋大学学报,1997,27(3):347～351 -
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