检测中国对虾非包涵体型杆状病毒的PCR方法的建立  

DEVELOPMENT OF POLYMERASE CHAIN REACTION ASSAY FOR DETECTION OF PENAEUS CHINENSIS NON-OCCLUDED BACULOVIRUS

作　　者：徐洪涛[1] 朴春爱[2] 王雷[1] 朱俊萍[1] 董杰[2] 王长安[2] 相建海[1] 金奇[2] 侯云德[2] 

机构地区：[1]中国科学院海洋研究所,山东青岛266071 [2]中国预防医学科学院病毒学研究所,北京100052

出　　处：《病毒学报》1999年第4期354-359,共6页Chinese Journal of Virology

基　　金：海洋"863";中国科学院EMBL 开放课题;病毒基因工程国家重点实验室客座课题

摘　　要：中国对虾非包涵体型杆状病毒（ＰｃＮＯＢＶ） 是中国大陆养殖的中国对虾（ Ｐｅｎａｅｕｓｃｈｉｎｅｎｓｉｓ） 暴发性流行病———白斑综合征的主要病原，与台湾流行的Ｐｍ ＮＯＢⅢ、泰国的Ｐｍ ＮＯＢⅡ及日本的ＲＶ－ ＰＪ（ ＝ ＰＲＤＶ） 有相似的致病特征、形态学及组织病理学特征。为了解ＰｃＮＯＢＶ 与Ｐｍ ＮＯＢⅢ两种地域分布邻近的毒株基因水平的关系，并建立早期、敏感、特异的检测技术，利用氯化铯密度梯度超速离心技术分离纯化了ＰｃＮＯＢＶ 核衣壳，根据台湾地区分离的Ｐｍ ＮＯＢⅢ的基因序列设计一对引物，从纯化的ＰｃＮＯＢＶ ＤＮＡ 及自然发病的中国对虾、日本对虾和斑节对虾组织ＤＮＡ中成功地扩增出长为３５５ｂｐ 的目的片段，可检测出３ ．４ｐｇ 的ＰｃＮＯＢＶ ＤＮＡ。从健康对虾组织提取的ＤＮＡ未能扩增出目的片段。扩增片段序列与Ｐｍ ＮＯＢⅢ相应的基因序列相同。该结果从分子水平证实ＰｃＮＯＢＶ 与Ｐｍ ＮＯＢⅢ存在基因同源性，同时表明聚合酶链式反应（ＰＣＲ） 可以作为检测ＰｃＮＯＢＶ 的一种敏感、特异、早期、快速的诊断手段。Penaeus chinensis non-occluded baculovirus(PcNOBV) is the causative agent of the widespread epizootic called white spot syndrome which has caused high shrimp mortality and great economic loss to Chinese shrimp industry. Preliminary research results indicated that PcNOBV belongs to members of white spot syndrome baculoviruses (WSBVs), representing a WSBV isolate from cultured P. chinensis in China's Mainland. It has been assumed that all of the WSBVs including PcNOBV isolated from P. chinensis in China's Mainland , PmNOBⅡ from P. monodon in Thailand, PmNOBⅢ from P. monodon and P. japonicus in Taiwan and RV-Pj(=PRDV)from P. japonicus in Japan are closely related and may represent different strains of a same virus. In order to obtain some information on the relationship between the two geographically adjacent PmNOBⅢ in Taiwan and PcNOBV in China's Mainland, a pair of primers producing 355bp amplification fragment was designed based on the sequence of PmNOBⅢ genome DNA SalⅠ fragment. The expected amplification product was obtained when DNA isolated from purified nucleocapsids of PcNOBV and DNAs from naturally diseased P. chinensis, P. japonicus and P. monodon tissues were used as templates. The detection limit of purified PcNOBV DNA is 3.4 pg. DNA isolated from healthy penaeid shrimps did not yield any amplification product. These results demonstrate that PcNOBV and PmNOBⅢ are homologous at least in part of the genome structure, and that this assay can be used for early, rapid, sensitive and specific detection of PcNOBV infection.

关 键 词：中国对虾 诊断 PCR PcNOBV WSBV 

分 类 号：S945[农业科学—水产养殖] Q93－33[农业科学—水产科学]