患病罗氏沼虾幼体可疑病原核酸随机克隆技术的探索-临床兽医学专业论文.docxVIP

入片段序列中，一段长 551bp 的克隆插入片段与西番莲属萎黄病毒(Passiflora chlorosis virus，PCV)序列有一定同源性，一段长 441bp 的克隆插入片段与轮状病 毒(Rotavirus，RV)序列有一定同源性。根据这两段克隆插入片段序列设计引物对样 品进行特异性鉴定的结果显示这些克隆插入片段序列确实是来源于采集的罗氏沼 虾幼体样本。 本文使用多重随机 PCR 技术和序列非依赖的单引物扩增技术，对罗氏沼虾幼 体样本进行分析的结果说明，可以应用这两种随机克隆技术对罗氏沼虾进行可疑 病毒的鉴定分析，对水产动物可疑病原的鉴定、流行病监控、养殖过程中病害的 早期防治可起到重要的作用。 关键词：罗氏沼虾，多重随机 PCR，序列非依赖的单引物扩增，可疑病毒病原， 鉴定与检测 The exploration of random cloning technology for suspicious pathogen nucleic acid in suffering Macrobrachium rosenbergii larvae ABSTRACT Macrobrachium rosenbergii, belongs to the family Palaemonidae，Macrobrachium, also known as the genus of white leg shrimp, Malaysia prawn shrimp, Wan prawn, originating in the India Pacific region, living in various types of freshwater or brackish waters, introduced to our country from Japan in 1976, is one of three prawn breed with a highest quantity in the present world and a basically breed variety of freshwater crustacean in China . But in recent years, with the continuous expansion of Macrobrachium rosenbergii culture area, ceaselessly intensive rising degree, virus disease is also frequent, bringing great threat for Macrobrachium rosenbergii culture. So far, the reported Macrobrachium rosenbergii virus has four types: WSSV, Macrobrachium rosenbergii Nodavirus(MrNV)causing Macrobrachium rosenbergii whitish muscle diseases ( whitish muscle disease, WTD)and XSV(extra small virus-like particles)may serving as a MrNV satellite virus or helper virus tiny virus, as well as a relatively rare parvo-like virus infecting epithelial cells of Macrobrachium rosenbergii liver pancreas ( parvo-like virus ). In March 2011, we collected a number of diseased larvae samples in a giant freshwater prawn culturing plant from Zhejiang. By homogenate, 0.45 micron membrane filtration, centrifugation, DNase I and RNase A processing, extraction of suspicious viral nucleic acid, we use multiple random PCR and sequence independent single primer amplification( SISPA ) for DNA and RNA random cloning and sequencin