一种水产动物病毒现场检测免疫芯片的制备与应用   总被引：4，自引：2，他引：2  

徐晓丽  绳秀珍  战文斌 《海洋与湖沼》2010,41(5):698-706

采用原子力显微镜对氨基化玻片及6种不同方法修饰玻片进行表面特征分析,并比较了硝酸纤维素(NC)膜、PVDF膜以及以上不同修饰玻片对抗体的固定效率、固定效果,最终选择琼脂糖修饰玻片作为芯片载体。将纯化后的兔抗血清(捕获抗体)点样至琼脂糖修饰玻片上,制备免疫芯片,与待检样品(病毒感染的靶器官组织)匀浆液孵育形成复合物,该复合物被辣根过氧化物酶(HRP)标记的特异性单克隆抗体(单抗)识别,经底物显色,得到肉眼可见的检测结果。通过改变芯片制备及应用过程中的具体条件参数,对各条件进行了优化,并采用生物素-链亲和素(BAS)标记特异性单抗作为检测抗体以提高检测灵敏度。基于此夹心免疫分析原理制备的WSSV、LCDV现场检测免疫芯片,病毒的最低检出量分别为82.50ng/ml、0.88μg/ml,在一定的抗原浓度范围内,病毒浓度的对数值与信号强度呈线性关系;采用BAS放大检测信号后,病毒的最低检出量为12.38ng/ml、0.22μg/ml。该免疫芯片与酶联免疫吸附法(ELISA)及免疫荧光法(IFAT)对同种样品的检测结果高度一致。  相似文献   

重点海水浴场主要肠道病毒的调查     

明红霞  樊景凤  关道明  梁玉波 《海洋学报》2010,32(3):138-145

海水浴场作为重要的娱乐场所,其水质卫生状况对保障公众健康极其重要。对全国重点海水浴场中轮状病毒、星状病毒和脊髓灰质炎病毒进行了调查,因为这3种病毒是引发腹泻性胃肠炎的重要病原。2007年8月选定全国10个重点海水浴场,于游泳区设定2个站位,无菌采集表层海水,用Millipore的病毒浓缩包进行海水中病毒的浓缩,用RT-PCR进行三种病毒的监测。调查结果显示:轮状病毒阳性检出率达40%(8/20)、星状病毒的为35%(7/20)、脊髓灰质炎病毒的为35%(7/20);在这十大重点海水浴场中,除北海一处无病毒检出外,其余均有不同程度的病毒阳性检出,表明我国的重点海水浴场水质已经遭受肠道病毒不同程度的污染。建议相关部门应该加强海水浴场的卫生管理,避免公众因在海水浴场娱乐导致疾病。  相似文献   

Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China （LCDV-cn） genome   总被引：1，自引：0，他引：1  

闫秀英  孙修勤 《中国海洋湖沼学报》2009,27(2):337-341

An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene. Supported by High Technology Research and Development Program of China (863 Program, No. 2006AA100309)  相似文献   

应用巢式逆转录聚合酶链反应检测鱼类病毒性出血性败血症病毒(VHSV)     

倪穗  余晓巍  王建平  雷爱莹  曾地刚  吴雄飞 《海洋与湖沼》2009,40(4):489-493

参照鱼类病毒性出血性败血症病毒序列,根据PCR引物设计的原则,设计巢式PCR引物,采用巢式逆转录聚合酶链反应(RT-PCR)方法,对如何快速检测鱼类病毒性出血性败血症病的方法进行了较为系统的研究,并对RT-PCR的反应条件进行了优化.结果表明,巢式RT-PCR扩增获得279bp的特异性片断,阴性对照无扩增条带.巢式RT-PCR扩增出的特异性片段经测序分析,结果证实与报道的序列完全一致.该方法最低可检测出0.1pg的鱼类病毒性出血性败血症病毒RNA.初步建立了VHSV的巢式RT-PCR检测方法,该方法灵敏、特异,可为VHSV的检测提供一个快速、有效的手段.  相似文献   

虾蟹混养塘中WSSV对三疣梭子蟹(Portunus trituberculatus)致死效应的定量研究   总被引：1，自引：0，他引：1  

王忠发  王志铮  许文军  何伟贤  顾松叶 《海洋与湖沼》2008,39(2):184-189

采用实时荧光定量PCR法,在对白斑综合症多发的虾蟹混养塘内三疣梭子蟹体内病毒含量进行定量跟踪检测的基础上,结合电子显微镜及病理切片,对室内经口服、注射两种回感模式处理已感染白斑综合症病毒的濒死三疣梭子蟹体内病毒含量进行实验验证,并观察其体内主要器官组织的病理改变。结果表明,受白斑综合症病毒感染的三疣梭子蟹存活与死亡取决于蟹体内病毒含量,当病毒含量≥109拷贝/ml时均进入濒死或死亡阶段,其主要器官组织均有明显的病理改变。  相似文献   

White spot syndrome virus envelope protein VP124 involved in the virus infection     

ZHU Yanbing  WU Chenglin  YANG Feng 《海洋学报(英文版)》2008,27(4):130-136

White spot syndrome virus （WSSV） is one of the major shrimp pathogens causing large economic losses to shrimp farming. In an attempt to identify the envelope proteins involved in the virus infection, purified WSSV virions were mixed with three antisera against WSSV envelope proteins （VP39, VP124 and VP187 ）, individually. And then they were injected intramuscularly into crayfish （Procambarus clarkii） to conduct in vivo neutralization assays. The results showed that for groups injected with virions only and groups injected with the mixture of virions and antiserum against VP124, the crayfish mortalities were 100% and 60% on the 8th day postinfection, individually. The virus infection could be delayed or neutralized by antibody against the envelope protein VP124. Quantitative PCR was used to further investigate the influence of three antisera described above on the virus infection. The results showed that the antiserum against VP124 could restrain the propagation of WSSV in crayfish. All of the results suggested that the viral envelope protein VP124 played a role in WSSV infection.  相似文献   

原位杂交技术在斜带石斑鱼神经坏死病毒检测中的应用   总被引：4，自引：0，他引：4  

陈晓艳  何建国 《海洋科学》2008,32(6):1-4

根据斜带石斑鱼（Epinephelus coioids）神经坏死病毒（orang-spotted nervous necrosis virus,0GNNV）的主衣壳蛋白（major capsid protein,MCP）基因的保守序列,设计一对引物,从感染0GNNV的斜带石斑鱼组织匀浆液提取RNA为模板进行RT-PCR扩增,得到426bp的cDNA片断。用得到的RT-PCR产物加上地高辛（DIG）标记作为核酸探针。通过注射病毒提取液人工感染一组斜带石斑鱼,解剖感染病毒的斜带石斑鱼,从中分离出脑和眼睛,运用原位杂交技术检测组织中的OGNNV。实验表明,原位杂交具有较高的灵敏性和特异性,可以用原位杂交的办法来检测养殖的石斑鱼是否有携带该病毒,达到监控和预防神经坏死病爆发的目的。本实验还采用了H＆E染色方法检测了感染NNV的石斑鱼脑部和眼部组织,观察细胞内的坏死部分。与原位杂交做对比,提高了检测的可靠性和准确性。本实验建立的石斑鱼神经坏死病毒的ISH检测方法有着较高的特异性和敏感性,易于操作,有助于石斑鱼神经坏死病毒的组织定位、发病机理的研究。  相似文献   

姬松茸提取物组分体外抗病毒感染活性的研究     

樊廷俊  钱冠兰  杜玉堂  袁文鹏  吕雅萌 《中国海洋大学学报(自然科学版)》2008,38(5)

为了探明姬松茸提取物的体外抗病毒感染活性及其作用方式,进而为水产养殖鱼类高效抗淋巴囊肿病毒(lymphocystis disease virus,LCDV)感染活性物质的开发和鲆蝶类淋巴囊肿病的防治奠定基础,利用热水浸提和酒精沉淀法得到了5种姬松茸提取物组分(E1～5),并利用MTT、细胞病变程度观察等方法研究了5种组分对LCDV感染体外培养大菱鲆鳍细胞(turbot fin cells,TF细胞)的影响作用.细胞毒性实验结果显示,5种组分对体外培养TF细胞均无毒性.细胞病变程度结果表明,本文所得5种姬松茸提取物组分尤其是E5组分具有显著的抗LCDV感染TF细胞的活性.不同方式感染的实验结果进一步显示,5种姬松茸提取物组分抗LCDV感染TF细胞的作用可能主要是通过直接灭活病毒和/或阻断病毒吸附细胞来实现的.  相似文献   

A STUDY ON PATHOGENS OF CHINESE PRAWN (PENAEUS CHINENSIS) VIRUS DISEASES     

孙修勤  张进兴 《中国海洋湖沼学报》1995,(3)

This pathogenic study shows that the viral diseases of Chinese prawns (Penaeus chinensis. O'sbeck)is due to three kinds of viruses: epithelium envelope baculovirus of Penaeus chinensis (EEBV-PC, de-tected by the authors in 1993), infections hypodermal and hematopoietic necrosis virus, andhepatopancreatic parvo-like virus, and that the first two viruses seem to be the main pathogens of theepidemic in the northern regions in 1993.  相似文献   

Flying-fox species and Hendra virus spillover risk: comment on ‘Factors explaining the dispersal of Hendra virus disease in South-East Queensland’ by Burnham and Chong (2016)     

Hume Field  Dan Edson  Craig Smith 《国际地球制图》2017,32(6):697-698

The approach taken by Burnham and Chong to explain the occurrence of Hendra virus incidents in Australia, while analytically valid, is compromised by erroneous and incomplete biological data underpinning the analyses. Firstly, they erroneously state the peak mating, pregnancy and birthing periods in black, grey-headed and little red flying-foxes, thus their related interpretations and conclusions are flawed. Secondly, the reported association between grey-headed flying-fox density and HeV incidents is at odds with recent findings suggesting grey-headed flying-foxes are an insubstantial host. While this broader epidemiological perspective was not within their stated aims, it is appropriate that it be canvassed in discussion of their geo-spatial analyses given the level of horse-industry, public and media interest in Hendra virus and the potential for confusion and mixed messages regarding HeV exposure risk management. We suggest that a broader collaborative cross-disciplinary approach offers synergies that produce robust, real-world ‘one health’ outputs.  相似文献