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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
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为了确定鱼类神经坏死病毒对军曹鱼(Rachycentron canadum)仔鱼的致病性,本研究通过肌肉注射、浸泡和共浴3种途径分别感染军曹鱼仔鱼,观察了由其导致的发病死亡情况和病理学变化。结果表明,3种感染途径都能造成军曹鱼仔鱼出现厌食、体色发黑和螺旋游泳等典型的病毒性神经坏死症症状,累计死亡率分别达到78%、44%和34%。肌肉注射18h后,利用RT-PCR方法在鱼的脑、眼、脊髓中均可检测到该病毒。患病鱼的脑、视网膜、脊髓等组织发生空泡化,与其他鱼类感染该病毒发生的变化一致。因此,军曹鱼仔鱼是鱼类诺达病毒的易感种群之一,病毒能通过水平传播感染军曹鱼仔鱼。 相似文献
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厦门海水网箱养殖石斑鱼春、夏季节暴发性疾病经过流行病学调查和病原检测，确认主要病原为病毒性神经坏死病毒，病鱼外观表现典型的神经异常症状。应用逆转录聚合酶链式反应（RT—PCR）技术检测5种石斑鱼：赤点石斑鱼（Epinephelus akaara）、青石斑鱼（E．awoara）、云纹石斑鱼（Emoara）、紫石斑鱼（E．lanceolatus）、马拉巴石斑鱼（E．malabaricus）.检测76条石斑鱼，检出率90％；病鱼的脑、视网膜、肾脏、肝脏、脾脏等组织均能检出神经坏死病毒（NNV），以脑和视网膜检出率最高，肾脏次之，肝脏、脾脏检出率较少，鳃未检出。 相似文献
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1 IntroductionNervous necrosis virus( NNV) is one of theworldwide pathogens in cultured species and breed-ing from the late 1980s (Bovo et al.,1999; Tan etal., 2001; Breuil et al., 2001; Munday et al.,2002; Lin et al., 2001). The virus is isometric andnon… 相似文献
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G. C. Hewitt 《新西兰海洋与淡水研究杂志》2013,47(4):463-468
Fourteen hundred trout, mainly Salmo gairdneri, supplied by the four Government and nine Acclimatisation Society hatcheries in New Zealand were examined for the presence of spores of Myxosoma cerebralis (Hofer, 1903) (Protozoa: Myxosporida) in their head cartilage. The only positive result obtained was of a 24% infection rate in fish from the Otago Acclimatisation hatchery at Waitati, near Dunedin, which has since been closed down. Sixty fish were examined from Acclimatisation Societies hatcheries and 200 from Government Hatcheries to give a 95% chance of detecting infections of 5% and 1.5%, respectively, of the fish in these hatcheries. A formula is given for estimating sample sizes on the bases of probable infection rates and the degree of certainty of detection that is required. 相似文献
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Sample preparation protocols for flow cytometry (FCM) analysis with five algal viruses were optimized: Heterocapsa circularisquama virus (HcV), Heterosigma akashiwo virus (HaV), Chaetoceros salsugineum nuclear inclusion virus (CsNIV), Rhizosolenia setigera RNA virus (RsRNAV) and H. circularisquama RNA virus (HcRNAV). The optimum staining protocols differed significantly among the viruses tested. FCM counts for the large
DNA algal viruses HaV and HcV (∼0.2 μm in diameter) were similar to numbers determined by epifluorescence microscopy (EFM).
In contrast, FCM counts of viruses smaller than 40 nm that harbor DNA (CsNIV) or RNA genomes (RsRNAV, HcRNAV) were comparable
to or lower than most probable number (MPN) values, which indicate only infectious virus number, suggesting that the FCM counts
were underestimates. This is presumably because their single particle fluorescence signals were below the detection limit
for the flow cytometer. These results indicate that a large portion of the smaller viruses in the aquatic plankton virus community
may be overlooked by FCM. 相似文献
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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
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锦鲤疱疹病毒实时荧光定量PCR检测方法的建立及应用 总被引：2，自引：0，他引：2
研究建立并完善了Taqman实时荧光定量PCR检测锦鲤疱疹病毒（KHV）的方法。首先通过选取KHV病毒TK基因的保守序列（GenBank AB182940）,利用PRIMER EXPRESS 2.0设计引物和探针。其中Taqman探针的5’端标记FAM,3’端标记TAMRA。然后利用梯度稀释的阳性样品克隆质粒进行定量PCR反应,制作标准曲线,得到病毒拷贝数与Ct值的关系为：Ct=-3.45 lgX＋42.73（相关系数R2=0.989）。通过试验检测得到实时荧光定量PCR对KHV的灵敏度为32个病毒核酸拷贝数,同时,设计的引物和探针对于鱼类细胞系和其它鱼类病毒没有扩增反应,表现出较好的特异性。实验结果表明,实时荧光定量PCR检测KHV的方法,具有特异性好、灵敏度高的特点,可以缩短检测时间,提高检测速度,非常适合口岸进出口检验检疫的要求。 相似文献
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福建海水鱼类人工繁殖和育苗技术的现状与展望 总被引：7，自引：0，他引：7
自90年代以来，福建海水鱼类人工繁殖和育苗技术已向着多品种和批量化方向发展，各特优种类不断增加，人工种苗为海水网箱和池塘养殖提供大量苗源，科研教学单位的多项育苗成果已转化为生产力，开始进入种苗规模化生产的可持续发展阶段。迄今为止，福建海水鱼类人工繁殖和育苗成功的种类至少有14科30种，1999年大黄鱼的育苗量已超过3亿尾，其他年产种苗千万尾以上的海水鱼类有美国红鱼、花鲈、免状黄姑针、真鲷（春季生殖群体）和花尾胡椒鲷；年产种苗数百万尾的有黑鲷、勒氏笛鲷、断斑石鲈、免鱼和斜带髭稠等种类。石首鱼科鱼类已成为人工繁殖和育苗的主要对象。本文还针对福建海水鱼类增养殖的问题，提出今后可持续发展的几点设想。 相似文献
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β诺达病毒利用其缺乏校正功能的聚合酶进行基因组复制, 易导致突变, 因而具有广泛的宿主感染性, 而且可引发致死率极高的病毒性神经坏死症, 需要有针对性地研究检测及防御方法。本研究以感染牙鲆(Paralichthys olivaceus)的β诺达病毒为对象, 利用引物设计, 将His标签编码序列连接到完整病毒衣壳蛋白C端, 构建原核表达载体; 采用SDS-PAGE及质谱对表达产物进行分离和鉴定; 重组衣壳蛋白经镍离子亲和柱纯化后进行复性条件的正交优化。结果表明4个肽段经质谱鉴
定与预期一致; 纯化产物产量可达36mg/L; 4°C条件下, 复性缓冲液中尿素和PBS的最适浓度为0.8mol/L和0.05mol/L。本研究建立的制备方案可为研制相关疫苗以及开发针对该病毒的快速检测产品等提供有价值的参考。 相似文献
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The relationship between trace elements in chum salmon otoliths and in their rearing water was investigated to develop ways to distinguish chum salmon stocks in Korea. Rearing water and otoliths of hatchery-reared chum salmon fry were collected from three major hatcheries (Yangyang, Samchuk, and Uljin) on the east coast of Korea in spring 2001 and 2002. Trace elements in the otoliths and rearing water were analyzed using inductively coupled plasma mass spectrometry (ICP-MS) at the Korea Basic Science Institute. The chemical composition of rearing water and otoliths of the salmon fry at specific sites did not vary significantly through the study period. The ratios of some trace elements to Ca in rearing water, such as Sr/Ca and Ba/Ca, was clearly reflected in the chemical composition of otoliths, although the absolute concentrations were different. These two elements may be useful in distinguishing between salmon hatcheries, which have different chemical environments. Discriminant analysis with the ratios of eight elements to Ca (Na/Ca, Mg/Ca, Al/Ca, Cu/Ca, Sr/Ca, Ba/ Ca, Mn/Ca, and Zn/Ca) revealed a distinct separation of natal area in stocks. The analysis of otolith chemistry may be an effective technique for identifying the origins of wild salmon caught at sea. 相似文献
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Evaluation of bioavailable hydrocarbon sources and their induction potential in Prince William Sound, Alaska. 总被引：2，自引：2，他引：0
Kathrine R Springman Jeffrey W Short Mandy Lindeberg Stanley D Rice 《Marine environmental research》2008,66(1):218-220
To realistically evaluate the consequences of exposure to a complex mixture, we modified a passive sampler technology, the semipermeable membrane device (SPMD), which absorbs the bioavailable hydrophobic organic compounds present in an environment. These samplers were deployed in Prince William Sound (PWS), Alaska, at locations selected as potential sites of hydrocarbon deposition, as well as in random sites for regional assessment.Some of these sites were affected by previous human activity, such as canneries and salmon hatcheries, while others were sites of oil discharge as a consequence of the 1964 earthquake or the oil spill of T/V Exxon Valdez in 1989. The SPMDs were deployed for 27-28 d, processed, and then split, with one aliquot dedicated to chemical analysis and the other injected into juvenile rainbow trout (Oncorhynchus mykiss), along with the proper controls including a solvent control, field blank, and positive control. Trout fry were sacrificed after 2 or 7d, and their livers assayed for CYP1A induction by the standard bioassay for hydrocarbon exposure, the ethoxyresorufin-o-deethylase (EROD) assay. The results of this study were consistent and reproducible and showed that oil, whether deposited in 1964 or 1989, is still bioavailable as it can elicit as sustained response. Also, the same oil deposited in different sites of the same region has degraded differently, which is demonstrated by this method. Other putative sources of hydrocarbons, such as oil seeps, were dismissed as regional sources of induction agents as the responses following injection of modified SPMD extract from those sites did not differ significantly from the solvent control. This is a flexible, sensitive method that assesses the response to site-specific bioavailable contaminants and does so within the normal physiological response range of the target. 相似文献
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俄国鱼类行为与感觉研究（Ⅲ） 总被引：2，自引：1，他引：2
本文简要介绍俄国鱼类高级神经活动的研究情况，着重介绍鱼在行为与感觉系统研究的应用，包括控制鱼类行为的一般原理。文中还介绍了俄国鱼类行为与感觉系统研究在捕捞，养殖及护鱼，过鱼设施和装置中的应用。 相似文献
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Ｄｅｔｅｃｔｉｏｎ ｏｆ Ｈｅｐａｔｉｔｉｓ Ａ ｖｉｒｕｓ ｉｎ ｓｈｅｌｌｆｉｓｈ ｂｙ ｒｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ ＰＣＲTXＤｅｔｅｃｔｉｏｎｏｆＨｅｐａｔｉｔｉｓＡｖｉｒｕｓｉｎｓｈｅｌｌｆｉｓｈｂｙｒｅｖｅｒｓｅｔｒａｎｓｃｒ... 相似文献
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Natural fisheries in most parts of the world are under pressure due to heavy fishing activities and pollution threats. The establishment of fish and shrimp hatcheries using native species provides an opportunity for replenishment of fish and shrimp stocks in an environmentally responsible manner. This paper provides guidelines for conducting an EIA of fish and shrimp hatcheries in a semi-enclosed water body, such as the Arabian Gulf and Red Sea. The purpose of the guidelines is to outline issues that may be relevant to the design and construction of hatcheries and to identify important factors for consideration in an EIA study. Effective consultations and technical discussions during the design stage with project proponent and relevant government agencies are recommended. Early evaluation of alternative sites and consideration of environmental factors in ascertaining the suitability of intended project site can help minimize potential impacts. Aspects to be considered in depth include impacts on water quality, sediment and habitat loss. The paper also provides suggested mitigation measures for minimizing potential impacts along with key elements to be considered in the development of an adequate Environmental Management and Monitoring Plan. 相似文献
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二温式RT-PCR检测对虾Taura综合征病毒的研究 总被引：15，自引：1，他引：15
设计了一对能扩增大小为231bp对虾Taura综合征病毒(TSV)某段基因的特异性引物，优化建立了能快速检测TSV的二温式RT-PCR。特异性和敏感性试验结果表明。二温式RT-PCR能对3个试验用TSV毒株的RNA进行扩增，并得到与预期大小一致的231bp的扩增产物，而其它3种对虾病原则无相同大小的特异性扩增产物出现；RT-PCR最低能检测到1pg的TSV RNA。应用RT-PCR对320份分别来自广西沿海不同对虾养殖场的对虾样品进行检测，结果共有85份检出TSV。说明TSV在广西沿海地区养殖对虾中已呈现出区域性流行趋势。同时也显示了RT-PCR在TSV临床检测中具有较高的实用性。 相似文献
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对2012—2013年从河北、福建和广东3个对虾养殖场采集的12份发生疑似早期死亡综合征/急性肝胰腺坏死综合征(EMS/AHPNS)的样品,采用组织病理学方法、套式RT-PCR检测方法以及快速灵敏检测试剂盒进行了检测。三地来源的养殖对虾样品症状均表现为肝胰腺颜色变淡,部分出现萎缩,空肠空胃,腹节肌肉略微白浊;组织病理切片结果表明,患病对虾肝胰腺血细胞浸润,肝胰腺盲管上皮细胞脱落,继发性细菌感染,淋巴器官和血淋巴细胞内存在核固缩、破裂和细胞质包涵体。按世界动物卫生组织(OIE)手册的套式RT-PCR方法检测,结果显示,黄头病毒(Yellow head virus,YHV)目的产物的阳性样品检出率为66.7%,该方法对这些样品的灵敏度可能很低。目的产物测序后经NCBI Blast,与已报道的YHV相似性为76.5%—89%;系统发育树显示这3个来源的样品位于同一个分支,与已知的YHV/鳃联病毒(gill-associated virus,GAV)株均不相同,其亲缘关系与YHV较近,而与GAV较远。用新研发的YHV快速高灵敏检测试剂盒检测,结果表明,所有样品均为YHV强阳性。以上表明患AHPNS的养殖对虾中存在一种YHV的新株型的感染,这也是我国首次检测到YHV的感染。 相似文献