斑节对虾白斑综合症杆状病毒感染的实验模式     

邱德全  林继辉  刘丽  张凌飞 《广东海洋大学学报》2001,21(1):10-13

在建立斑节对虾实验室养殖模式的基础上，对野外采集的有白斑综合症病毒（WSSV）病典型症状的斑节对虾初步纯化其病毒，并进行WSSV的PCR检测，得到WSSV的感染用样品。对健康斑节对虾分别进行浸浴感染，投喂感染和注射感染。对感染死亡个体进行WSSA的PCR检测和细菌检测，证实WSSV感染性和致死性。浸浴感染、投喂感染和注射感染的感染量分别为4mL/L、0.2g/10g虾体、1/2稀释液0.05mL/10g虾体，死亡开始时间分别为16d、42h、28h，三种感染模式最终死亡率100%，从开始死亡到全部死亡延续时间分别为15d、82h、44h。  相似文献   

对虾一种无包涵体杆状病毒病原的PCR检测   总被引：4，自引：0，他引：4  

石拓  孔杰  刘萍  包振民  韩玲玲  徐怀恕 《海洋学报》2000,22(4):96-100

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.  相似文献   

Development of a real-time PCR method for Thalassiosira rotula rapid detection     

HE Shanying  YU Zhigang  MI Tiezhu 《海洋学报(英文版)》2007,26(2):133-139

Gene specific primers and DNA probe were designed based on the sequence of 18S rDNA cloned from the red tide alga Thalassiosira rotula. A real-time fluorescent quantitative PCR (RFQ-PCR) method was developed for quantitative detection of T.rotula. The RFQ-PCR assay data showed that the results obtained with the RFQ-PCR quite good agreement with those with the light microscope (LM) counting method, which suggested that the RFQ-PCR could be a useful method for red tide alga detection.  相似文献   

Cloning of a chub metallothionein cDNA and development of competitive RT-PCR of chub metallothionein mRNA as a potential biomarker of heavy metal exposure   总被引：3，自引：0，他引：3  

Hayes RA  Regondi S  Winter MJ  Butler PJ  Agradi E  Taylor EW  Kevin Chipman J 《Marine environmental research》2004,58(2-5):665-669

Metallothionein has been assayed in a range of aquatic animal tissues as an indicator of metal exposure. We sequenced chub (Leuciscus cephalus) metallothionein cDNA which showed over 90% homology to common carp, goldfish and stone loach and 77% homology to rainbow trout sequences for metallothionein. We then used the extended primer method to develop an accurate quantitative competitive RT-PCR assay for metallothionein mRNA. RT-PCR was used to measure metallothionein mRNA in feral chub from a range of field sites, with different levels of heavy metal pollution, in the West Midlands, UK. Measurements were complemented by analysis of liver and gill metallothionein protein by capillary electrophoresis. There was no significant difference in the metallothionein protein levels between fish of different rivers and there was no evidence of elevation of mRNA at the sites of highest metal exposure. The level of metal exposure (e.g. zinc, nickel and cadmium each ranging between 15 and 28 microg/l ) at the pH (7.5-8.5) of these rivers appears insufficient to elevate hepatic or gill metallothionein in chub. A lack of elevation of hepatic metallothionein mRNA in chub exposed to zinc, copper and manganese for 24 h and 10 days in the laboratory also suggests a non-responsiveness of this species.  相似文献   

用PCR方法快速检测水产品中的副溶血弧菌   总被引：10，自引：0，他引：10  

寇运同  马洪明  刘晨光 《海洋科学》2002,26(9):66-69

建立了水产品中副溶血弧菌快速,敏感,特异的PCR检测方法;选择的引物具有良好的副溶血弧菌的种特异性,对人工污染在冷冻水产品中副溶血弧菌的检测低限是12-21CFU/10g,对其增菌液的检测低限是7600-9100GFU/ml。每PCR反应体系的检测低限为91-109CFU,对400份自然污染样品的检测结果显示,PCR方法的检测结果同常规培养法的结果完全相符。  相似文献   

用16S rRNA基因的内切酶图谱快速鉴别几种对虾病原菌   总被引：4，自引：0，他引：4  

孔杰  刘萍  张岩  杨丛海  叶军  徐怀恕  郭华荣 《海洋与湖沼》1997,28(5):449-452

坎普氏弧菌是青岛海洋大学生物系微生物实验室于１９８９－１９９０年自对虾养殖场中国对虾红腿病心脏及血淋巴中分离并鉴定的菌株，副溶血菌和溶藻胶弧菌两菌株于１９９４年９月得自中国科学院微生物研究所，为建立快速、准确的中国对虾病原菌的诊断技术，根据几种细菌的１６ＳｒＲＮＡ基因的序列，设计并合成该基因的多聚酶反应的引物ＰＬ１和ＰＬ２。并用该对引物分别从坎普氏弧菌、副溶血弧菌和溶藻胶弧菌的ＤＮＡ要品中扩增出分  相似文献   

Diagnosis of iridovirus in large yellow croaker by PCR   总被引：1，自引：0，他引：1  

Chen Xinhu  Wang Xiaowen  Wu Wenzhong 《海洋学报(英文版)》2003,22(4):635-642

A rapid and sensitive PCR-based method for the detection of the large yellow croaker iridovirus(LYCIV) is described,which involves the amplification of a 295 bp fragment of the LYCIV ATPase gene from DNA isolated from naturally infected fish spleen.Sequencing of LYCIV ATPase gene fragment showed it shared 100% nucleotide sequence homology with the corresponding region of the ATPase gene of red sea bream iridovirus(RSIV) and sea bass iridovirus(SBIV),suggesting that LYCIV was homologous with RSIV and SBIV at least in part of the gemone.The specificity and sensitivity of the PCR procedure were tested on the iridovirus-infected fishes,the expected fragment was detected from spleen DNA samples of infected fishes,whereas no fragments were amplified from healthy fish spleen DNA,white spot syndrome baculoviruses(WSBV) DNA and pseudorabies virus(PRV) DNA.Detection limit of this method was 10^-7 ng positive plasmid DNA containing target sequence,equal to about 100 virions.In the infected experiment,first positive detection(1/4) appeared at Day 3 post-infection,all fish(4/4) tested positive at Day 7,however obvious symptoms were observed at Day 8,so LYCIV infection could be detected prior to the appearance of obvious symptoms.These results indicate that this PCR method could be used for early,rapid and specific detection of LYCIV infection.  相似文献   

东海原甲藻cDNA文库构建及尝试性EST分析   总被引：2，自引：0，他引：2  

张秀芳  杨官品  刘永建  李瑞香  朱明远 《中国海洋大学学报(自然科学版)》2006,36(3):361-364

东海原甲藻(Prorocentrumdonghaiense)是中国沿海频繁发生的大规模赤潮的原因种之一,大规模分离鉴定东海原甲藻功能基因是理解东海原甲藻赤潮形成过程和机理的重要基础。取对数生长期藻体,经微量总RNA抽提、cDNA合成、cDNA扩增和克隆等步骤,构建了东海原甲藻cDNA文库并进行了尝试性表达序列标签分析。从含有约5000个转化子的文库中随机取150个测序,获得126条EST序列。经网上BlastN及BlastX分析,共发现11个是已知功能的基因的标签。这些基因与东海原甲藻生长发育、物质转换和能量代谢相关。  相似文献   

青蛤(Cyclina sinensis) TRAF6基因克隆及其在Poly I:C胁迫下的免疫应答     

高玮玮  潘宝平  闫春财 《海洋与湖沼》2016,47(5):1040-1046

对青蛤(Cyclina sinensis)转录组文库筛选并克隆得到青蛤TRAF6(Cs TRAF6)基因的c DNA序列,其开放阅读框为2337bp,编码778个氨基酸,分子量为86.59k Da。Cs TRAF6蛋白包含RING型锌指结构,两个TRAF型锌指结构,coiled-coil结构域以及MATH结构域。经生物信息学及分子系统学分析表明Cs TRAF6属于TRAF6家族的一员。利用实时荧光定量PCR方法检测了Cs TRAF6基因在青蛤各个组织以及在Poly I:C侵染下在血淋巴中的时序性表达情况。结果表明,Cs TRAF6基因在青蛤血淋巴、闭壳肌、鳃、性腺、外套膜和肝脏中普遍表达,其中在血淋巴中的表达水平最高。在Poly I:C刺激后的3h,青蛤血淋巴中Cs TRAF6的表达量迅速升高,在6h达到最大值,说明青蛤的Cs TRAF6基因参与了病毒类似物侵染下的免疫应答反应。  相似文献   

白缘(鱼央)5S rDNA的扩增分析及其在染色体上的FISH定位     

张伟伟  付元帅  邵燕  王宝琴  石东里  张兰 《海洋学报》2016,38(6):82-88

本文分别对雌雄白缘(鱼央)的5S rDNA进行了PCR扩增和序列分析,并采用双色荧光原位杂交(FISH)技术,以白缘(鱼央)的5S rDNA序列和小麦的45S rDNA为探针,对其在白缘(鱼央)雌雄中期染色体上进行了FISH定位研究。结果表明,白缘(鱼央)5S rDNA序列无雌雄差异;5S rDNA的保守区序列为117 bp;5S rDNA和45S rDNA分别被定位于白缘(鱼央)的性染色体和第5号染色体上。同时从GenBank中获得了22种鱼的5S rDNA,运用DNAman软件构建了23种鱼的系统发育树,对白缘(鱼央)的进化地位进行了初步研究。本研究为阐明白缘(鱼央)在鱼类系统进化中的位置、重复序列在脊椎动物性染色体上的分布状况以及与性别决定与分化的关系,提供了资料积累和分析依据。  相似文献