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1.
草鱼出血病是由草鱼呼肠孤病毒(GCRV)引起的草鱼养殖业一种严重的病毒性出血病,给草鱼养殖产业造成重大损失。该病毒是一种双链分节段RNA病毒,根据GCRV多个病毒分离株基因序列分析及临床发病特点,共有三种基因型GCRV,其中基因Ⅰ型和Ⅱ型较为流行。通过细胞分离培养基因Ⅰ型(GCRV-ZV8909)和Ⅱ型(GCRV-HZ13) GCRV,研制出草鱼出血病二价细胞灭活疫苗,通过绝对定量的方法,测定基因Ⅰ型和Ⅱ型的病毒含量分别为2.0×109及1.5×106 copies/mL。用生理盐水将其分别进行50倍稀释和100倍稀释,包括原液及生理盐水对照共4组,分别腹腔注射免疫(20±5)g健康草鱼,剂量均为0.2mL/尾,每组40尾,各组于免疫后3、5、7、14、21、28、42、56、70、84 d分别取3尾采集血液制备血清。分别利用经原核表达并纯化制备两种基因型GCRV的VP7蛋白以及实验室保存的草鱼IgM单抗,建立了三抗体夹心酶联免疫方法(TAS-ELISA)分析评价疫苗体液免疫效果。结果表明,草鱼经腹腔注射不同剂量的细胞灭活疫苗后,与对照组相比,各免疫组血清抗体均有不同程度的提高,且原液组与50倍稀释组基本持平,但都高于100倍稀释组,各免疫组在免疫后5d即可检测到两种基因型GCRV抗体,且抗体水平持续到84d,但两种基因型抗体水平达到高峰的时间不一致,抗基因Ⅰ型GCRV抗体达到高峰是在28d,抗基因Ⅱ型GCRV抗体是在14d达到高峰。两种基因型抗体水平达到高峰时的血清效价分别为1︰800、1︰600。免疫保护率实验结果表明,原液组保护率最高,为67%,50倍稀释组为60%,100倍稀释组只有33%。 相似文献
2.
采用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)进行核酸扩增,凭借横向流动试纸条(lateral flow dipstick,LFD)完成扩增产物检测,建立了可快速检测孔石莼(Ulva pertusa)的LAMP-LFD方法。该方法首先在孔石莼的内转录间隔区序列(internal transcribed spacer,ITS)的8个种内保守区域设计6条特异性引物(上游内引物由生物素标记),进行由生物素标记的LAMP反应;同时,在两条外引物的有效扩增区段内设计1条异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的探针,生物素标记的LAMP产物与FITC标记的探针特异性杂交后,在LFD上完成结果显示。优化后LAMP的反应条件为63°C反应50min,加上探针杂交与LFD检测共需60min。结果表明,利用该LAMP-LFD方法可特异性地检出孔石莼,对浒苔(Ulva prolifera)等9种常见藻类的检测均呈阴性。利用该方法最低可检测到3.04×10~(–2) pg/μL的孔石莼基因组DNA,是以LAMP外引物进行的常规PCR方法的1000倍。针对一定数量的实际样本的检测结果表明,LAMP-LFD方法与传统的形态学观察的结果一致。因此,本研究建立的孔石莼LAMP-LFD快速检测方法,具有特异性强、灵敏度高、操作简便等优点,有望成为我国东部沿海孔石莼快速检测和定期监测的有效技术手段。 相似文献
3.
我国的养殖香鱼正面临着严重的香鱼格留虫（Glugea plecoglossi）感染。本研究联合运用环介导等温扩增技术（LAMP）和横向流动试纸条（LFD）的检测技术，建立了快速便捷地检测G. plecoglossi的LAMP-LFD方法。该方法以G. plecoglossi的β微管蛋白基因为检测靶标，在其保守区域设计并筛得6条特异性引物（其中上游内引物用生物素标记），进行LAMP反应，产物再与异硫氰酸荧光素（FITC）标记的特异性探针杂交，在LFD上进行结果判断。结果表明，LAMP-LFD方法能够特异性地检出G. plecoglossi，对梅氏新贝尼登虫、刺激隐核虫、肝肠胞虫阳性的虾组织、派氏异尖线虫、内弯宫脂线虫、鳗弧菌香鱼分离株、杀香鱼假单胞菌，以及香鱼组织等的检测均呈阴性。优化后，LAMP的反应条件为65℃反应45min，与探针杂交的条件为65℃反应5min，加之5min的显色时间，整个检测时程为55min。利用该方法能够检测到2.0fg/μL的含β微管蛋白的质粒DNA，针对G. plecoglossi基因组DNA的检测灵敏度为14.0pg/μL；能够从感染强度达到100个虫体/克的香鱼肝组织中稳定地检测到虫体。该方法可在简单的恒温加热设备（如水浴锅）中完成核酸扩增和探针杂交，无需昂贵的仪器装置。综上，香鱼格留虫LAMP-LFD方法操作便捷、灵敏度高、特异性好、检测快速，而且设备依赖性低，完全适合于基层检测的需求。 相似文献
4.
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
5.
环介导等温扩增联合横向流动试纸条快速检测扁浒苔(Ulva compressa)的研究 总被引：2，自引：1，他引：1
本研究将环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)与横向流动试纸条(lateral flow dipstick,LFD)的可视化检测方法结合,建立了扁浒苔(Ulva compressa)的LAMPLFD快速检测技术。该方法以扁浒苔的内转录间隔区(ITS1-5.8S-ITS2)序列为检测靶标,设计了3对特异性引物(其中,上游内引物由生物素标记)和1条异硫氰酸荧光素标记的探针。结果表明,LAMP最适反应温度为63°C,扩增时间为60 min,从核酸扩增到LFD结果判读需70 min。利用LAMP-LFD可特异性检出扁浒苔,对浒苔、曲浒苔、缘管浒苔和孔石莼等石莼属绿藻以及塔玛亚历山大藻、无纹环沟藻、东海原甲藻、锥状斯克里普藻和赤潮异弯藻等常见微藻的检测结果为阴性。该方法最低可检测到0.1 pg的扁浒苔基因组DNA,是以Uco ITS-F3和Uco ITS-B3为特异性引物的PCR方法的100倍。对实际样品的检测结果表明,LAMP-LFD方法检测扁浒苔与传统的形态学观察的结果一致。因此,该方法可快速、特异地检测出扁浒苔,而且操作简单,仪器设备依赖性低,有潜力成为扁浒苔现场检测的常规技术手段。 相似文献
6.
为筛选与草鱼呼肠孤病毒GCRV096 VP6发生相互作用的宿主蛋白,先将VP6基因的PCR扩增产物,克隆至酵母表达载体p GBKT7以构建其诱饵质粒(p GBKT7-VP6);再将酵母菌Y2HGold(含p GBKT7-VP6)与酵母菌Y187[含草鱼肾脏细胞(CIK)c DNA文库质粒]进行人工诱导融合,然后经选择培养筛选阳性克隆。结果共筛选到4株阳性克隆,经测序、生物信息学分析,分别属于两条不同的核苷酸序列,其中之一所编码的产物与GAPDH(3-磷酸甘油醛脱氢酶)同源性较高。可见,该酶极可能在GCRV096侵染宿主的初期发挥着重要作用。 相似文献
7.
应用LAMP-LFD技术可视化检测河流弧菌(Vibrio fluvialis)的研究 总被引：2，自引：0，他引：2
以河流弧菌(Vibrio fluvialis)为检测对象,将环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)与横向流动试纸条(lateral flow dipstick,LFD)联合应用,建立了快速便捷的LAMP-LFD方法。该方法以河流弧菌的外膜蛋白omp U基因作为检测靶标,在其保守区域设计6条特异性引物(上游内引物由生物素标记),并在两条外引物的有效扩增区段内设计1条特异性探针,由异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记。使用引物进行有生物素标记的LAMP反应,产物与FITC标记的探针完成杂交,杂交产物在LFD上进行结果展示。结果表明,LAMP-LFD方法可特异性地检出河流弧菌,对近似物种如创伤弧菌等弧菌病原以及迟缓爱德华菌等其他水产养殖病原的检测均呈阴性。经优化,LAMP的反应条件为63°C反应25min,加上5min的探针杂交和3—5min的LFD显色,整个检测时程约35min。利用该方法最低可检测到1.0×10~2 CFU/m L的河流弧菌纯培养物,能够从污染强度为5.0×10~2 CFU/m L的组织样品中检测到该病原。该方法设备依赖性更低,仅需简单的恒温加热设备即可完成。因此,本研究建立的河流弧菌LAMP-LFD方法,具有操作便捷,设备依赖性低、灵敏度高和检测快速等优点,在河流弧菌的基层检测中具有良好的应用前景。 相似文献
8.
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
9.
本研究将交叉引物恒温扩增技术(cross priming amplification,CPA)与核酸试纸条相结合建立一种副溶血性弧菌(Vibrio parahaemolyticus)快速可视化检测方法。针对副溶血性弧菌特有的不耐热溶血素基因tlh的六个不同区域设计两对特异性引物和一对检测探针,通过优化反应条件确定了最佳反应体系。CPA-核酸试纸条方法对副溶血性弧菌的检测具有较强的特异性,对纯培养物的检测灵敏度达到58 cfu/m L,对污染牡蛎中副溶血性弧菌的检测灵敏度为5.2 cfu/g,比传统的PCR技术灵敏度提高了10倍,且具有较高的稳定性。交叉引物恒温扩增技术与核酸试纸条相结合的方法操作简便、特异性强、灵敏度高且能有效防止污染,可用于现场及基层单位副溶血性弧菌的快速检测。 相似文献
10.
以东海原甲藻的ITS序列(Internal transcribed spacer,ITS)为检测靶标,将生物素标记的环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP)扩增产物与经异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记的探针特异性杂交,并结合横向流动试纸条(Lateral flow dipstick,LFD)肉眼直接观察检测结果,建立了有害赤潮藻东海原甲藻(Prorocentrum donghaiense)的快速检测技术。经优化后的最适条件为63°C、30min,较常规PCR扩增缩短约2h。结果表明:LAMPLFD可特异性检出东海原甲藻,对常见赤潮藻检测结果均为阴性;其对东海原甲藻基因组DNA的检测最低限为47pg/μL,是常规PCR技术(以F3/B3为引物)的10倍。LAMP-LFD技术能高效、特异地检出东海原甲藻,仪器设备依赖性低,结果可视化,有望成为赤潮原因种检测监控的常规方法。 相似文献
11.
港口内靠码头系泊船运动的计算 总被引：11，自引：1，他引：11
本文以英国南海岸处Ｓｈｏｒｅｈａｍ港内系泊船为例，研究了港口内靠码头系泊船运动的数值计算问题。该船的实船实验和模型实验已经完成，为验证本文计算结果提供了依据。运动方程在时域内求解。在计算船体脉冲响应函数时，引入了船体阻尼系数在频率很低时的渐近表达式和一迭代算法。分析了港内共振波浪和其对船体的作用力。讨论了靠码头系泊船运动的非线性特征。计算结果与实验结果符合很好。 相似文献
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13.
An elliptic jet and a square jet flowing into a counterflow with different jet-to-current velocity ratios are investigated by using realizable k–ε model. Some computed mean velocity and scalar features agree reasonably well with experimental measurements, and more features are obtained by analyzing the computed results. After fluid issues from a nozzle, it entrains ambient fluid, and its velocity and concentration on the centerline decay with the distance downstream from the potential core (l0). The decay ratio increases with the decreasing jet-to-current velocity ratio α. For an elliptic jet, the evolution of the excess velocity half-width b and the concentration half-width bc merely remains constant near the jet exit on major-axis plane while they increase linearly on the minor-axis plane. However, the half-widths on the major-axis and minor-axis plane become proportional to the axial distance downstream after equaling each other. For a square jet, b and bc increase linearly with the distance downstream from the jet exit, but the spread ratio is larger on the middle plane than that on the diagonal plane before they equal each other. The radial extent of the dividing streamline rs or the mixing boundary rsc increases linearly downstream, and decreases exponentially after reaching a peak at xb. The ratio on the minor-axis plane is larger than that on the major-axis plane for an elliptic jet. The characteristics are the same for the square jet. b, bc, rs, and rsc on two corresponding planes become equal to each other more rapidly for the square jet than for the elliptic jet, because the sharp corner of the square nozzle induces secondary structures that are more intense. The distributions of the excess axial velocity and scalar concentration exhibit self-similarity for either the elliptic jet or square jet in the region of l0 < x < xb. On the cross section, four counter-rotating pairs of vortices, which enhance the entrainment between the jet and counterflow, form at the four corners of the square jet or at the two ends of the major-axis plane of the elliptic jet. The recirculation pattern formed by these axial vortices is more complex for the square jet than that for the elliptic jet. The turbulent kinetic energy k have large value in the region near the jet exit and stagnation point. The maximum value of k for the square jet is larger than that of the elliptic jet near the jet exit. This results in the square jet mixing more strongly than the elliptic jet. 相似文献
14.
提出一种基于序贯预测误差方法（ＳＰＥ）的多层神经网络（ＭＮＮ）的学习算法。经模拟计算，它比传统的基于最陡下降方法的误差反传（ＳＤＢＥＰ）算法具有更好的收敛性能。并对这两种算法进行了模拟计算的比较． 相似文献
15.
This paper deals with the study of moisture budget of the typhoon No. 7507. The results show that the mass convergence produced by divergent wind has made the maximum contribution to precipitation, especially at the lower level, accounting for about 70% of the total precipitation, and that the advection term of water vapor is very small. The vertical flux term is to transport water vapor from the lower level to the middle and upper levels, resulting in the increase of water vapor accumulation at these levels. Evaporation term also plays an important role in precipitation, accounting for about 13% of the total precipitation and 23% of the horizontal moisture convergence. The moisture of the typhoon comes mainly from its southern boundary and the secondary inflow of moisture is from its western or eastern boundary. All the inflow layers of these three boundaries are very deep, reaching up to 300 hPa, while the strongest inflow occurs below 700 hPa. In the northern boundary of the typhoon, we obtained that 相似文献
16.
A Time-Domain Coupled Model for Nonlinear Wave Forces on A Fixed Box-Shaped Ship in A Harbor 总被引：2，自引：1，他引：1
A 2-D time-domain numerical coupled model is developed to obtain an efficient method for nonlinear wave forces on a fixed box-shaped ship in a harbor.The domain is divided into an inner domain and an outer domain.The inner domain is the area beneath the ship and the flow is described by the simplified Euler equations.The other area is the outer domain and the flow is defined by the higher-order Boussinesq equations in order to consider the nonlinearity of the wave motions.Along the interface boundaries between the inner domain and the outer domain,the volume flux is assumed to be continuous and the wave pressures are equal.Relevant physical experiment is conducted to validate the present model.It is shown that the numerical results agree with the experimental data.Compared with the coupled model with the flow in the inner domain governed by the Laplace equation,the present coupled model is more efficient and its solution procedure is more simple,which is particularly useful for the study on the effect of the nonlinear wave forces on a fixed box-shaped ship in a large harbor. 相似文献
17.
中国隆头鱼之一新属新种——尖尾新丝隆头鱼 总被引：1，自引：0，他引：1
在过去几年的西沙群岛鱼类区系调查中,我们曾在西沙群岛的东岛和永兴岛附近水域,先后三次采到5尾前所未曾见过的隆头鱼类标本,经过比较研究后,认为是一新属、新种,定名为“尖尾新丝隆头鱼”,(Neocirrhilabrus oxyurus gen. et sp. nov.)现描述如下: 相似文献
18.
王荣良 《中国海洋大学学报(自然科学版)》1988,(1)
本文对J.Maynard Smith提出的物种竞争模式: x=x(a-bx-cy-kxy) y=y(e-fx-gy-lxy)作了定性分析,并给出两物种共存或其中任一物种死亡,而另一物种趋于确定值的条件。 相似文献
19.
An edge wave is a kind of surface gravity wave basically travelling along a shoaling beach. Based on the periodic assumption in the longshore direction, a second order ordinary differential equation is obtained for numerical simulation of the cross-shore surface elevation. Given parameters at the shoreline, a cross-shore elevation profile is obtained through integration with fourth-order Runge-Kutta technique. For a compound slope, a longshore wavenumber is obtained by following a geometrical approach and solving a transcendental equation with an asymptotic method. Numerical results on uniform and compound sloping beaches with different wave periods, slope angles, modes and turning point positions are presented. Some special scenarios, which cannot be predicted by analytical models are also discussed. 相似文献
20.
This is a numerical study on the time development of surface waves generated by a submerged body moving steadily in a two-layer fluid system, in which a layer of water is underlain by a layer of viscous mud. The fully nonlinear Navier-Stokes equations are solved on FLUENT with the Volume-of-Fluid (VOF) multiphase scheme in order to simulate the free surface waves as well as the water-mud interface waves as functions of time. The numerical model is validated by mimicking a reported experiment in a one-layer ... 相似文献