共查询到20条相似文献，搜索用时 218 毫秒
1.
为建立Taura综合征病毒（Taura syndrome virus,简称TSV）的TaqMan—MGB探针荧光定量RT-PC方法,根据TSV衣壳蛋白基因保守序列设计引物和TaqMan—MGB探针,扩增产物长度为118bp。将PCR产物克隆到pGM—T载体,重组质粒经筛选鉴定、SalⅠ单酶切,得到线性化转录模板DNA,经体外转录获得标准品RNA。以体外转录的RNA作为模板,进行一步法荧光定量RT-PCR反应,根据RNA模板拷贝数与Ct（Cycle threshold,Ct）值制备了标准曲线,制作的标准曲线在2×10^1拷贝／μL≤TSV RNA≤2×10^7拷贝／此的范围内具良好的线性关系,可以用于TSV的定量分析,检测灵敏度为20个拷贝数。特异性、重复性试验结果表明,该方法对SPF对虾组织RNA没有扩增反应,表现出良好的特异性;组内和组间实验变异系数分别为0．32％～1．84％和0．79％-2．71％。利用TaqMan—MGB探针建立检测TSV的一步法荧光定量RT-PCR方法可用于对虾TSV的检测。 相似文献
2.
东海原甲藻线粒体细胞色素b(Cytb)基因的定量检测 总被引：3，自引：0，他引：3
建立了检测东海原甲藻线粒体细胞色素b(Cytb)基因mRNA含量的实时荧光定量PCR方法.以甲藻Cytb基因的简并引物扩增得到984 bp长的东海原甲藻Cytb基因片段,经克隆、测序,制备并纯化质粒.以光度法测定质粒浓度并作为标准品.对上述基因片段,利用PRIMER EXPRESS 3.0 设计引物,以梯度稀释的质粒标准品进行定量PCR反应,以SYBR Green I为荧光染料,制作标准曲线,得到基因拷贝数X与Ct值的关系为:Ct=-3.50 lg X+39.35(相关系数r为0.999).通过对不同生长时期的东海原甲藻样品的Cytb基因mRNA含量检测,发现该基因的表达量较稳定,平均值为(45.4±4.7)拷贝/细胞,受生长状态影响不大,可作为实时荧光定量PCR的内参基因. 相似文献
3.
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
4.
本文根据GenBank中对虾传染性皮下及造血组织坏死病毒(IHHNV)基因组上的保守序列,采用Taqman-MGB荧光定量PCR方法,设计了一组特异性引物和探针.利用阳性标准模板进行了定量PCR条件的优化研究.结果显示,在101～108拷贝范围内标准曲线的线性关系为R=0.999 4,检测灵敏度达到10拷贝,重复检测的变异系数均小于2％.利用上述建立的方法和条件,测定39份不同来源的对虾样品中IHHNV,检出率为49％.因此所建立的荧光定量PCR具有较高的灵敏度、特异性和重复性,可作为IHHNV快速的定量检测方法. 相似文献
5.
利用SYBR荧光定量PCR技术，制备用于花鲈（Lateolabrax japonicus）PPARα基因实时荧光定量PCR检测的质粒标准品．通过PCR扩增出目的片断344-bp，纯化后连接至pMDI8-T载体，转化宿主菌E．coli DH5α．通过PCR鉴定、双酶切鉴定和测序分析后抽提重组质粒，测定其拷贝浓度，10倍梯度稀释成标准品模板10^8～10^4 copy／mm^3，进行荧光定量PCR分析．结果显示，循环阈值（Ct）与起始模板量的对数值之间有着良好的线性关系． 相似文献
6.
淋巴囊肿病毒实时定量PCR检测方法的建立和应用 总被引：2，自引：0，他引：2
选取淋巴囊肿病毒(Lymphocystis disease virus,LCDV)主衣壳蛋白(MCP)基因保守序列,利用Primer Express2.0软件设计引物,通过优化反应条件,建立了SYBR GreenⅠ实时定量PCR的检测方法,并进行了敏感性、特异性分析.SYBR GreenⅠ实时定量PCR反应体系的最佳引物浓度为0.5 μmol/L,方法的检测限为30个病毒拷贝,扩增产物的Tm值为74.5 ℃.该方法对淋巴囊肿病毒有特异性,不能扩增流行性造血器官坏死病毒(EHNV)、虎纹蛙病毒(TFV)、蛙虹彩病毒(BIV)、新加坡石斑虹彩病毒(SGIV)、鳜鱼传染性脾肾坏死病毒(ISKNV)及真鲷虹彩病毒(RSIV).对12份发病牙鲆鱼样品进行检测,均感染淋巴囊肿病毒;20份无临床症状的牙鲆经检测不含淋巴囊肿病毒.建立的LCDV的SYBR GreenⅠ实时定量PCR方法,具有成本低、特异、灵敏、快速等优点,可用于淋巴囊肿病毒的早期诊断. 相似文献
7.
根据包纳米虫（Bonamia sp.）SSU rDNA和派琴虫（Perkinsus sp.）ITS rDNA的保守区序列,设计特异性引物和Taqman MGB探针,建立了同时检测上述两种贝类原虫的双重实时荧光PCR方法.该方法可检测到包纳米虫基因的446拷贝质粒,以及派琴虫基因的171拷贝质粒,具有较高的灵敏度.以10倍系列稀释的包纳米虫阳性标准品质粒和派琴虫阳性标准品质粒为模板,测得该方法的定量标准曲线相关系数分别为0.999和1.000,显示出很好的扩增效率.同时该方法与尼氏单孢子虫（Haplosporidium nelsoni）、沿岸单孢子虫（H.costale）等其他贝类原虫,以及副溶血性弧菌（Vibrio parahaemolyticus）、迟缓爱德华氏菌（Edwardsiella tarda）和鮰爱德华氏菌（E.ictaluri）等海水养殖常见致病菌之间无交叉反应,表现出较高的特异性.福建莆田的湄州湾、平海湾、兴化湾等地采集的296份贝类临床样本的检测证明,该方法是一种有效的快速检测鉴定上述2种贝类原虫的方法,具有良好的灵敏度和特异性,可广泛应用于海洋贝类原虫感染情况调查,以及贝类疫病检疫监控等领域. 相似文献
8.
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
9.
10.
3种水产病原菌简型基因芯片检测技术的建立 总被引：1，自引：0，他引：1
根据水产养殖中常见的3种病原菌鳗弧菌(Vibrio anguillarum)、嗜水气单胞菌(Aeromonas hydrophila)及迟缓爱德华氏菌(Edwardsiella tarda)的研究资料,筛选3个毒力相关基因toxR、aerA、evpA设计引物和探针,构建简型基因芯片,并使用对虾白斑病综合征病毒(WSSV)variable region的PCR荧光标记产物作为表面化学质控。通过已构建和优化的多重PCR反应条件,获得了3个目的基因的PCR产物;经过芯片制作过程的优化,将探针以终浓度20μmol/L溶于50%DMSO,在室温、相对湿度45%的条件下点印于醛基基片表面。扩增的PCR产物与杂交液混合后,在42℃杂交2 h就可检测到理想的杂交信号。芯片的灵敏度试验结果表明,可以检测到的3种水产病原菌的最低模板DNA为:鳗弧菌3×102拷贝、嗜水气单胞菌5×103拷贝、迟缓爱德华氏菌6×101拷贝。该芯片可成功应用于患病大菱鲆内脏的细菌分离物的鉴定。 相似文献
11.
Chandralata Raghukumar G. Sheelu P. A. Loka Bharathi Shanta Nair C. Mohandass 《Marine Georesources & Geotechnology》2001,19(1):1-16
In order to assess the impact of deep-sea mining on the in situ benthic life, we measured the microbial standing stock and concentration of organic nutrients in the deep-sea sediments of the Central Indian Ocean Basin in the Indian pioneer area. Sediments were collected using box core and grab samples during September 1996. The total bacterial numbers ranged from 10 10 -10 11 cells per g -1 dry weight sediment. There was a marginal decrease in the number of bacteria from surface to 30 cm depth, though the subsurface section registered a higher number than did the surface. The highest numbers were encountered at depths of 4-8 cm. The retrievable number of bacteria were two orders less in comparison with the direct total counts of bacteria. An almost homogeneous distribution of bacteria, total organic carbon, living biomass, and lipids throughout the depth of cores indicates active microbial and benthic processes in the deep sea sediments. On the other hand, a uniform distribution of total counts of bacteria, carbohydrates, and total organic carbon in all the cores indicates their stable nature and suggests that they can serve as useful parameters for long-term monitoring of the area after the benthic disturbance. Further studies on temporal variability in this region would not only verify the observed norms of distribution of these variables but would also help to understand restabilization processes after the simulated benthic disturbance. 相似文献
12.
Arne Schnack-Friedrichsen Angela M. Davis Jim D. Bennell Dei G. Huws 《Marine Georesources & Geotechnology》2001,19(4):221-243
The interdependence between the seismo-acoustic properties of a marine sediment and its geotechnical/physical parameters has been known for many years, and it has been postulated that this should allow the extraction of geotechnical information from seismic data. Though in the literature many correlations have been published for the surficial layer, there is a lack of information for greater sediment depths. In this article, a desktop study on a synthetic seafloor model illustrates how the application of published near-surface prediction equations to subsurface sediments (up to several tens of meters burial depth) can lead to spurious predictions. To test this further, acoustic and geotechnical properties were measured on a number of sediment core samples, some of which were subjected to loading in acoustically-equipped consolidation cells (oedometers) to simulate greater burial depth conditions. For low effective pressures (representing small burial depths extending to around 10 meters subsurface), the general applicability of established relationships was confirmed: the prediction of porosity, bulk density, and mean grain size from acoustic velocity and impedance appears generally possible for the investigated sedimentary environments. As effective pressure increases through, the observed relationships deviate more and more from the established ones for the near-surface area. For the samples tested in this study, in some instances increasing pressure even resulted in decreasing velocities. There are several possible explanations for this abnormal behavior, including the presence of gas, overconsolidation, or bimodal grain size distribution. The results indicate that an appropriate depth correction must be introduced into the published prediction equations in order to obtain reliable estimates of physical sediment properties for greater subsurface depths. 相似文献
13.
Derek V. Ellis 《Marine Georesources & Geotechnology》2001,19(1):51-63
This article reviews information recently available from existing marine and coastal mining for responses to environmental issues affecting marine mining at different depths. It is particularly but not exclusively concerned with those issues affecting seabed biodiversity impact and recovery. Much information has been gathered in the past 10 years from shallow mining operations for construction aggregate, diamonds, and gold, from coastal mines discharging tailings to shallow and deep water, and from experimental deep mining tests. The responses to issues identified are summarized in a series of eight tables intended to facilitate site-specific consideration. Since impacts can spread widely in the surface mixing layer SML, and can affect the biologically productive euphotic zone, the main issues considered arise from the depth of mining relative to the SML of the sea. Where mining is below the SML, the issue is whether it is environmentally better to bring the extraction products to the surface vessel for processing (and waste discharge), or to process the extraction products as much as possible on the seabed. Responses to the issues need to be sitespecific, and dependent on adequate preoperational environmental impact and recovery prediction. For deep tailings disposal from a surface vessel, there are four important environmental unknowns: (1) the possible growth of "marine snow" (bacterial flocs) utilizing the enormous quantities of fine tailings particles (hundreds or thousands of metric tons per day) as nuclei for growth, (2) the possibility that local keystone plankton and nekton species may migrate diurnally down to and beyond the depth of deep discharge and hence be subjected to tailings impact at depth, (3) the burrow-up capability of deep benthos and their ability to survive high rates of tailings deposition, and (4) the pattern and rate of dispersion of a tailings density current through the deep water column from discharge point to seabed. Actions to obtain relevant information in general and site-specifically are suggested. 相似文献
14.
G. Parthiban 《Marine Georesources & Geotechnology》2000,18(3):223-235
Particle fluxes were measured 7 m above the sea bottom during the predisturbance, disturbance, and postdisturbance periods by using time series sediment traps attached to seven deep-sea moorings deployed in the INDEX experiment site in the Central Indian Basin. The predisturbance particle fluxes varied between 22.3 to 55.1 mg m -2 day -1 . Increased and variable particle fluxes were recorded by the sediment traps during the disturbance period. The increase observed was 0.5 to 4 times more than the background predisturbance fluxes. The increases in particle fluxes (~4 times) recorded by the sediment trap located in the southwestern direction (DMS-1) were the greatest, which could be the result of preferential movement of resuspended particles generated during the deep-sea benthic disturbance along the general current direction prevailing in this area during the experimental period. Also, the traps located closer to the disturbance area recorded greater fluxes than did the traps far away, across the Deep Sea Sediment Resuspension System path. This variability in recorded particle fluxes by the traps around the disturbance area clearly indicates that physical characteristics such as grain size and density of the resuspended particles produced during the disturbance had an important effect on particle movement. The postdisturbance measurements during ~5 days showed a reduction in particle fluxes of ~50%, indicating rapid particle settlement. 相似文献
15.
海上大直径钢管桩打桩过程中,桩周土体受到强烈扰动而发生强度弱化,掌握桩周土体强度弱化规律对于准确预测打桩过程、保证工程安全具有重要意义。为研究土体强度弱化规律,开展了环剪试验模拟打桩对桩周土体的扰动,测试土体强度随剪切速率的变化规律,建立了描述土体强度弱化规律的拟合公式,引入到打桩分析软件中。研究结果表明：土体的强度折减程度不仅与土体本身的性质有关还受到土体的埋深和剪切速率的影响,埋深越深土体强度折减程度越低,剪切速率越高土体强度折减越高,在打桩分析中可采用这里推荐的线性折减方法来模拟不同深度处土体强度的折减规律。 相似文献
16.
The advanced piston corer (APC) has been used by the Ocean Drilling Program since 1985 for recovering soft sediments from the ocean floor. The pullout force measured on extracting the core barrel from the sediment is shown to correlate with the average shear strength of the sediment core measured in the ship's laboratory. A simple rule of thumb is derived relating the shear strength of the sediment to the pullout force. Multiple APC holes at individual sites allow the consistency of the pullout measurements to be assessed. The effects of different operational procedures during APC coring are also explored. Although generally applicable, the correlation between pullout force and laboratory measurements of shear strength breaks down for some APC holes, possibly because of the disturbance of some sediment types during the APC coring process. A better understanding of the physical process of APC coring, and its effect on the properties of the sediment both inside and immediately outside the core barrel, would indicate what confidence can be put on the measurement of pullout force as a way of evaluating the in situ shear strength of deep sea sediments. 相似文献
17.
Geoacoustic Inversion via Genetic Algorithm and Its Application to Manganese Sediment Identification
An acoustic inversion method using a wide-band signal and two near field receivers is proposed and applied to multiple layered seabed models including a manganese sediment. The inversion problem can be formulated into a probabilistic model comprised of signals, a forward model, and additive noise. The forward model simulates wide-band signals, such as chirp signals, and is chosen to be the source-waveletconvolution plane wave modeling method. The wavelet matching technique, using weighted least-squares fitting, estimates the sediment sound-speed and thickness on which determination of the possible numerical ranges for a priori uniform distribution is based. The genetic algorithm is applied to a global optimization problem to find a maximum a posteriori solution for determined a priori search space. Here the object function is defined by an L 2 norm of the difference between measured and modeled signals. Not only the marginal pdf but also its statistics are calculated by numerical evaluation of integrals using the samples selected during importance sampling process of the genetic algorithm. 相似文献
18.
Shigehisa Nakamura 《Marine Geodesy》2000,23(1):55-61
Specific properties of the interannual sea level variations and annual tides in the Northwestern Pacific were studied. Several tide stations were monitored. The monthly mean sea level for the year of 1995 was analyzed at each tide station. A seismic event in 1995, some tectonic activity around the subject area, and the Kuroshio (the oceanic western boundary current) may possibly contaminate results which would have occurred from the astronomical annual tide alone. 相似文献
19.
The effect of different fixation and storage protocols on the flow cytometric (FCM) simultaneous analysis of bacterioplankton and phytoplankton in coastal seawater samples (Mediterranean coastal lagoons) was investigated. FCM measurements (cell number, fluorescence and scatter characteristics) were obtained through DAPI staining. Three fixatives [glutaraldehyde (GA), formaldehyde (FA) and paraformaldehyde (PFA)] and two storage (3 months duration) methods (5 °C and −196 °C) were tested. Two dominant populations were detected in studied samples: bacteria and eukaryotic picophytoplankton. Adding fixatives (2% final concentration) appears necessary to obtain FCM exhaustive counts of all the bacteria and phytoplanktonic cells. This was related to the permeation effect of fixatives which allowed a better DAPI staining of the cells. Maximum fluorescence, i.e. optimal staining of the cells was obtained with FA or PFA, and significant lower fluorescences with GA. Fixed samples stored at 5 °C induced rapid cell loss. Only storage in liquid nitrogen of samples fixed with FA or PFA, allows mid-term (≥4 months) preservation of bacteria or picophytoplankton cell numbers, and limited evolution of DAPI-induced fluorescence and scatter characteristics. 相似文献
20.