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渤海湾肠道病毒的季节分布及其污染类型分析 总被引：1，自引：0，他引：1
肠道病毒是一类水传播胃肠道疾病的重要病原体。为了解感染性肠道病毒在海水环境中的季节分布规律、污染类型以及与流行病学的关系,于2010年12月到2011年10月应用能够提供病毒感染性信息的细胞培养结合实时定量PCR（ICC-qPCR）方法,分4个季节对渤海湾天津近岸海域表层海水中肠道病毒进行了监测。500 mL海水经超滤浓缩,48 h细胞培养,然后用qPCR检测得到四季海水样品中感染性肠道病毒的浓度为0.2~196 PFU/L,平均值为60 PFU/L,其中夏季和秋季肠道病毒浓度较高,其平均值分别为82 PFU/L和110 PFU/L。秋季阳性检出率最高（85.7%）,其次是冬季（71.4%）。经测序分析,其主要污染类型是脊髓灰质炎 Ⅰ 型疫苗株,此外,还检测出危及公众健康的一株类口服脊灰 Ⅰ 型疫苗脊灰和一株脊灰疫苗衍生株。由此可见,肠道病毒的季节分布和主要污染类型与临床上的流行趋势基本一致,海洋作为一个天然的受纳水体为病毒提供了良好的栖息场所和传播机会,对公众健康存在着潜在的威胁。因此,为减少相关疾病的暴发,应重点加强肠道病毒流行季节的海洋水质监测。 相似文献
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为分析全国海水浴场、贝类增养殖区人类肠道腺病毒的污染状况,采用超滤浓缩方法,TaqMan探针实时定量PCR技术对其进行了定量。结果表明,全国10个海水浴场表层海水中腺病毒的阳性检出率为35%,含量为1.1×107~2.8×107 copies/L。在离岸100m和1 000m处腺病毒的分布无显著差别(p0.05);在调查的162份贝类样品中,腺病毒的阳性检出率为11.7%,含量为2.4×104~3.1×105 copies/g。6类经济贝类中牡蛎带病毒量最高。由此可见,全国范围内海水浴场表层海水和主要增养殖区经济贝类中腺病毒污染比较普遍,尤其以山东、辽宁和浙江三省最为严重,给洗浴者和消费者健康造成潜在威胁。人类肠道腺病毒可以很好地指示粪便污染,调查其在海水浴场和经济贝类中的分布,可以帮助公众预警胃肠道病毒的暴发流行,本研究为开展全国腺病毒的环境风险评估和分子流行病学调查奠定了良好的基础。 相似文献
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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
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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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建立了Taqman 实时定量RT-PCR方法检测诺沃克样病毒(Norwalk-like Virus,NLV)的方法,并对中国的牡蛎样品进行了分析.选取GⅡ型NLV的RNA聚合酶区域,利用Primer Express 2.0软件设计引物和探针,建立了定量RT-PCR反应体系.将含有NLV扩增片段的质粒10倍梯度稀释,作为标准品进行反应以确定检测灵敏度并制备标准曲线.结果表明,质粒密度在6×106,6×105,6×104,6×103,6×102,6×101,6个拷贝之间,共7个数量级的范围内,定量RT-PCR反应都有"S"型扩增曲线,检测灵敏度为6个拷贝.制备的标准曲线中,病毒拷贝数(X)与Ct值的关系为Ct = -3.36lg X 37.11,相关系数R2=0.998.对中国37批太平洋牡蛎(Crassostrea gigas Thunberg)样品进行了定量检测,有6批样品为阳性,并根据标准曲线测定了NLV的含量. 相似文献
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应用实时荧光定量PCR技术研究九龙江口水域米氏凯伦藻的分布 总被引：1，自引：0，他引：1
为快速精确监测九龙江口小型有毒赤潮藻的分布,本工作应用实时荧光定量PCR技术检测了2009年春(5月)、夏(8月)、秋(11月)3个季节九龙江口18个站位水样中米氏凯伦藻(Kareniamikimotoi)的密度.这3个季节分别对应九龙江口水域的丰水期、平水期、枯水期.结果表明,在九龙江口水中米氏凯伦藻的检出范围为未检出至2.3×104cells/dm3;其空间分布差异比较大,主要分布在厦门西港海域,其次是在高潮时的海门岛附近海域;海门岛以西水域几乎未监测到该藻的存在,仅5号站位有1个航次检出.米氏凯伦藻密度的季节分布差异也很明显,春、夏季的密度(最高检出值都达到了2.3×104cells/dm3)明显高于秋季的密度(最高检出值仅为5.4×103cells/dm3).本研究结果可为厦门西港以及九龙江口水域赤潮的研究与监测提供参考.同步进行的显微镜镜检技术没有观测到米氏凯伦藻的存在,可见在藻密度较低(低于103cells/dm3)的情况下,实时荧光定量PCR技术较传统镜检技术(检出限一般在103cells/dm3以上)可能更灵敏.该技术特异性好且操作简便,使对大样本的检测具有可行性,为实现沿岸海域赤潮的动态监测和深入研究奠定了基础. 相似文献
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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
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聚合酶链式反应(Polymerase chain reaction,PCR)较容易受到某些抑制成分的影响,尤其是在对复杂环境样品进行的荧光定量PCR反应中,定量结果的准确性更易受到影响。为减轻这种抑制作用,以采自长江口邻近海域沉积物的DNA为研究对象,采用稀释模板法和添加BSA法对荧光定量PCR反应进行了优化。结果表明2种方法都有减轻抑制的作用,但若考虑方法的准确性和检测的便捷性,则以添加BSA的方法更为优越,最适添加浓度为0.1～0.5μg/μL。这一检测方法的改进,为研究沉积物中微生物在硝化过程中的重要作用提供了可靠的技术途径。 相似文献
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C.M.G Van Den Berg 《Marine Chemistry》1982,11(4):323-342
The MnO2 adsorption method combined with voltammetry is proposed for the direct determination of metal complexation in seawater of various salinities as a more satisfactory alternative to direct voltammetric measurements and bioassay methods. A small quantity of MnO2 is equilibrated with copper ions in filtered seawater. Natural organic ligands in the seawater compete for copper with the MnO2. Total dissolved copper is measured by differential pulse anodic stripping voltammetry after filtration and acidification of the sample. Preconcentration of natural water samples is unnecessary and measurement is performed at the natural equilibrium pH of the aerated sample. The analytical limit of detection of the method depends on contamination from the filtration step, and for copper complexation a ligand concentration of 5 × 10?8 M was obtained. The sensitivity can be increased by use of radioisotopes as tracers. The method is very versatile in that complexation of various metals may be determined by any analytical method that measures total dissolved metal concentrations. Neither organic ligands nor their complexes with copper adsorb on the MnO2 at pH8, but at pH 1.8 MnO2 is an efficient scavenger for electroactive organic material.Samples of surface water from the Irish Sea and the Atlantic Ocean were found to contain ligand concentrations of 1.7 × 10?7 and 1.1 × 10?7 M, with conditional stability constants (log values) of 9.84 ± 0.13 and 9.86 ± 0.23, respectively, at pH 8.0. 相似文献
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A detailed examination of a high-temperature catalytic oxidation method for liquid samples in the analysis of total nitrogen dissolved in seawater is reported. The nitrogenous compounds in liquid samples are oxidized on a platinum catalyzer at 680°C under oxygen atmosphere and the generated NO2 is absorbed into a chromogenic reagent, followed by a spectrophotometric determination. The results of this method are much higher than those of wet oxidation methods. Molecular size dependency of the results clearly indicates that the above discrepancy is caused by the low oxidation capacity of the wet oxidation method against high-polymer organic matter dissolved in seawater. The results revealed that the concentration of total nitrogen in seawater is nearly constant from surface to bottom, ranging from 30 to 40 μM 1?1, which organic nitrogen concentration higher in the surface layer, and a rapid decrease with depth. An examination of molecular size distribution indicates that the concentration of high-polymer organic nitrogen decreases rapidly from surface to deeper layers, with molecular sizes ranging from 5 × 103 to 3 × 104. Because of the well-defined principle of the oxidation process, its reliability, ease of sample handling and of analytical procedure on board or in the land laboratory, the present method is much more suitable for the marine analytical chemistry of total and organic nitrogen than the other previous methods. 相似文献
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Pulleniatina obliquiloculata shells from 16 core-top samples from the tropical Indo-Pacific Oceans are analyzed for the ratios of boron and cadmium to calcium(B/Ca and Cd/Ca). The B/Ca ratios show a very weak positive relationship with [B(OH)_4~-] and the dissolved carbonate species at the apparent calcification depth of P.obliquiloculata. The boron partition coefficients(K_D) between P. obliquiloculata B/Ca and seawater[B(OH)_4~-]/[HCO_3~-] distribute around 1.1×10~(-3)-1.3×10~(-3) with a mean value of(1.19±0.12)×10~(-3),and are significantly related to the nutrient concentration, especially phosphate. The lack of any clear correlation between the P. obliquiloculata B/Ca and seawater carbonate chemical parameters suggests that the physiochemical controls on boron incorporation are masked by the complexity of natural seawater condition. But the significant dependence of Ku on nutrient may likely be explained by a nutrient related growth-rate effect. Cd/Ca of P.obliquiloculata shows significant correlation with seawater phosphate concentration, and its partition coefficients(D_(cd)) are significantly related to temperature. A first-principle methodology of P. obliquiloculata B/Ca is applied;with the aid of Cd/Ca as a phosphate proxy and a constraint on K_D, to estimating sea water carbonate chemistry(e.g., pH). The results are fairly promising and allow us to propose the possibility to apply the combination of B/Ca and Cd/Ca proxies(and also Mg/Ca and δ~(18)O for estimating temperature and salinity) for the paleoreconstruction of seawater carbonate chemistry. 相似文献
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A method for the determination of nanomolar concentrations of orthophosphate in oligotrophic seawater developed by Liang et al. (2007) has been modified to make it fully feasible for shipboard application and for faster sample throughput with minimized
sample volume. The technique is based on the flow injection method with solid phase extraction on a Sep-Pak C18 cartridge
and colorimetric detector. The Schlieren effect was minimized by rinsing the cartridge sequentially with 5 mL water and 2
mL 95% ethanol solution. With three micro pumps in parallel, savings of up to 80% in amount of reagents and 25% volume of
seawater samples could be achieved in comparison to the previous method. Variation of stopped flow time and sample loading
time gave 3 different standard curves, which corresponded to 3 linear ranges within 3.4 and 515 nM. The modified method permits
the analysis of samples over a wide range of concentrations, and has been successfully applied to shipboard determination
of trace orthophosphate in more than 200 seawater samples during a one-month cruise in the South China Sea. For seawater at
concentrations of 20.6, 82.5, 206.2 nM orthophosphate, the relative standard deviations (RSD) (n = 6), determined daily for 6 days on board ship were 4.45%, 4.73% and 6.75%, respectively. Five seawater samples collected
in the Station SEATS (South East Asia Time Series Station at 18°N, 116°E) were analyzed using the present method both on board
and in a land-based laboratory, as well as with the magnesium hydroxide-induced coprecipitation (MAGIC) method, and showed
no significant difference according to the statistical t-test. 相似文献
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The distribution of Al, Mn, Fe, Co, Ni, Cu, Zn, Cd and Pb in seawater was investigated in the Chukchi and Beaufort Seas of the western Arctic Ocean in September 2000. The unfiltered and filtered seawater samples were used for determination of total dissolvable metal (TDM) and dissolved metal (DM), respectively. The concentration of labile particulate metal (LPM) was estimated with the difference between that of TDM and DM. The concentrations of TDAl, TDMn, TDFe, TDCo and TDPb varied substantially in the study area. The high concentrations occurred at stations near the Bering Strait, in the Mackenzie delta, and above reductive sediments on the shelf and slope. These elements were mostly dominated by labile particulate species, such as Fe?CMn oxides and species adsorbed on terrestrial clay. DCo was correlated with DMn over the study area (r?=?0.78, n?=?135), and the slope of the regression line was 27 times higher at a pelagic station than at a shelf station. TDNi, TDCu, TDZn and TDCd showed relatively small variations and were generally dominated by dissolved species. There was a moderate correlation between DCd and phosphate for all samples (r?=?0.79), whereas there were no significant correlation between the other DMs and nutrients. TDNi and TDCu showed a remarkable linearity for most stations except those near the Bering Strait (R 2?=?0.95, n?=?126). These results suggest that biogeochemical cycling including uptake by phytoplankton and remineralization from settling particles has only minor control over the distribution of trace metals in this area. Using the present data, the annual input of bioactive trace metals form the Bering Strait and the Mackenzie River was estimated. Also, the trace metal compositions of major water masses were evaluated. The dissolved elemental ratio was P:Al:Mn:Fe:Co:Ni:Cu:Zn:Cd?=?1:1.2?×?10?2:4.4?×?10?4:1.4?×?10?3:3.7?×?10?5:3.7?×?10?3:1.4?×?10?3:4.5?×?10?3:2.2?×?10?4 for Canada Basin deep water (CBDW). This ratio was significantly different from that for Pacific deep water and Bering Sea water, suggesting substantial modification of the trace metal compositions of seawater in the study area. 相似文献
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Effects of exposure to polychlorinated biphenyls (PCBs) on Sebastiscus marmoratus were investigated using a suppression subtractive hybridization method. A total of 108 gene sequences were identified as having the potential for being differentially expressed, and 45 could be identified with homologous database sequences. Functions with which they were associated included long-term potentiation and neurotransmitter release, neuroendocrine, mitosis and cell proliferation, energy-related metabolism, general metabolism, signal protein, hemopoiesis system, immune system, and structure. The expression of 17 of these genes was analyzed in the brain using real time fluorescent quantitative PCR. The present study provided a basis for studying the response of fish to PCB exposure and allowed the characterization of new potential neurotoxicol biomarkers of PCB contamination in seawater. 相似文献
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为分析海洋微表层这一特殊生境中的细菌类群,于2010—2011年4个航次对北黄海微表层和次表层海水中的总菌丰度、可培养细菌丰度和群落结构进行了分析。采用流式细胞仪测定总菌丰度,平板计数法测定可培养细菌丰度,PCR-16SrDNA分析可培养细菌的群落结构。结果表明,调查海域微表层海水总菌均值为1.76×106 cell/mL,次表层海水总菌均值为1.07×106 cell/mL。可培养细菌丰度范围是1.00×102~1.70×106 CFU/mL,微表层和次表层可培养细菌所占总菌数量的百分比分别为13.05%和0.45%。微表层对总菌的富集因子(EF)均值为2.02,可培养细菌的EF均值为74.16。PCR-16SrDNA序列分析结果表明,该海域可培养细菌分属变形菌门(Proteobacteria)(94.34%)、厚壁菌门(Firmicutes)(1.89%)、拟杆菌门(Bacteroidetes)(1.89%)和放线菌门(Actinobacteria)(1.89%)4个类群。本研究初步发现,微表层对细菌具有较强的聚集作用,尤其对可培养细菌聚集作用更为明显。微表层中可培养细菌的群落结构与次表层有所不同,其种类丰富,来源和功能多样。由此可见,微表层独特的生境成就了其独特的微生物类群,其生态功能有待于进一步研究。 相似文献