氢化物原子荧光法测定水产品中的砷、汞

采用氢化物原子荧光法测定水产品中的砷、汞。在0～12μg／mL浓度范围内As、Hg标准曲线相关系数达0．999。As检出限为0．2μg／L，Hg为0．3μg／L，As回收率在100％-162％之间，Hg回收率在124％～152％之间。     

荧光原位杂交概述

荧光原位杂交(fluorescence in situ hybridization，FISH)是在20世纪80年代末在放射性原位杂交技术的基础上发展起来的一种非放射性分子细胞遗传技术，以荧光标记取代同位素标记而形成的一种新的原位杂交方法，探针首先与某种介导分子(reporter molecule)结合，杂交后再通过免疫细胞化学过程连接上荧光染料。FISH的基本原理是将DNA(或RNA)探针用特殊的核苷酸分子标记，     

利用试剂盒检测对虾白斑综合症病毒

对虾白斑综合症病毒(wssv),又称对虾杆状病毒或皮下造血组织坏死杆状病毒,是迄今为止危害对虾养殖最为严重的一种病害.发病期一般在幼体期到生长期,死亡率可达100%,该病毒的流行给我国对虾养殖业造成了巨大的经济损失,成为对虾养殖业可持续发展的主要障碍之一.     

用生物素标记寡核苷酸DNA探针快速检测鱼传染性胰脏坏死病病毒IPNV

研究了用生物素标记的寡核苷酸ＤＮＡ探针快速检测鱼传染性胰脏坏死病病毒ＩＰＮＶ，取得了较好的结果。对探针的灵敏度，特异性进行了灵敏度、特异性进行了测定，并与其它快速检测技术作了比较。     

Morphogenesis of koi herpesvirus observed by electron microscopy

Koi herpesvirus (KHV or cyprinid herpesvirus 3) was inoculated onto three fish cell lines derived from carp, Cyprinus carpio L., and the process of virion formation observed with electron microscopy. Essentially, similar features of virus particles were observed in all three cell lines. The nucleus of infected cells was characterized by margination of chromatin and often contained many capsids of about 110 nm in diameter with varying morphology. The morphological variation of the capsids was very similar to that of mammalian herpesviruses. Some capsids protruded from the inner nuclear membrane, and others, with envelopes, were located in the perinuclear space between the inner and outer nuclear membrane, suggesting budding of capsids at the inner nuclear membrane. Naked capsids and envelopment of capsids by budding into vesicles were also observed in the cytoplasm. Mature, enveloped virions 170-200 nm in diameter were seen in cytoplasmic vesicles or outside the cell. These observations suggest KHV virions mature through a complex morphological pathway including two distinct envelopments, which have been found only in herpesviruses. These observations support the inclusion of KHV in the family Herpesviridae.     

罗非鱼暴发性流行病病毒病原的电镜观察初报

利用电子显微镜技术,首次在国内从患暴发性流行病罗非鱼体内发现病毒病原。电镜观察结果显示,在患病罗非鱼脾脏、心脏以及鳃组织细胞质中观察到大量病毒颗粒。病毒颗粒呈球形,无囊膜,直径约30~40 nm,与鱼类神经坏死病毒相近。     

用地高辛标记的PCR－ELISA技术快速检测转基因鱼

取含有小鼠重金属螯合蛋白（mMT)基因启动子及人生长激素（hGH)基因的鱼样品，以Qiagene核酸纯化技术对鱼组织DNA进行纯化。常规PCR检测显示，mMT启动基因和hGH基因的PCR产物分别为240bp和130bp，与设计相符。PCR产物的核酸测序结果证实其扩增产物具特异性。地高辛-PCR（Dig-PCR)标记反应显示，2个基因均产生1条Dig标记的特异产物带。Dig-PCR-ELISA检测敏感性试验显示，对mMT启动子和hGH基因的检测敏感性可达10^-3，与常规PCR结合琼脂糖胶电泳检测方法相比，检测敏感性可提高至100-1000倍。试验证明，针对mMT和hGH基因所建立的Dig-PCR-ELISA技术特异可靠。     

Fluorescence of Extracts from Argentine Hake (Merluccius hubbsi) Muscle and Its Correlation with Quality Indices During Chilled Storage

Argentine hake (Merluccius hubbsi) was stored chilled for 8 days. The evolution of fluorescence of the aqueous and organic extracts of muscle during storage at 4°C was studied and correlated with common damage indices; pH, total volatile basic nitrogen, free fatty acids, and the fluorescence intensity of the aqueous phase showed a good correlation with storage time. The excitation-emission matrices of the aqueous extracts were also obtained. A fluorescence landscape that could be attributed to tryptophan decreased with storage time, meanwhile a peak (around 340 and 400 nm for excitation and emission wavelengths, respectively) is developed during chilled storage.     

基于钨丝原子阱与直接进样技术的原子荧光光谱法快速测定贝类中的镉

本研究利用集成多孔石墨管电热蒸发器和钨丝捕获阱技术的原子荧光光谱分析系统,基于固体进样技术建立了适用于现场快速检测贝类中镉(Cd)的测定方法,实现了贝类中Cd的快速灵敏检测.结果显示,仪器在最佳参数条件下稳定性良好,采用“在线原子阱”捕获后释放的信号可以消除大多数基体干扰.贝类样品匀浆后在酸性条件下直接进样可快速测定Cd,且使用1％硝酸进样效果最好.采用标准曲线和标准加入法验证,均线性良好(R2≥0.995),样品加标回收率分别为98.1％-110.6％和96.4％-105.5％,相对标准偏差(RSD)分别为3.3％-11.6％和7.5％-8.7％,方法检出限分别为3 pg和0.6 pg,满足贝类样品的现场快速检测要求.但相对标准曲线法,标准加入法具有更好的准确度、精密度和稳定性,用标准物质验证,检测结果在参考值范围内.因此,标准加入法更适合贝类中Cd的快速测定.     

不同氮素对冠盘藻生长和光合荧光特性的影响

硅藻水华广泛存在于富营养化水体中，常造成恶劣的环境影响，氮素输入是造成硅藻水华现象的主要因素之一。为明确氮素营养对硅藻水华的影响，揭示其对氮素的响应机理，以常见硅藻水华优势种冠盘藻(Stephanodiscus hantzschii)为研究对象，分别以硝酸钠、氯化铵和尿素作为氮源，分析了不同氮素条件下冠盘藻生长和叶绿素荧光参数（Fv/Fm、α、Ik、ETRmax）的变化特征。结果显示，不同形态氮素对冠盘藻的生长及叶绿素荧光参数影响显著，一定程度上提高水体氮素浓度能够提升冠盘藻的叶绿素荧光，促进其生长。相比于氨氮和尿素，硝氮营养更有利于冠盘藻的生长，高浓度硝氮条件下，冠盘藻生长良好，而氨氮浓度大于5 mg/L、尿素浓度达到10 mg/L时，冠盘藻叶绿素荧光参数显著降低，生长受到抑制。较高浓度氨氮及尿素对冠盘藻叶绿素荧光参数的抑制作用主要发生在其生长的前中期，随着氮素消耗，冠盘藻的光合参数Fv/Fm、Ik、ETRmax均随氮素浓度的增加而增加，低氮条件下的冠盘藻会提高α值来增大对于光能的利用效率，从而提高环境适应性。研究表明，氮素一定程度上能够通过影响冠盘藻的光合作用而影响其在水环境中的生长。     

超高效液相色谱荧光法检测贝类中三种高毒性麻痹性贝毒

建立灵敏可靠的麻痹性贝类毒素(PSP)检测技术是保障我国贝类水产品质量安全的有力手段。本研究选取3-(2-呋喃甲酰基)-喹啉-2-羰醛(FQ)为荧光衍生试剂,利用超高效液相色谱(UPLC)和柱前衍生荧光检测技术,建立了贝类中3种高毒性PSP毒素成分(STX、GTX1及NEO)的检测方法,对影响荧光衍生效率和色谱分离效果的各主要实验因素分别进行了优化。结果表明,在优化后的最佳实验条件下,3种PSP毒素成分线性方程的相关系数(r)均大于0.998,保留时间(tm)及峰面积(PA)的日内及日间精密度RSD值分别小于3.1%和5.6%,当信噪比(S/N)等于3时,检测限范围为7～14μg.kg-1,样品加标回收率为82%～92%,RSD值小于5.2%。该方法灵敏、稳定且可靠,可用于贝类中三种高毒性PSP毒素成分的日常分析检测。     

大黄鱼幼鱼虹彩病毒感染的电镜研究

目的:探索大黄鱼幼鱼夏季高温病流行的病毒性病原体。方法:应用电子显微镜观察大黄鱼幼鱼内脏组织超薄切片。结果:发现大黄鱼幼鱼脾脏细胞肿胀,细胞内存在大量虹彩病毒颗粒。病毒颗粒直径约120nm,呈六角形,有一层蛋白质外壳,核心为核酸。病毒感染主要位于细胞质内。结论:大黄鱼幼鱼脾细胞感染的病毒是虹彩病毒,虹彩病毒感染及环境因素可能是导致大黄鱼幼鱼夏季高温病暴发流行并致死的主要原因。     

Fluorescence localization of epidermal cathepsins L and B in the Japanese eel

Histochemical localization of proteolytic activities in the dorsal epidermis of Japanese eel was demonstrated by fluorescent microscopy utilizing 4-methoxy-2-naphthylamide (4M$\beta$NA) derivatives as substrates and 5-nitrosalicylaldehyde as a trapping agent. Carbobenzoxy-L-phenylalanyl-L-arginyl-4M$\beta$NA (Cbz-Phe-Arg-4M$\beta$NA) and Cbz-Arg-Arg-4M$\beta$NA were used for direct detection of cathepsins L and B activities, respectively, in fresh frozen sections and unfixed cells of the eel epidermis. The fluorescing areas, where Cbz-Phe-Arg-4M$\beta$NA was hydrolyzed by cathepsin L, were shown in mucus secretory cells and club cells and broadly around skin surface. The fluorescing areas due to Cbz-Arg-Arg-4M$\beta$NA hydrolysis by cathepsin B were localized similarly in these tissues. The fluorescing intensity for both catheptic activities in mucus secretory cells was higher than that in club cells, where small fluorescing granules were distributed. These results indicate that eel cathepsins L and B are stored in epidermal secretory cells at different levels and probably serve as defense factors before or after secretion by these cells. Abbreviations: Cbz – carbobenzoxy; 4M$\beta$NA – 4-methoxy-2-naphthylamide; NSA – 5-nitrosalicylaldehyde.     

虾夷扇贝精子形态结构和超低温冷冻损伤的电镜观察

应用透射电镜和扫描电镜技术,研究了超低温冷冻保存前后虾夷扇贝精子超微结构和形态的变化。结果显示,虾夷扇贝精子由头部、中段和尾部3部分组成,外被光滑质膜;顶体位于头部最前端,呈倒"v"形,细胞核近似圆柱状,电子密度较高;4个线粒体和两个相互垂直的中心粒构成了精子的中段;鞭毛细长,轴丝为典型的"9 2"结构。经超低温冷冻保存后,冷冻损伤的精子表现为:精子被膜肿胀、被膜与核膜分离、丢失;顶体破裂、内容物流出;线粒体解体、线粒体嵴变形;鞭毛被膜肿胀、部分精子鞭毛脱落。可以推测,超低温冷冻保存对精子膜、顶体、线粒体和鞭毛的损伤可导致冻精活力和受精能力的下降。     

Morphology of Ichthyophonus hoferi assessed by light and scanning electron microscopy

Abstract. The morphology of Ichthyophonus hoferi in vitro at pH 3.5 and 7.0 is described using light and scanning electron microscopy. Only vegetative growth was observed. At pH 3.5, hyphal growth was seen. The hyphae of I. hoferi are characterized by evacuated hyphal walls with the cytoplasm migrating to the apex and no septation. In contrast, the growth at pH 7.0 is mainly seen as spherical bodies which vary in size and are uni- to multinucleate. Amoeboid bodies showing slow movements were observed within 3–6 h of transfer to pH 7.0. We propose a life-cycle involving the germination of thick-walled multinucleate spores in the fish stomach as a response to the low pH. The hyphae then penetrate the digestive tract and rupture when they reach a blood vessel (neutral pH), whereby uni- and binucleate bodies and/or amoeboid bodies are released. The small cells are transported in the blood vessels and spread in the organs richly supplied with blood (heart, kidney, spleen, liver and muscle tissue) where they grow to form multinucleate spores.     

鲈鱼组织中阿维菌素、伊维菌素残留的高效液相色谱荧光检测法研究

采用高效液相色谱分离,荧光检测器检测,建立鲈鱼组织中阿维菌素和伊维菌素残留的检测方法.样品用乙腈提取,碱性氧化铝SPE柱净化,N-甲基咪唑和三氟乙酸酐的乙腈溶液为衍生化试剂,常温衍生化15 rain.荧光检测器激发波长为365 nm,发射波长为475 nm.结果表明,阿维菌素和伊维菌素衍生化产物的浓度与峰面积在0.5～200 ng/ml范围内有很好的线性关系.添加水平为1～10 μg/kg,阿维菌素和伊维菌素的平均回收率在80.6%～88.0%和78.8%～82.8%之间,相对标准偏差均小于10%.阿维菌素的检出限为0.1/μg/kg,伊维菌素为0.2 μg/kg.该方法简便、灵敏、可靠,可用于鲈鱼组织中阿维菌素和伊维菌素残留的检测.     

Identification and detection of Pseudomonas plecoglossicida isolates with PCR primers targeting the gyrB region

Pseudomonas plecoglossicida is the agent of bacterial haemorrhagic ascites (BHA) in freshwater fish farming in Japan. To develop a rapid identification and detection method for P. plecoglossicida, a PCR amplification technique targeting the chromosomal DNA region coding the B subunit of the DNA gyrase (gyrB) was used. The nucleotide sequences of gyrB were determined in nine isolates of P. plecoglossicida and two other Pseudomonas species. On the basis of these determined sequences and the gyrB sequences of other Pseudomonas species or fish pathogenic bacteria deposited in international nucleotide sequence databases (GenBank/EMBL/DDBJ), PCR primers PL-G1F, PL-G1R, PL-G2F and PL-G2R were designed for specific amplification of the partial gyrB of P. plecoglossicida. The specificity of these primers in amplifying the gyrB of P. plecoglossicida was verified using selected strains of related bacterial species. The nested PCR technique was used to detect P. plecoglossicida from kidney and intestine of ayu. Primer pair PL-G1F and PL-G1R was used for the external PCR, and primer pair PL-G2F and PL-G2R for the internal PCR. Of 10 ayu juveniles, expected size PCR products were observed from intestine and kidney samples in one and two specimens, respectively. The PCR technique with primers based on the gyrB sequence is thus useful for the diagnosis of BHA.     

水产品中呋喃唑酮含量的高效液相色谱检测法

在传统法基础上 ,研究了测定草鱼、鳜、对虾、中华鳖等水产养殖动物肌肉中呋喃唑酮残留的快速HPLC法。用二氯甲烷提取样品中的呋喃唑酮 ,浓缩提取液至干 ,用乙腈水溶液溶解残渣并去脂肪后过滤 ,清液供HPLC分析 ,草鱼、鳜、对虾、中华鳖等水产养殖动物肌肉中杂峰都能很好地与药物峰分离 ,方法回收率稳定在70 %～ 75 % ,日内精密度与日间精密度均小于 3% ,本方法可检出的样品中呋喃唑酮低限为 0 0 0 1μg/g。