猴痘病毒PCR检测方法的建立

从GenBank上调取猴痘病毒的基因序列,经过分析,找出了猴痘病毒的特异性靶基因序列F3L片段,人工合成猴痘病毒MPV(AF380138)F3L基因片段(48048-48509bp)并插入质粒,作为病毒检测的模拟阳性模板。根据该序列设计并合成PCR引物,对模拟的阳性模板进行扩增,结果能扩增出与目的片段大小一致的条带。建立了PCR检测方法,经条件优化后,对鸡痘、禽痘、羊痘等14种相似病毒核酸进行PCR扩增,发现具有良好的特异性。PCR方法能扩增出0.3pg的阳性模板,显示建立的PCR方法具有高效、快速、特异、灵敏的特点,可用于口岸猴痘病毒的检疫。     

A Novel Particle Contact Assay with the Yeast Saccharomyces cerevisiae (8 pp)

Goal, Scope and Background   Numerous xenobiotics released into surface waters are transferred to suspended particulate matter and finally attached to sediments. Aquatic organisms may be exposed to them by direct particle feeding, by physical contact with contaminated surfaces as an exposure route, and by the uptake of dissolved contaminants after equilibration via the free water phase. In order to assess potential sediment toxicity, each of these exposure routes has to be addressed. This paper presents a newly developed particle contact assay that uses the fermentation performance of a specific Saccharomyces cerevisiae strain for the assessment of toxic effects in sediments. The test procedure is based on the characteristic feature of growing yeast cells to attach to sediment particles, which are also relevant for the accumulation of contaminants. The physical contact with lipophilic contaminants mirrors an exposition pathway for the direct uptake into the cells. In order to quantitatively characterize the toxic effects of particle attached pollutants on the fermentation performance, unpolluted native reference sediment was spiked with representatives for widely distributed anthropogenic contaminants. Methods   Saccharomyces cerevisiae was established as sensitive eukaryotic microorganism for the ecotoxicological assessment of particle attached anthropogenic contaminants in freshwater sediments. For this purpose, yeast cells were cultivated in sediment samples and the resulting fermentation performance was continuously measured. Sediments artifically spiked with HCB, PCB, g-HCH, DDT, and benzo(a)pyrene and solutions of each contaminant were comparatively investigated by means of their adverse effects on yeast fermentation performance. Additionally, four native river sediments characterized by increasing levels of pollution were assessed by the yeast particle contact assay, and simultaneously by standard aquatic tests with algae, daphniae, and luminescent bacteria using pore water and elutriates. Results of the bioassays were related to specific sediment contamination with respect to metals and organic priority pollutants. Results and Discussion   In sediments spiked with PCB and benzo(a)pyrene fermentation, performance was affected extensively below concentrations inhibiting fermentation in contaminant solutions. This suggests a high efficiency of the exposure route by physical contact. The fermentation performance was only slightly affected by single lipophilic pollutants, whereas mixtures of individually spiked sediments caused critically reduced fermentation performance suggesting additive synergistic effects. Native river sediments modestly to critically polluted by hazardous organic compounds lead to a slightly to dangerously reduced fermentation performance in the yeast contact assay. These inhibitory effects were much less pronounced in the standard bioassays conducted with algae, daphniae and luminescent bacteria, applying pore waters and elutriates as sample matrices. Using pore water, inhibition was measured only in the most polluted sediment, elutriates lead to a slight inhibition of the algal growth in the undiluted sample only. These results indicate an improved sensitivity of the yeast particle contact assay compared to the standard assays, due to uptake and physical cell contact as additional routes of exposure. Conclusion   The yeast particle contact assay is a valuable tool for the assessment of ecotoxicological potential in freshwater sediments. Since the assay addresses physical contact as an exposure route, it indicates bioavailability of lipophilic compounds in sediments. Outlook   The sensitive indication of bioavailable contaminants associated to sediment particles by the newly developed yeast particle contact assay recommends it as a complementary microbial bioassay in a test battery for assessing major pathways of contaminants in whole sediments.     

Seasonal changes in periphyton nitrogen fixation in a protected tropical wetland

Cyanobacteria are important for global nitrogen cycle and often form complex associations referred to as cyanobacterial mats or periphyton that are common in tropical, limestone-based wetlands. The objective of this study was to monitor the nitrogen fixation rate using the acetylene reduction assay of these cyanobacterial mats in a tropical, unfertilized, and protected wetland. To account for temporal and spatial variation of nitrogenase activity, we were interested in seasons in a hydrological cycle (dry, rains, and end of rains), sites with different vascular vegetation, and rates of nitrogenase activity in a 24-h cycle. The annual average of nitrogenase activity was 22 nmol C₂H₄ cm⁻² h⁻¹, with a range of <6 to 35 nmol C₂H₄ cm⁻² h⁻¹, and the annual nitrogen fixation rate of our study site (9.0 g N m⁻² year⁻¹) is higher than similar estimates from other freshwater wetlands. There was a clear temporal pattern in nitrogenase activity with a maximum rate occurring during the rainy season (August) and a maximum nitrogenase activity occurring between 0600 and 1200 hours. We found spatial differences in nitrogenase activity among the four sites that could be attributed to variations in species composition within the periphyton.     

Molybdenum reserves of seed,and growth and N2 fixation by Phaseolus vulgaris L.

Summary Plants grown from seed with high (1.5–7.3 g Mo seed^-1) and low (0.07–1.4 g Mo seed^-1) Mo contents were grown in the presence and absence of Mo in growth media (perlite) or in a flowing-solution culture, in a controlled environment. Neither the high (1.5 g Mo seed^-1) nor the low (0.1 g Mo seed^-1) Mo content in seed from a small-seeded genotype (BAT 1297) was able to prevent Mo deficiency (reduced shoot, root and nodule dry weight, N₂ fixation and seed production) in growth media without an external supply of Mo, whereas both the high (7.3 g Mo seed^-1) and the low (0.07 g Mo seed^-1) contents in seed were able to prevent Mo deficiency in a large-seeded genotype (Canadian Wonder). Responses to Mo treatment by the Two genotypes were inconsistent between the growth media and solution culture experiments. Seed with a large Mo content (3.5 g Mo seed^-1) from the Canadian Wonder genotype was unable to prevent Mo deficiency (reduced shoot and nodule dry weight and N₂-fixation) in a solution culture without an external source of Mo, whereas both the large (1.7 g Mo seed^-1) and the small (0.13 g Mo seed^-1) contents in seed prevented a deficiency in BAT 1297. Growing plants from seed with a small Mo content, without additional Mo, reduced the seed Mo content by 83–85% and seed production by up to 38% in both genotypes. Changes in seed size and increases in shoot, root and nodule dry weight occurred, but varied with the genotype and growth conditions. These effects were also observed in some cases where plants were grown with additional Mo, demonstrating that the amount of Mo in the seed sown can influence plant nutrition irrespective of the external Mo supply. Nodule dry weight, total N content of shoots and seed production were improved by using seed with a small Mo content (1.64–3.57 g Mo seed^-1) on acid tropical soils in Northern Zambia. Plants of both the large- and small-seeded genotypes grown from seed with a small Mo content (<1.41 g Mo seed^-1) had a smaller nodule weight, accumulated less N and produced less seed. The viability of seed with a small Mo content was lower (germination up to 50% less) than that of seed with a large Mo content.     

Effect of high temperature on growth,nitrogen fixation,and chlorophyll content of five species of Azolla Anabaena symbiosis

Summary Cultures of Azolla sp. ST-SI, A. microphylla BR-GI, A. mexicana BR-GL, A. caroliniana WT-V, and A. filiculoides BR -H were grown in N-free International Rice Research Institute growth medium in the glasshouse at 38±1 °C (day) and 25±1 °C (night) under a light intensity of 350 Em²s^–1 for 27 days. Biomass, chlorophyll contents and nitrogenase activity (acetylene reduction assay) were recorded on the 19th and 27th day. For comparison the same parameters were studied in Azolla spp. under normal growth conditions at 26±1 °C (day) and 19±1 °C (night). Azolla sp. STSI and A. microphylla BR-GI had produced a larger biomass by the 19th and the 27th day of incubation than A. caroliniana WTV and A. filiculoides which showed poor growth. Under normal growth conditions A. caroliniana WTV and A. filiculoides BRH produced less biomass than the other Azolla spp. cultures tested. A. mexicana BR-GL had a higher total chlorophyll content in both incubation periods than A. caroliniana WT-V and A. filiculoides BR-H. The N content was high in Azolla sp. ST-SI, A. microphylla BR-GI, and A. mexicana BR-GL compared with the low N content of A. filiculoides BR-H and A. caroliniana WT-V. At the higher temperature (38±1 °C/25±1 °C) Azolla sp. ST-SI and A. microphylla BR-GI consistently showed a higher growth rate than A. filiculoides BR-H and A. caroliniana WTV, while the growth rate of A. mexicana BR-GL was intermediate.The study was carried out at C.F. Kettering Research Laboratory, Yellowsprings, OH - 45387, USA     

Estimation of associative nitrogen fixation in relation to water regime and plant nutrition in a long-term pot experiment with rice (Oryza sativa L.)

Summary The contribution of associative N₂ fixation to the N nutrition of lowland rice was estimated in a long-term pot experiment with ten consecutive crops of rice. The experiment comprised two N and two K levels with wet (WF) and dry fallow (DF) between the cropping seasons. Growth of N₂-fixing cyanobacteria was prevented. Greatest yields were obtained in the high NK fertilizer treatment, but with continuation of the experiment yields responsed more to DF than to WF. Nitrogenase activity, however, was favoured by WF. Higher K application increased and higher N application decreased nitrogenase activity. Under WF treatments the organic C and total N contents of the soil remained unchanged during the experimental period, but alternate drying and flooding in DF treatments caused a decline. Lower N fertilizer rates in the second five-crop period did not affect yields, but increased the ratio of N removed to N applied. For the ten-crop period the estimated N balance was positive in the low-N and negative in the high-N treatments. N balances were also established separately for both the first and the second halves of the ten-crop period. In the first period N losses were higher, and the N balance was mostly negative. In the second period only high-N combined with low-K fertilization resulted in a negative N balance. DF favoured N losses in the first but not in the second period. The highest N gain in the second period was found in the DF treatment with low-N and high-K application. In this treatment, nearly one-quarter of the N taken up by the above-ground parts of the plants could be ascribed to associative N₂ fixation. In the corresponding treatment with the higher N level and a 49% higher yield, the contribution of fixed N declined to less than 5%. When harvested straw contained more than 10 mg N g^–1, the N balance was mostly negative, while at N contents less than 10 mg N g^–1, the N balance was generally positive.     

酶联免疫吸附分析技术在农药残留分析中的应用

简述酶联免疫吸附分析技术的原理、方法类型以及关键的技术要点,并对该技术在农药残留分析检测中的应用前景进行了展望。     

太湖微囊藻毒素在罗非鱼体内累积及生物降解的初步研究

为了解蓝藻水华期间微囊藻毒素在罗非鱼体内的分布及累积传递过程,2008年6～8月采集了高密度蓝藻池塘及太湖网箱内的鱼样及水样,用ELISA法对鱼样和水样进行微囊藻毒素MC-LR含量的检测。结果表明,池塘水体微囊藻毒素MC-LR含量在0.123～0.514μg/L,MC-LR含量随着藻密度的下降而降低,对照组水体MC-LR浓度显著高于实验组MC-LR含量。池塘鱼体肌肉组织微囊藻毒素MC-LR累积含量在1.194～3.615ng/g,肝脏组织微囊藻毒素MC-LR累积含量显著高于肌肉组织。将池塘与网箱罗非鱼转至无微囊藻水体中暂养,跟踪检测MC-LR含量变化,池塘和网箱鱼体肌肉组织微囊藻毒素MC-LR含量均低于人体每日可耐受摄入量,而肝脏组织藻毒素MC-LR含量则分别需要经过10～20d自然生物降解后降低至安全摄入量之下。讨论了微囊藻毒素在鱼体组织分布与食物链中的累积传递。     

Development of immunodot blot assay for the detection of white spot syndrome virus infection in shrimps (Penaeus monodon)

The VP 28 gene encoding a structural envelope protein of the white spot syndrome virus (WSSV) was cloned into a pET32a(+) expression vector for the production of the recombinant VP28 protein. A purified recombinant protein of 39.9 kDa size was used for polyclonal antibody production in rabbit. Specific immunoreactivity of the rabbit anti rVP28 antiserum to the viral antigen was confirmed by a Western blot. The specificity of this polyclonal anti‐rVP28 antiserum to detect the presence of the virus in WSSV‐infected Penaeus monodon was verified using a immunodot blot assay. Immunodot blot showed a positive reaction in infected shrimp tissues with prominent colour development using 3,3′,5,5′‐tetramethylbenzidine (TMB) as a chromogenic substrate when compared with 3–3′ diaminobenzidine tetrahydrochloride (DAB). Highest signal intensities of the immunodots were observed in infected shrimp pleopod extracts and haemolymph. On comparison with polymerase chain reaction (PCR), immunodot blot could detect 76% of PCR‐positive WSSV‐infected shrimp samples. Immunodot blot was found to be equivalent to first‐step PCR sensitivity to detect WSSV particles estimated to contain 1.0 × 10⁵ viral DNA copies.