Evaluation of test kits for the determination of aflatoxins in meat products

A study was conducted to evaluate the performance of 4 different assays for rapidly screening samples of artificially contaminated hams and salami for the presence of aflatoxins (B₁+B₂+G₁+G₂) at concentrations ≥5 μg/kg. Test samples were contaminated in the range of 0 – 100μg/kg. At 0 μg/kg level no false positive (all <5 μg/kg) were found for all commodities by the kits tested; all test samples spiked at level >20 μg/kg were found positive by each kit, while most of the errors associated in the assays occurred on samples containing <10μg/kg. For samples either negative or contaminated above 20μg/kg all the methods were suited for use as rapid screening tests.     

Heterotrophic high-cell-density fed-batch and continuous-flow cultures of <Emphasis Type="Italic">Galdieria sulphuraria</Emphasis> and production of phycocyanin

Production of biomass and phycocyanin (PC) were investigated in highly pigmented variants of the unicellular rhodophyte Galdieria sulphuraria, which maintained high specific pigment concentrations when grown heterotrophically in darkness. The parental culture, G. sulphuraria 074G was grown on solidified growth media, and intensely coloured colonies were isolated and grown in high-cell-density fed-batch and continuous-flow cultures. These cultures contained 80–110 g L⁻¹ biomass and 1.4–2.9 g L⁻¹ PC. The volumetric PC production rates were 0.5–0.9 g L⁻¹ day⁻¹. The PC production rates were 11–21 times higher than previously reported for heterotrophic G. sulphuraria 074G grown on glucose and 20–287 times higher than found in phototrophic cultures of Spirulina platensis, the organism presently used for commercial production of PC.     

Heterotrophic cultivation of Euglena gracilis Z for efficient production of α-tocopherol

Effects of hydrodynamic stress, dissolved oxygen (DO) concentration and carbon sources on heterotrophic α-tocopherol production by Euglena gracilis were investigated. In a jar fermentor without baffle plates, increasing the agitation speed up to 500 rpm had no significant effect on cell growth and α-tocopherol production. However, in a jar fermentor equipped with baffle plates, both the cell growth and α-tocopherol production were highly suppressed at 500 rpm. At high hydrodynamic stress, the cells secreted nucleic acid-related substances to the culture broth and the shape of the cells shifted from elongated toward spherical. High DO concentration had adverse effects on both cell growth and α-tocopherol production, the optimum DO concentration being below 0.8 ppm. In comparison with glucose, the growth rate was lower but the α-tocopherol content of the cells was almost four times higher when ethanol was used as the organic carbon source. In a fed-batch culture with ethanol, a very high cell concentration of 39.5 g L^-1 was obtained with α-tocopherol content of 1200 μg g-cell^-1. This α-tocopherol content is very close to the values reported for photoautotrophic and photoheterotrophic cultures. A very high α-tocopherol productivity of 102 μg L^-1 h^-1 was obtained, indicating that heterotrophic cultivation of E. gracilis has a very high potential as a substitute for the current method of extraction from vegetable oils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Octadecylsilyl silica column method for extraction of messenger RNA

We have developed a simple and rapid method for the purification of poly(A) tail-messenger RNA (mRNA) from total RNA by using a solid phase extraction column filled with a small amount of octadecylsilyl silica. The method is based on a hydrophobic interaction between the poly(A) tail and the octadecyl unit on the silica particle in a water/dimethyl sulfoxide mixed solution. By using this column, mRNA can be separated from 100 μg of total RNA in less than 10 min with high yields (>80%).     

不同亲子鉴定试剂盒对D7S820基因座稀有等位基因检测结果比较

目的：观察分析用不同亲子鉴定试剂盒对D7S820基因座稀有等位基因检测结果的差异。方法：280名不同个体的血样DNA提取和基因型检测,按中华人民共和国公共安全行业标准GA／T382—2002和GA／T383—2002进行;分别用Identifiler试剂盒（美国AB公司）、PowerPlex 18Dsystem试剂盒（美国普洛麦格公司）、GoldeneyeTM20A试剂盒（北京基点认知技术有限公司）,经PCR复合扩增STR基因座,用AB公司DNA序列分析仪电泳分离扩增产物和激光扫描分析。结果：检测到280名不同个体的常用常染色体STR基因座的等位基因。,其中六份样本在D7S820基因座上,Identifiler试剂盒检测的基因型与PowerPlex 18D—system和GoldeneyeTM20A试剂盒检测的基因型结果有差异。结论：不同厂家生产的试剂盒检测常用常染色体D7S820基因座的稀有等位基因存在有一定误差。     

Evaluation of three field test kits to detect microcystins from a public health perspective

Cyanobacteria blooms may present a public health concern in sources of drinking water and recreational water due to the production of toxins by some species, microcystins being the most commonly found. It is possible to detect microcystins using instrumental analyses and field test kits. While instrumental analysis methods are accurate, they are also costly, and in regions with a high incidence of blooms the time to report is lengthy (days). On the other hand, the use of commercially available test kits may provide quicker results at a lower cost. The purpose of this work was to evaluate three commercially available kits: the Immunochromatographic Strip Test for the Detection of Microcystins and Nodularins in Source Drinking Water at 1 μg/L (Abraxis strip test), the Abraxis Microcystin Tube Kit and the Envirologix QualiTube Kit. The evaluation of each kit focussed on the interpretation of the results by the end-user and the validity of a test kit was based on four indices: sensitivity, specificity, positive predictive rate (PPR) and negative predictive rate (NPR) (false positive/negative) based on the manufacturer's specifications. The results indicate that there are challenges in the visual interpretation of the results at levels close to the threshold value for each kit. The scope of each kit must be understood: free vs. total, qualitative vs. semiquantitative. For instance, the Envirologix Qualitube Kit does not provide a lysing agent, therefore it will underestimate the levels of total microcystin if a lysing step is not included. In the case of the Abraxis strip test, the kit provides information on the absence/presence of microcystin at a threshold value of 1 μg/L, but false positives were encountered.     

Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells

Human 293S cells, a cell line adapted to suspension culture, were grown to 5×10⁶ cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×10⁵ cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×10⁶ cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM calcium-free DMEM - CS bovine calf serum - hpi hours post-infection - J+ enriched Joklik medium - MLP major late promoter - MOI multiplicity of infection (# of infectious viral particle/cell) - q specific consumption rate (mole/cell.h) - pfu plaque forming unit (# of infectious viral particle) - Y yield (g/E6 cells or mole/cell)     

Validation of a simple,rapid and cost effective method for the estimation of caprylic acid and sodium caprylate from biological products using NEFA-C kit

The method for the determination of caprylic acid and sodium caprylate from biological products was systematically validated using NEFA-C kit. The results obtained demonstrated that the kit method was simple, rapid, reliable, sensitive, reproducible and cost effective in comparison to the current methods i.e. colorimetric, High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) methods. The assay exhibited excellent linearity, accuracy, precision and robustness. Mean recoveries ranged between 95 and 101.3% (n = 6). The proposed method was linear over the concentration range of 0.05–10 mM of caprylate with values of coefficient of regression being >0.99. Method showed sensitivity of 0.05 mM (7.21 μg/ml for caprylic acid and 8.31 μg/ml for sodium caprylate). The % Relative standard Deviation (%RSD) for intra and interprecision studies was less than 5%. In conclusion the validated method was successfully used in monitoring of processed bulk and final products generated during production of biological products thus laying emphasis on strict control of release criteria for biological products fractionated using caprylic acid.     

An improved protocol for electroporation in members of the genus Gordonia

Gordonia are high GC gram-positive bacteria that have not yet been exploited well for biotechnological purposes because of the limited genetic tools. Described here is an improved protocol for electroporation, which is useful for several Gordonia species. The maximum transformation efficiency obtained was 2.8 × 10⁴/μg (Gordonia rubropertinctus, Gordonia sp), and 1.7 × 10³/μg (Gordonia amarae).     

Phorid fly, Pseudacteon tricuspis, response to alkylpyrazine analogs of a fire ant, Solenopsis invicta, alarm pheromone

The phorid fly, Pseudacteon tricuspis Borgmeier, is a parasitoid of the red imported fire ant, Solenopsis invicta Buren. This fly has been reported to use fire ant chemicals, specifically venom alkaloids and possibly alarm pheromone to locate its host. A recent study identified 2-ethyl-3,6-dimethyl pyrazine as a component of the alarm pheromone of S. invicta. To determine the possible involvement of this fire ant alarm pheromone component in mediating fire ant-phorid fly interactions, we tested electroantennogram (EAG) and behavioral responses of P. tricuspis females to the commercially available mixture of 2-ethyl-3,6-dimethyl pyrazine and its 3,5-dimethyl isomer, as well as six structurally related alkylpyrazine analogs at varying doses. Pseudacteon tricuspis females showed significant EAG response to 2-ethyl-3,6(or 5)-dimethyl pyrazine (herein referred to as pheromone-isomer) at all doses, 0.001-10 μg. Among the tested alkylpyrazine analogs, 2,3-diethyl-5-methyl pyrazine showed significant EAG activity at 0.1 and 1 μg. 2,3-dimethyl pyrazine also showed significant EAG activity at 0.1 μg. Results of four-choice olfactometer bioassays demonstrated significant attraction of P. tricuspis females to the pheromone-isomer (2-ethyl-3,6(or 5)-dimethyl pyrazine) at all tested doses (0.01, 0.1, 1 and 10 μg). The analogs, 2,3-diethyl-5-methyl pyrazine and 2,3-dimethyl pyrazine were significantly better than the control at the higher doses (0.1, 1 and 10 μg). The pheromone-isomer was significantly better than both analogs at two doses, 0.1 and 1 μg. These results confirm that the reported fire ant alarm pheromone component plays a role in mediating attraction of phorid flies to host workers. Venom alkaloids were previously shown to attract P. tricuspis; therefore, we propose that fire ant alarm pheromones may act in tandem or synergistically with venom alkaloids to attract phorid fly parasitoids to fire ant workers.     

糖类和植物生长调节剂对冬虫夏草子实体人工培养的影响

为提高人工培养冬虫夏草子实体的产量,需要优化其培养参数。本实验测定培养基中糖类和植物生长调节剂对冬虫夏草子实体产量的影响。于大米小麦作为主要组分的培养基中接入冬虫夏草菌,在9~13℃下培养60 d,转入4℃培养。葡萄糖培养基中,冬虫夏草子实体干重是麦芽糖培养基中的7.6倍,出现菌丝和收获子实体的时间也比麦芽糖培养基中至少快2个月;蔗糖培养基中未获得子实体。不同种类和浓度植物生长调节剂对冬虫夏草子实体产量影响显著。于菌液中加入环磷腺苷、三十烷醇和玉米素的培养瓶均未发现菌丝生长。加入100μg/mL 6-苄氨基腺嘌呤的培养瓶可见菌丝生长,但未见原基分化。转入4℃下6个月后,与对照相比,加入100μg/mL吲哚乙酸、1μg/mL和100μg/mL吲哚丁酸、10μg/mL赤霉素、1μg/mL和10μg/mL乙烯利,以及1μg/mL 2,4-D的培养瓶中子实体干重均显著提高,其中加入1μg/mL吲哚丁酸和1μg/mL 2,4-D的培养瓶的子实体干重是对照的15倍。实验结果为优化冬虫夏草子实体人工培育提供了支撑。     

甘肃兴隆山养麝场圈养高山麝的产香量

兴隆山国家级自然保护区(E103。50'-104。10',N35。38-58')位于甘肃省兰州市榆中县境内,是高山麝(Moschus chrysogaster)的自然分布区.     

Comparison and evaluation of RNA quantification methods using viral, prokaryotic, and eukaryotic RNA over a 10 concentration range

Quantification of RNA is essential for various molecular biology studies. In this work, three quantification methods were evaluated: ultraviolet (UV) absorbance, microcapillary electrophoresis (MCE), and fluorescence-based quantification. Viral, bacterial, and eukaryotic RNA were measured in the 500 to 0.05-ng μl⁻¹ range via an ND-1000 spectrophotometer (UV), Agilent RNA 6000 kits (MCE), and Quant-iT RiboGreen assay (fluorescence). The precision and accuracy of each method were assessed and compared with a concentration derived independently using inductively coupled plasma-optical emission spectroscopy (ICP-OES). Cost, operator time and skill, and required sample volumes were also considered in the evaluation. Results indicate an ideal concentration range for each quantification technique to optimize accuracy and precision. The ND-1000 spectrophotometer exhibits high precision and accurately quantifies a 1-μl sample in the 500 to 5-ng μl⁻¹ range. The Quant-iT RiboGreen assay demonstrates high precision in the 1 to 0.05-ng μl⁻¹ range but is limited to lower RNA concentrations and is more costly than the ND-1000 spectrophotometer. The Agilent kits exhibit less precision than the ND-1000 spectrophotometer and Quant-iT RiboGreen assays in the 500 to 0.05-ng μl⁻¹ range. However, the Agilent kits require 1 μl of sample and can determine the integrity of the RNA, a useful feature for verifying whether the isolation process was successful.     

Intra- and extra-cellular organophosphorus hydrolase production with recombinant <Emphasis Type="Italic">E. coli</Emphasis> using fed-batch fermentation

A fed-batch fermentation process for the production of organophosphorus hydrolase (OPH) (EC 3.1.8.1) by E. coli pET812 was developed in this research. With batch fermentation, the maximum OPH concentrations attained by batch fermentation were as low as 4 × 10⁵ U/l because cell growth and OPH production were inhibited by a high initial concentration of glucose. To develop a fed-batch fermentation process for obtaining higher concentrations of OPH, highly concentrated glucose solution (500 g/l) was added intermittently or continuously to increase the carbon source concentration. Eventually, 3.2 × 10⁶ U/l of OPH was produced with fed-batch fermentation in 24 h. This was eight times higher than the yield with conventional batch fermentation. A total concentration of 399–441 mg of OPH was produced/l, which was four times higher than that reported when using E. coli. Nearly half (44%) of the produced OPH was secreted into the culture solution.     

Antibacterial and anti-biofilm effects of cathelicidin peptides against pathogens isolated from cystic fibrosis patients

Six different cathelicidin-derived peptides were compared to tobramycin for antibacterial and anti-biofilm effects against S. aureus, P. aeruginosa, and S. maltophilia strains isolated from cystic fibrosis patients. Overall, SMAP-29, BMAP-28, and BMAP-27 showed relevant antibacterial activity (MIC₅₀ 4-8 μg/ml), and in some cases higher than tobramycin. In contrast, indolicidin, LL-37, and Bac7(1-35) showed no significant antimicrobial activity (MIC₅₀ > 32 μg/ml). Killing kinetics experiments showed that in contrast to tobramycin the active cathelicidin peptides exert a rapid bactericidal activity regardless of the species tested. All three peptides significantly reduced biofilm formation by S. maltophilia and P. aeruginosa strains at 1/2× MIC, although at a lower extent than tobramycin. In addition, BMAP-28, as well as tobramycin, was also active against S. aureus biofilm formation. Preformed biofilms were significantly affected by bactericidal SMAP-29, BMAP-27 and BMAP-28 concentrations, although at a lesser extent than tobramycin. Overall, our results indicate the potential of some cathelicidin-derived peptides for the development of novel therapeutic agents for cystic fibrosis lung disease.