Rapid detection of Clostridium botulinum toxins A, B, E, and F in clinical samples, selected food matrices, and buffer using paramagnetic bead-based electrochemiluminescence detection

Sensitive and specific electrochemiluminescence (ECL) assays were used to detect Clostridium botulinum neurotoxins serotypes A, B, E, and F in undiluted human serum, undiluted human urine, assay buffer, and selected food matrices (whole milk, apple juice, ground beef, pastry, and raw eggs). These novel assays used paramagnetic bead-based electrochemiluminescent technology in which biotinylated serotype-specific antibodies were bound to streptavidin-coated paramagnetic beads. The beads acted as the solid support and captured analyte from solution. Electrochemiluminescent detection relied on the use of ruthenium chelate-labeled anti-serotype antibodies and analysis with a BioVeris M-Series M1R analyzer. The sensitivities of the assays in clinically relevant matrices were 50 pg/ml for serotypes A and E, 100 pg/ml for serotype B, and 400 pg/ml for serotype F. The detection limits in selected food matrices ranged from 50 pg/ml for serotype A to 50 to 100 pg/ml for serotypes B, E, and F. The antibodies used for capture and detection exhibited no cross-reactivity when tested with the other serotypes. When purified native toxin was compared with toxins complexed to neurotoxin-associated proteins, no significant differences in assay response were noted for serotypes A, B, and F. Interestingly, the native form of serotype E exhibited reduced signal and limit of detection compared with the complexed form of the protein. We suspect that this difference may be due to trypsin activation of this particular serotype. The assays described in this article demonstrate limits of detection similar in range to the gold standard mouse bioassay, but with greatly reduced time to data. These rapid sensitive assays may have potential use in clinical settings, research studies, and screening of food products for botulinum toxins.     

Species-specific DNA probe and development of a quantitative PCR assay for the detection of Morganella morganii

Aims: To develop a SYBR Green quantitative PCR assay (qPCR) for the specific detection of Morganella morganii, a fish pathogen responsible for the Histamine Fish Poisoning. Methods and Results: A new primer set, amplifying a 179‐bp fragment of the 16S rRNA gene, was selected for specificity, and 14 M. morganii strains and 32 non‐Morganella strains were evaluated. The melting temperature of 84°C was consistently specific for the amplicon. Two standard curves were constructed: the minimum detection sensitivity was 0·563 pg of pure DNA, corresponding to DNA extracted from nine cells of M. morganii. The qPCR assay was evaluated in experiments with seeded fish samples, and the regression coefficient values were calculated. Conclusions: A highly specific and rapid assay was developed for the detection of M. morganii in tuna fish samples. Significance and Impact of the Study: This method represents the first study about the quantification of pathogenic M. morganii in fish products. This approach can be utilized to prevent the presence of this undesirable species in the food chain.     

The allele-frequency spectrum in a decoupled Moran model with mutation, drift, and directional selection, assuming small mutation rates

We analyze a decoupled Moran model with haploid population size N, a biallelic locus under mutation and drift with scaled forward and backward mutation rates θ₁=μ₁N and θ₀=μ₀N, and directional selection with scaled strength γ=sN. With small scaled mutation rates θ₀ and θ₁, which is appropriate for single nucleotide polymorphism data in highly recombining regions, we derive a simple approximate equilibrium distribution for polymorphic alleles with a constant of proportionality. We also put forth an even simpler model, where all mutations originate from monomorphic states. Using this model we derive the sojourn times, conditional on the ancestral and fixed allele, and under equilibrium the distributions of fixed and polymorphic alleles and fixation rates. Furthermore, we also derive the distribution of small samples in the diffusion limit and provide convenient recurrence relations for calculating this distribution. This enables us to give formulas analogous to the Ewens-Watterson estimator of θ for biased mutation rates and selection. We apply this theory to a polymorphism dataset of fourfold degenerate sites in Drosophila melanogaster.     

Do-It-Yourself device for recovery of cryopreserved samples accidentally dropped into cryogenic storage tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 °C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues (1,2). The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn' (2), which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces (2). In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks (1). These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials (3). However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use. In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.     

Isotope Ratio Measurements of Iron in Blood Samples by Multi-collector ICP-MS to Support Nutritional Investigations in Humans

With the perspective of embarking on a human study using a double iron (Fe) stable isotope tracer protocol to assess iron bioavailability, investigations were conducted on Fe isotope ratios in blood samples using a VG Axiom Multi-collector ICP-MS. The factors affecting the precision and accuracy of Fe isotopic ratios, such as spectral- and matrix-induced interferences and Fe recoveries from sample preparation, have been identified and optimized. Major polyatomic interferences (e.g., Ar-O, Ar-OH, and FeH) were significantly reduced by using an Aridus nebulizer and desolvating system. Isobaric metal (e.g., ⁵⁴Cr⁺ on ⁵⁴Fe⁺ and ⁵⁸Ni⁺ on ⁵⁸Fe⁺) interferences and Ca-oxides and hydroxides were quantitatively removed during chemical purification of blood samples and selective isolation of Fe by anion-exchange resin, after mineralization of the blood samples by microwave digestion. Quantitative recoveries of Fe from different steps of sample preparation were verified using whole blood reference material. Fe isotopic compositions of the samples were corrected for instrumental mass bias by the standard-sample bracketing method using the certified reference standard IRMM-014. External precisions on the order of 0.008–0.05 (% RSD), 0.007–0.015 (% RSD), and 0.03–0.09 (% RSD) were obtained for ⁵⁴Fe/⁵⁶Fe, ⁵⁷Fe/⁵⁶Fe, and ⁵⁸Fe/⁵⁶Fe, respectively, in the blood for three replicate measurements. The level of precision obtained in this work enables the detection of low enrichments of Fe in blood, which is highly desired in nutrition tracer studies.     

Screening for Tannin Degradation by Rumen and Faecal Samples of Wild and Domestic Animals in Ethiopia

Summary Tannins limit the use of fodder trees as feed for ruminants. Removal of the effects of tannins would thus improve the nutritional quality of these trees. This prompted the study to evaluate the effect of rumen or faecal mixed cultures from different animals on tannin degradation. Tannin extracts, tannic acid and gallic acid were used to enrich media to assess if rumen or faecal mixed cultures could degrade the phenolic compounds. Rumen fluid of Acacia-adapted sheep, sheep fed on wheat bran, bush duikers (Sylvicapra grimmia) and goats fed on Leucaena pallida and Sesbania goetzei were separately inoculated into Growth Study Medium (GSM) and incubated for 5-15 days. Faecal samples from dikdik (Madoqua guentheri), camel (Camelus dromedarius), zebra (Equus quagga), Grant’s gazelle (Gazella granti) and hartebeest (Alcelaphus buselaphus) were also separately inoculated into GSM media and incubated from 3-5 days. TLC results showed that mixed cultures from rumen fluids of Acacia-adapted sheep, sheep on wheat bran, goats on Leucaena pallida and Sesbania goetzei partially hydrolysed tannic acid to pyrogallol. Complete degradation of the heterocyclic ring in tannic acid and gallic acid was achieved by the mixed cultures from the faecal samples of dikdik and this was confirmed by HPLC. Mixed cultures from faecal samples of camel hydrolysed gallic acid to phloroglucinol. This study has demonstrated that faecal microorganisms of Ethiopian dikdik could completely degrade hydrolysable tannin.     

A new technique for simultaneous collection of macroalgal propagules in the water column

A Simultaneous Collection Module (SCM) was designed to concurrently collect multiple samples of macroalgal propagules, and to evaluate their abundance and spatial distribution in the water column. The basic sampling units are water collectors (tubes), held together to form an evenly spaced grid forming a spatial array with three dimensions. Each collector in the module possesses a simultaneous closing mechanism. The water collected in each tube is then filtered and the propagules retained on each filter cultivated under controlled conditions. The sampling system was deployed in the field in central Chile. Results suggest the system is an efficient collector, able to provide data on time-space changes of macroalgal propagules in the water column.     

环境样品中DNA的分离纯化和文库构建

采用研磨 /冻融和SDS/蛋白酶K热处理等理化方法 ,直接从性质不同的环境样品中提取和纯化混合基因组DNA。所获得纯品DNA的产量为每克样品 2～ 1 6μg。对纯品DNA进行限制性内切酶处理后 ,构建了以pUC1 8为载体的DNA文库。建库效率为从每克环境样品获得约 1 0 3～ 1 0 4 个含 3～ 8kb外源随机插入片段的克隆。通过DNA序列测定和基因注释 ,对从文库中随机选取的克隆进行了分析 ,发现外源插入片段均含序列未见报道的新基因。本文所做的尝试对于保存、研究和开发未培养微生物基因资源具有意义     

Micro-distribution of Soil Inhabiting Tardigrades (Tardigrada) in a Sub-alpine Coniferous Forest of Japan

Micro-distribution of soil inhabiting tardigrades was studied in a sub-alpine coniferous forest (alt. 1,840 m a. s. l.) on the eastern slope of Mt. Yatsugatake, Central Japan. Eight cylindrical soil samples were taken from the forest floor. Each sample was divided into 10 slice samples of 1 cm thick. Tardigrades were extracted from samples by Baermann funnels, identified to specific level and counted. The average number of tardigrades in the study site is 74,058/m². High densities sometimes occurred at depths greater than 5 cm. Consequently for investigating tardigrade populations, 5cm depth core sampling is insufficient in this type of habitat. Composition of the main groups of species reveals that shallow layers (1, 2 or 3 cm depth) are frequently dominated by the Diphascon-group. Macrobiotus-group species generally occurred in higher abundance in the deeper layers. It is very interesting that there are some different patterns of soil micro-distribution in tardigrade species.