Different Procedures For Preparation Research Articles

Detection of Southern tomato virus by molecular hybridisation

AbstractSouthern tomato virus (STV) is a double‐stranded RNA (dsRNA) virus belonging to the genus Amalgavirus from the family Amalgamaviridae. STV has been detected in tomato plants showing symptoms of stunting, fruit discoloration and size reduction, although its role on symptom development is unclear. Also, little is known about the incidence and epidemiology of this virus and how it spreads in tomato crops. In this work, we developed a molecular hybridisation method by using a digoxigenin‐labelled RNA probe based on the nucleotide sequence of the STV putative coat protein which was tested with different procedures for preparation of plant material. This technique was sensitive enough to detect STV from sap extracts (obtained just by grinding in buffer) from different plant tissues such as leaves, fruits, roots and seeds. This procedure is suitable for field surveys since it allows a cheap and quick processing of a high number of samples. Surveys performed in three important tomato production areas (Peninsular Spain, the Canary Islands and Sicily) showed that STV is widely spread, with incidences ranging from 18% to 74% in different local and commercial tomato varieties.

Evaluation of the COBAS® AmpliPrep/COBAS® TaqMan® HCV Test v2.0 for HCV viral load monitoring using dried blood spot specimens

This study evaluated the use of dried blood spot (DBS) for HCV viral load quantification using the COBAS® AmpliPrep/COBAS® Taqman® HCV Quantitative Test v2.0 (CAP/CTM HCV v2), and compared two different procedures for preparation of DBS samples with a Specimen Pre-Extraction (SPEX) reagent (either heated [SPEX with SH] for 10min at 56°C on a thermomixer, or incubated for 1h at room temperature [SPEX at RT]) against the standard plasma input. Whole blood specimens from 48 patients with chronic HCV infection and Whatman® 903 Protein Saver Cards were used to prepare 35μL DBS. An aliquot of plasma was spun and frozen from each draw. Mean DBS viral load results were compared to the corresponding results from plasma. Correlation between DBS to plasma was linear for both SPEX with SH (R2=0.96) and SPEX at RT (R2=0.97) procedures, with a constant negative offset of approximately 2.0log10IU/mL between whole blood DBS without any adjustments and plasma results. After volume corrections, the mean offset to plasma decreased to −0.39 and −0.36 for the two procedures, respectively. The study demonstrated the use of DBS for HCV viral load correlates well with plasma with a constant offset.

A critical overview of Cr speciation analysis based on high performance liquid chromatography and spectrometric techniques

The role and impact of chromium (Cr) on the environment and living organisms depends primarily on its chemical form. High toxicity of hexavalent Cr is well documented, while trivalent Cr is an essential micronutrient. In the last decades numerous analytical procedures have been developed for the determination of Cr(VI) in different sample matrices. To obtain reliable speciation data it is important to preserve species integrity during the sample storage, pre-treatment, extraction and the determination of Cr species. Among different speciation methods combination of high performance liquid chromatography (HPLC) with atomic spectrometry techniques provides comprehensive information on the presence of Cr species in a variety of sample matrices, while hyphenation of HPLC to inductively coupled plasma mass spectrometry (ICP-MS) represents the most powerful and the most sensitive analytical tool for Cr speciation. Precise isotope ratio measurement enables the application of isotope dilution techniques for the quantification of trace amounts of individual Cr species in various environmental and biological samples. Furthermore, enriched stable isotopes can be introduced as tracers to investigations on the fate and role of Cr in the environment and living organisms or to monitor the species transformation during the analytical procedure. Despite general understanding of Cr chemistry, which is closely related to its trivalent and hexavalent oxidation states and knowledge on conditions that may influence species transformation, there are still open questions that should be addressed to obtain reliable speciation analysis data. So, this article is focused mostly to overview recent developments in methodological approaches for Cr speciation in different sample matrices by applying HPLC and spectrometric techniques. Different procedures for preparation of isotopically enriched Cr spike solutions are critically evaluated. The advantages of their use as tracers to follow and account for species transformation during sample preparation and for the quantification of Cr species by HPLC-ID-ICP-MS are discussed. The importance of the use of adequate analytical methodologies and speciation analysis in the determination of Cr(VI) is highlighted in order to avoid inadequate conclusions to be made based on wrongly applied analytical methodologies. An increasing need to develop speciation procedures for selective determination of Cr(III) species is also emphasized.

SEM–EDX and XRD characterization of zeolite NaA, synthesized from rice husk and aluminium scrap by different procedures for preparation of the initial hydrogel

A number of investigations have demonstrated that zeolite NaA could be synthesized using Si, extracted from rice husk ash; however, experiments on direct extraction of Si from rice husk (RH) are scarce. The main objective of the present study was to explore the possibility to synthesize high-quality zeolite NaA from RH and waste aluminium cans (as a source of Al), applying different procedures for the preparation of initial hydrogel and a unified procedure for crystallization of zeolite NaA. Products were characterized by SEM–EDX and XRD analyses. The investigation demonstrated that Si could be extracted directly from RH, avoiding the process of RH burning. Practically complete dissolution of Si from RH was achieved by alkali treatment (with 10 % NaOH for 7 h) at boiling temperature and atmospheric pressure, i.e. using refluxing system instead of autoclave for the preparation of Si-gel. Zeolite NaA samples synthesized from such Si-gels were pure, highly crystalline and white. Furthermore, it was found that the direct dissolution of Al in Si-gel did not affect the quality of the final product. Although this investigation was not focused on the mechanism of zeolite NaA crystallization, the results obtained indicated clearly that the history of Si-gel preparation played an important role in the nucleation and growth of zeolite NaA crystals and influenced their yield, size, and shape. Therefore, the optimization of Si-gel preparation procedure has to be considered as essential not only for the economy of the synthesis of NaA from RH, but also for the quality of the final product.

Lipid peroxidation in human milk and infant formula: effect of storage, tube feeding and exposure to phototherapy

Preformed lipid peroxidation products present in the feed may contribute to the total reactive oxygen radical load infants have to deal with and may play a role in the pathogenesis of necrotizing enterocolitis and bronchopulmonary dysplasia. In this study, the occurrence of lipid peroxidation in human milk and feeding formulas for preterm babies was evaluated in vitro. Free linoleic acid (18:2) and its hydroperoxide (18:2OOH) were measured by gas chromatography-mass spectrometry and the concentration of 18:2OOH and the 18:2OOH/18:2 ratio were used as indices of peroxidation. In all feeds peroxidation products were present, but the proportion of peroxidized 18:2 was greater in infant formula. Storage of human milk (+4 degrees C for four days) increased lipid peroxidation. Exposure to light during tube feeding increased peroxidation in infant formula but not in human milk. Different procedures for preparation, storage and feeding may decrease the concentration of these potentially toxic peroxidized lipids in human milk and infant formula.

Homocysteine and other thiols determined in plasma by HPLC and thiol-specific postcolumn derivatization

We describe a versatile high-performance liquid-chromatographic method for determining homocysteine and other plasma sulfhydryls. Using three different procedures for preparation of plasma, we determined total, free (non-protein-bound), and reduced forms of homocysteine, cysteine, glutathione, cysteinylglycine, and gamma-glutamylcysteine in human plasma. Sample preparation involves disulfide reduction with dithiothreitol and protein precipitation with sulfosalicylic acid. The assay utilizes isocratic reversed-phase ion-pair liquid chromatography at pH 2.4, postcolumn derivatization with 4,4'-dithiodipyridine, and colorimetric detection at 324 nm. The intra-assay precision (CV) of the method for total homocysteine is 1.5%; the interassay precision over 2.5 months is 2.5%. The detection limit for homocysteine is < 50 nmol/L plasma.