Simultaneous detection of the main black aspergilli responsible for ochratoxin A (OTA) contamination in grapes by multiplex real-time polymerase chain reaction

9 pages.This paper reports a duplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of members of the Aspergillus niger aggregate and A. carbonarius, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the β-ketosynthase and the acyl transferase domains of the poliketide synthase of A. carbonarius and the A. niger aggregate, respectively. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic Tm-values demonstrating the specific, efficient and balanced amplification of the two PCR fragments. Subsequently, a TaqMan real-time PCR approach was settled, using 6-carboxy-fluorescein group (FAM) and VIC®-labelled specific probes for automated detection. Results indicated no differences in sensitivity when using either the two sets of primers and probes in separate or in the same reaction. However, when both targets are in very different amounts, there is a preferential amplification of the target which is in more concentration. CT-values obtained in the presence of grape DNA were very similar to those observed when only fungal purified DNA was present, indicating that the grape DNA does not interfere in the real-time PCR reaction. This procedure provides a fast and accurate tool to monitor, in a single reaction, the presence of OTA-producing species in grapes which, to some extent, will facilitate OTA contamination surveys to guarantee food safety in the wine industry.Peer reviewe

<title language="por">Amplification of the cap20 pathogenicity gene and genetic characterization using different markers molecular in Colletotrichum gloeosporioides isolates

Studies were performed to analyze the genetic characterization using RFLP-ITS and Intron (primer EI1) markers and the amplification of the cap20 pathogenicity gene by PCR in Colletotrichum gloeosporioides isolates of different hosts plant. The genetic variability was accessed using RFLP-ITS and Intron markers and grouping by UPGMA method. Primers to cap20 gene were constructed using selected sequences of the GenBank (National Center of Biotechnology Information, http://www.ncbi.nlm.nih.gov) with the Primer 3 program. The dendrograms analysis showed that the RFLP-ITS marker was more informative to separate the Colletotrichum sp, and that primer EI1 demonstrated greater genetic diversity. The amplification of the DNA of the Colletotrichum isolates to the cap20 gene with primers P1 and P2 indicated that this gene could present variations into C. gloeosporioides related with the host, and also that it was present in other Colletotrichum sp.<br>Estudos foram realizados para analisar a caracterização genética usando marcadores de RFLP-ITS e ISSP e a amplicação do gene de patogenicidade cap20 por PCR em isolados de Colletotrichum gloeosporioides de diferentes hospedeiros. Primers para o gene cap20 foram construídos a partir de seqüências selecionadas do GenBank (National Center of Biotechnology Information, http://www.ncbi.nlm.nih.gov) com o programa Primer 3. A análise dos dendrogramas revelou que o marcador RFLP-ITS foi mais informativo em separar as espécies de Colletotrichum, e que o primer EI1 evidenciou maior diversidade genética. A amplificação do DNA dos isolados de Colletotrichum para o gene cap20 com os primers P1 e P2 indicou que este gene pode apresentar variações dentro de C. gloeosporioides relacionada ao hospedeiro, e que também está presente em outras espécies de Colletotrichum

Dynamics of hydration water in gelatin and hyaluronic acid hydrogels

[EN] We employed broadband dielectric spectroscopy (BDS), for the investigation of the water dynamics in partially hydrated hyaluronic acid (HA), and gelatin (Gel), enzymatically crosslinked hydrogels, in the water fraction ranges [Formula: see text]. Our results indicate that at low hydrations ([Formula: see text]), where the dielectric response of the hydrogels is identical during cooling and heating, water plasticizes strongly the polymeric matrix and is organized in clusters giving rise to [Formula: see text]-process, secondary water relaxation and to an additional slower relaxation process. This later process has been found to be related with the dc charge conductivity and can be described in terms of the conduction current relaxation mechanism. At slightly higher hydrations, however, always below the hydration level where ice is formed during cooling, we have recorded in HA hydrogel a strong water dielectric relaxation process, [Formula: see text], which has Arrhenius-like temperature dependence and large time scale resembling relaxation processes recorded in bulk low density amorphous solid water structures. This relaxation process shows a strong-to-fragile transition at [Formula: see text]C and our data suggest that the VTF-like process recorded at [Formula: see text]C is controlled by the same molecular process like long range charge transport. In addition, our data imply that the crossover temperature is related with the onset of structural rearrangements (increase in configurational entropy) of the macromolecules. In partially crystallized hydrogels ([Formula: see text]) HA exhibits at low temperatures the ice dielectric process consistent with the bulk hexagonal ice, whereas Gel hydrogel exhibits as main low temperature process a slow relaxation process that refers to open tetrahedral structures of water similar to low density amorphous ice structures and to bulk cubic ice. Regarding the water secondary relaxation processes, we have shown that the [Formula: see text]-process and the [Formula: see text] process are activated in water hydrogen bond networks with different structures.The support from Ministerio de Economia, Industria y Competitividad (MINECO) through the MAT2016-76039-C4-1-R project (including the FEDER funds) is acknowledged. The CIBER-BBN initiative is funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. 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The interest of the Spanish network of investigators in back pain for rehabilitation physician

Background: The Spanish Back Pain Research Network (REIDE) brings together teams of researchers and clinicians who are interested in nonspecific neck and back pain (BP). Its objective is to improve the efficacy, safety, effectiveness, and efficiency of the clinical management of BP. Method: The Network welcomes clinicians and researchers interested in BP. The only requirement to become a member of REIDE is to take part in one of its research projects, and any member can propose a new one. The Network supports those projects that are of interest to two or more groups by assuming their administration and management, which allows the researchers to focus on their task. Its working method ensures methodological quality, a multidisciplinary approach, and the clinical relevance of those projects that are carried out. Results: 179 researchers from 11 areas in Spain are involved in REIDE, including experts in all of the relevant fields of BP research. Most Spanish studies on BP that have been published in international scientific journals come from the teams involved in REIDE, and it currently has 13 ongoing research projects. Conclusions: The Network can help to enhance research among rehabilitation specialists who are interested in BP, and can contribute to the development of research projects which are of interest to the specialty. © 2005 Sociedad Española de Rehabilitación y Medicina Física (SERMEF) y Elsevier España, S.L