Comparison of real-time PCR methods for quantification of European hake (Merluccius merluccius) in processed food samples.

The quantification of species in commercial products is limited by analytical shortcomings, as most of them provide semiquantitative results. An exception is real-time PCR, which can provide quantitative results using hybridization probes. In the present work, this technique has been applied to the absolute, absolute-relative and relative quantification of the most valued hake species in European markets, Merluccius merluccius (European Hake). The best quantification results for this species in binary mixtures with non-target species (Merluccius capensis) and using a species-specific real-time PCR MMER_VIC system was achieved using a relative quantification approach (MLL as reference system). Absolute quantification using the MLL nuclear system has been demonstrated as appropriate for the quantification of the Merluccius genus in food model samples. This study reveals the impact of different reference systems (MLL and HAKE) in the absolute-relative and relative quantification approaches, showing that the nuclear MLL system performed better than the mitochondrial HAKE system.

Identification of European Hake species (Merluccius merluccius) using real-time PCR.

A rapid and precise method for identifying European hake (Merluccius merluccius) based on TaqMan technology is presented. The method can be applied to fresh, frozen, and processed fish products to detect the fraudulent or unintentional mislabeling of this species. Specific primers and a minor groove binding (MGB) TaqMan probe were designed for this purpose based on partial sequences of the mitochondrial DNA control region. Combinations of primers and probe concentrations that gave the lowest Ct value and the highest final fluorescence value were selected to carry out efficiency, specificity, and cross-reactivity assays. The method was successfully tested on 31 commercial hake samples. A Ct value of about 16 was obtained when Merluccius merluccius was present; however, the fluorescence signal was not detected most of the time (Ct value 40) or presented significantly higher Ct values (38.2 +/- 0.96) for the nonhake species.

The action of pulse-modulated GSM radiation increases regional changes in brain activity and c-Fos expression in cortical and subcortical areas in a rat model of picrotoxin-induced seizure proneness.

The action of the pulse-modulated GSM radiofrequency of mobile phones has been suggested as a physical phenomenon that might have biological effects on the mammalian central nervous system. In the present study, GSM-exposed picrotoxin-pretreated rats showed differences in clinical and EEG signs, and in c-Fos expression in the brain, with respect to picrotoxin-treated rats exposed to an equivalent dose of unmodulated radiation. Neither radiation treatment caused tissue heating, so thermal effects can be ruled out. The most marked effects of GSM radiation on c-Fos expression in picrotoxin-treated rats were observed in limbic structures, olfactory cortex areas and subcortical areas, the dentate gyrus, and the central lateral nucleus of the thalamic intralaminar nucleus group. Nonpicrotoxin-treated animals exposed to unmodulated radiation showed the highest levels of neuronal c-Fos expression in cortical areas. These results suggest a specific effect of the pulse modulation of GSM radiation on brain activity of a picrotoxin-induced seizure-proneness rat model and indicate that this mobile-phone-type radiation might induce regional changes in previous preexcitability conditions of neuronal activation.

[Psychiatric and cognitive complications arising from subthalamic stimulation in Parkinson's disease]. / Complicaciones psiquiátricas y cognitivas de la estimulación subtalámica en la enfermedad de Parkinson.

INTRODUCTION AND DEVELOPMENT: Subthalamic stimulation is a therapeutic option that can be used to treat advanced cases of Parkinson's disease. However, psychiatric or cognitive disorders have been reported in some patients treated using this technique. Age and a long disease history are two important risk factors for the appearance of these problems. The complications that have been reported include cases of depression, apathy, manias and psychosis. Surgery can also exacerbate the syndrome of addiction to levodopa that is sometimes observed. In contrast, sleep disorders usually improve with this technique. As far as the cognitive sphere is concerned, verbal fluency has been seen to deteriorate and the executive functions become impaired in patients over 69 years of age. These disorders are usually due to a number of different causes and have been attributed to the action of stimulating areas close to the subthalamic nucleus, to the presence of previously existing cognitive or psychiatric problems, to unrealistic expectations about this technique or to the individual's inability to adapt to the functional situation after surgery. CONCLUSIONS: Although generally speaking these disorders are not usually serious, they must be borne in mind so that adequate treatment can be indicated.

Identification of Hake species (Merluccius Genus) using sequencing and PCR-RFLP analysis of mitochondrial DNA control region sequences.

The use of DNA-based methodologies in identification of hake species belonging to the Merluccius genus was shown to be successful. A short fragment of the left hypervariable domain of the mitochondrial control region was amplified, sequenced, and digested from 11 hake species. The hake-specific PCR product, due to its limited size, was obtained in a variety of tissue samples with different levels of DNA concentration and degradation, including sterilized food products. On the basis of this phylogenetically informative 156-bp sequence were selected four restriction enzymes (ApoI, DdeI, DraIII, and MboII) that allow the hake species discrimination. Species identification by phylogenetic analysis of sequences or by PCR-RFLP methodologies is useful in a variety of scenarios including authentication of thermally processed food, detection of food components, and species determination of individuals whose morphological characters are removed.

Identification of flatfish (Pleuronectiforme) species using DNA-based techniques.

Identification of flatfish species using a DNA-based methodology was studied. The polymerase chain reaction was employed to obtain a 464 bp amplicon from mitochondrial cytochrome b gene. The sequences from this fragment belonging to 24 species were analyzed using a genetic distance method, and polymorphic sites were determined. The fragment was found to be highly polymorphic (231 sites), and this permitted the differentiation of most of the species. Phylogenetic tree construction was employed to allow the identification of flatfish species. As a result, each species was grouped in a well-differentiated clade, except for two pairs: Limanda ferruginea and L. limanda, and Solea impar and S. lascaris, which could not be differentiated. On the basis of the sequences obtained, restriction enzymes were selected to provide specific restriction profiles, which allow the differentiation of 21 species of flatfish in a faster and less expensive manner than sequencing. This polymerase chain reaction-restriction fragment length polymorphism methodology (PCR-RFLP) was tested using commercial samples.

Development of a DNA-based method aimed at identifying the fish species present in food products.

Analysis of restriction fragment length polymorphism (RFLP) profiles of a 464 bp amplicon obtained from the mitochondrial cytochrome b gene was used to differentiate between several different fish species. The method was tested by a collaborative study in which 12 European laboratories participated to ascertain whether the method was reproducible. Each laboratory was required to identify 10 unknown samples by comparison with RFLP profiles from authentic species. From a total of 120 tests performed, unknown samples were correctly identified in 96% of cases. Further work attempting to use the method to analyze mixed and processed fish samples was also performed. In all cases the species contained within mixed samples were correctly identified, indicating the efficacy of the method for detecting fraudulent substitution of fish species in food products.

Use of restriction fragment length polymorphism to distinguish between salmon species.

Identification of 10 salmon species using DNA-based methodology was investigated. Amplification of DNA was carried out using a primer set which amplified a region of the mitochondrial cytochrome b gene. Sequences of PCR-amplified DNA from the salmon species were used to select six restriction enzymes allowing species to be uniquely classified. RFLP patterns generated following analysis with each enzyme were resolved using polyacrylamide gel electrophoresis and visualized by silver staining. Results indicate that it is possible to differentiate between all 10 salmon species and that the technique could be easily adopted by the food industry for analysis of processed salmon products.

Comparison of different methods to produce single-strand DNA for identification of canned tuna by single-strand conformation polymorphism analysis.

By using single-strand conformation polymorphism (SSCP) analysis of three amplicons of the cytochrome b gene obtained by the polymerase chain reaction (PCR) it was possible to differentiate between various species of tunas and bonitos processed as canned fish. Four different techniques were used to produce single-strand DNA (ssDNA): (i) Denaturation of double-strand DNA (dsDNA) by formamide and alkali, (ii) two-step asymmetrical PCR, (iii) one-step asymmetrical PCR, and (iv) exonuclease digestion of the phosphorylated strand of dsDNA. The technique rendering optimal results depended on the type of amplicon (i.e. the sequence).

[Use of flumazenil to reverse the effects of midazolam in children]. / Utilidad del flumazenil para revertir las acciones del midazolam en niños.

We have studied the clinical usefulness of Flumazenil to reverse the sedative action of Midazolam in 12 children admitted in a Pediatric Intensive Care Unit. Two groups were established, one treated with individual dose and the other treated with continuous infusion. In four cases the indication of Flumazenil was the reversion of secondary effects and in 8 cases it was elective. The average reversion time was 1.22 +/- 0.42 minutes. Flumazenil is able to reverse immediately the effects of Midazolam.