Disposable Amperometric Polymerase Chain Reaction-Free Biosensor for Direct Detection of Adulteration with Horsemeat in Raw Lysates Targeting Mitochondrial DNA

A novel electrochemical disposable nucleic acid biosensor for simple, rapid, and specific detection of adulterations with horsemeat is reported in this work. The biosensing platform involves immobilization of a 40-mer RNA probe specific for a characteristic fragment of the mitochondrial DNA D-loop region of horse onto the surface of magnetic microcarriers. In addition, signal amplification was accomplished by using a commercial antibody specific to RNA/ DNA duplexes and a bacterial protein conjugated with a horseradish peroxidase homopolymer (ProtA-HRP40). Amperometric detection at −0.20 V vs Ag pseudoreference electrode was carried out at disposable screen-printed carbon electrodes. The methodology achieved a limit of detection (LOD) of 0.12 pM (3.0 attomoles) for the synthetic target and showed ability to discriminate between raw beef and horsemeat using just 50 ng of total extracted mitochondrial DNA (∼16 660 bp in length) without previous fragmentation. The biosensor also allowed discrimination between 100% raw beef and beef meat samples spiked with only 0.5% (w/w) horse meat (levels established by the European Commission) using raw mitochondrial lysates without DNA extraction or polymerase chain reaction (PCR) amplification in just 75 min. These interesting features made the developed methodology an extremely interesting tool for beef meat screening, and it can be easily adapted to the determination of other meat adulterations by selection of the appropriate specific fragments of the mitochondrial DNA region and capture probes

Del color de los ojos al interior del genoma. Nuevas tecnologías aplicadas a la educación: una experiencia en la enseñanza de la Genética

Depto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasFALSEsubmitte

Herramientas de aprendizaje para estudiantes de secundaria: Aplicación de la Genética a una situación real

Depto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasFALSEsubmitte

Herramientas de aprendizaje para estudiantes de secundaria en el campo de la Genética

Se ha diseñado una actividad: la caracterización molecular de la mutación de un gen que modifica el color de los ojos en Drosophila melanogaster, partiendo de un carácter morfológico, el color de los ojos, se obtendrá la secuencia del gen responsable y su localización en el genoma de la especie. Se pretende desarrollar una actividad práctica que permita a los alumnos de segundo ciclo de la ESO comprender la genética y la genómica y cómo estos conocimientos se pueden aplicar a distintas áreas: salud, biotecnología o impacto ambiental

Insights into the abundance, expression and diversity of key denitrification genes in an ecologically managed greenhouse agricultural soil

Understanding the bacteria associated with nitrification and denitrification is crucial for comprehending the processes that lead to nitrous oxide emissions in agricultural greenhouse soils. Therefore, it is important to determine their abundance and expression to gain insight into these processes. The aim of this study was to explore the bacterial communities associated with denitrification in a greenhouse agricultural soil amended with crop residues and manure for six years. For this purpose, we proceeded to detect and quantify the genes nirK and nirS and the gene nosZ through clone library construction, sequencing, phylogenetic analysis, and quantitative polymerase chain reaction (qPCR). Sequence analysis based on the clone library revealed that many of the nirS or nirK genes detected were not closely related to known denitrifier bacteria, but some of the nosZ sequences were related to the genera such as Pseudomonas, Halomonas, and Marinobacter. Furthermore, the qPCR revealed a high abundance of DNA copies in nirK, 6.08 × 109 ± 1.16 × 109, while nirS and nosZ showed lower values, 9.05 × 106 ± 1.65 × 106 and 8.71 × 106 ± 1.44 × 106, respectively. However, the highest expression rate was observed for nirS (mRNA/DNA ratio = 3.10 × 10− 3), while nirK and nosZ showed 10-fold lower expression rates (4.4 × 10− 4 and 3.5 × 10− 4, respectively). The results of this work provide a preliminary overview of the diversity, abundance and expression of key genes associated with the denitrification process in this type of soil and are a starting point for further studies to understand how this type of soil management can influence the denitrification process.Ministerio de Ciencia, Innovación y Universidades (España)European Union. NextGeneration (EU/PRTR)Depto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEpu

Influence of boiling and autoclave processing on the phenolic content, antioxidant activity and functional properties of pistachio, cashew and chestnut flours

Nuts are a rich source of nutritional compounds with beneficial effect on health. The objective of this study was to evaluate the effect of heat and/or pressure processing on protein and phenolic compounds (anthocyanins, flavonols, tartaric esters and total phenols) content, antioxidant activity and functional properties of pistachio, cashew and chestnut flours. Phenolic compounds content was increased in pistachio, cashew and chestnut after heat combined with pressure processing. Antioxidant activity resulted almost not reduced by harshest autoclave condition (138 °C, 2.56 atm, 30 min) and even, was increased in chestnut. The antioxidant activity, determined by dimethyl-ρ-phenylenediamine (DMPD) as well as oxygen radical absorbance capacity (ORAC) assays, was positively correlated with phenolic compounds content. Boiling and autoclave treatments increased water holding capacity (WHC) and water absorption capacity (WAC) in the three analysed tree nuts, and oil holding capacity (OHC) raised in processed pistachio and cashew defatted flours. Emulsifying and foaming capacities of pistachio, cashew and chestnut were negatively affected by processing as well as chestnut gelation capacity. Therefore, pistachio, cashew and chestnut flours maintaining, or even increasing, their phenolic content and antioxidant activity can be obtained by combining heat and pressure processing

Amperometric determination of hazelnut traces by means of Express PCR coupled to magnetic beads assembled on disposable DNA sensing scaffolds

A disposable amperometric sensor using magnetic microcarriers has been designed and implemented to be used in combination with the so called Express PCR to detect the presence of hazelnut traces in foodstuffs through the detection of Cor a 9 allergen coding sequence. The developed procedure involves the use of streptavidin-modified magnetic microbeads (Strep-MBs), specific biotinylated capture and detector probes which hybridize with a specific region of the gene encoding the protein Cor a 9, and appropriate primers for PCR amplification. A 50-mer synthetic target DNA or unmodified 100-bp PCR products were selective captured via sandwich hybridization with capture probe modified MBs and biotinylated signaling probes. The resulting biotinylated hybrids were coupled with a commercial streptavidin–peroxidase (Strep-HRP) conjugate and the final modified MBs were magnetically captured onto a screen-printed carbon electrode to perform amperometric detection using the H2O2/HQ system. A LOD of 0.72 pM was obtained for the synthetic target and the applicability studies demonstrated the possibility to detect the denatured PCR amplified samples obtained using only 20 pg of genomic DNA extracted from hazelnut. RSD values obtained, below 10% in all cases, confirmed the good reliability of extraction, amplification and quantification protocols involved in the developed methodology. The strict specificity of the designed primers and selected probes for hazelnut was demonstrated by performing PCR amplification of genomic DNA extracted from different hazelnut varieties and other species of similar families (pistachio, cashew, walnut and tangerine) and analyzing the resultant amplicons by the developed electrochemical sensor. The reliable and sensitive results achieved indicate that Express PCR in conjunction with an electrochemical DNA sensor, used for the first time in this work, provides a suitable sensitive, specific, and cost-effective method for routine food allergens determinations, particularly useful for resource-limited settings