Teresa García Lacarra

Development of a PCR-culture technique for rapid detection of yeast species in vacuum packed ham

A rapid assay for detection of yeast species in vacuum packed ham has been developed based on the polymerase chain reaction (PCR) coupled to a 24 h pre-enrichment at 25 °C. DNA was isolated from yeast inoculated ham samples and amplified using primers specific for the 18S rRNA gene sequences of yeasts. A detection limit of 10(2) CFU/cm(2) was achieved following enrichment of samples experimentally inoculated with three yeast species frequently associated with meat products spoilage: Debaryomyces hansenii, Yarrowia lipolytica, and Kluyveromyces marxianus. Likewise, commercial sliced and vacuum packed ham samples were analysed using the PCR-culture technique. The results obtained in this work show that PCR amplification of a conserved region of the 18S rRNA gene in the yeast species could be potentially used as a rapid tool for detection of low levels of viable spoilage yeasts in meat products.

ITS-based detection and quantification of Alternaria spp. in raw and processed vegetables by real-time quantitative PCR.

A TaqMan real-time polymerase chain reaction (PCR) method was developed for specific detection of Alternaria spp. in foodstuffs. The method uses Alternaria-specific primers and probe targeting the internal transcribed spacer regions ITS1 and ITS2 of the rRNA gene, and a positive amplification control based on 18S rRNA gene. The applicability of the real-time PCR protocol was assessed through analysis of 190 commercial food samples, including 80 fresh fruit and vegetable samples and 110 processed foodstuffs. The assay demonstrated the presence of Alternaria spp. DNA in 46 out of the 80 raw samples (57.5%) and in 66 out of the 110 processed samples (60%), enabling quantitative detection of Alternaria spp. DNA at levels as low as 1 CFU/g. The estimated Alternaria counts obtained by real-time PCR showed a good relationship (R(2) = 0.9006, P < 0.01) with the Alternaria counts obtained by plating on Potato Carrot Agar (PCA). The developed real-time PCR assay provides a useful tool for early detection of Alternaria spp. and could be applied as a quality and biosecurity marker of raw materials and final products in the fruits and vegetables processing industries.

The use of high-performance liquid chromatography to detect ochratoxin A in dried figs from the Spanish market

BACKGROUND Detection and quantification of ochratoxin A (OTA) in dried fig samples purchased in Spain has been carried out using high-performance liquid chromatography with fluorescence detection after extraction with methanol and sodium bicarbonate, and clean-up by using an immunoaffinity column. RESULTS The detection limit of the method was 0.06 ng g(-1), and the limit of quantification 0.18 ng g(-1) . OTA was detected in 31 (88.6%) out of 35 samples of dried figs analysed, with concentrations that ranged from < 0.1 to 277 ng g(-1). However, only three samples contained OTA concentrations above the tolerable level set by European Commission regulations for dried vine fruits (10 ng g(-1)). CONCLUSION The results of this survey show the value of monitoring OTA in dried figs especially if they are home grown.

Diferenciación de grupos de especies del género Alternaria mediante la reacción en cadena de la Polimerasa (PCR)

A polymerase chain reaction (PCR) method, based on oligonucleotide primers targeting the Alt a 1 gene, has been developed for the specific identification of several species-groups within the genus Alternaria. The introduction of a third oligonucleotide in the multiplex PCR allows amplification of a common DNA fragment in all Alternaria species, besides the specific fragment of each group.

Autentificación de carnes procedentes de aves de caza y de la avicultura alternativa por PCR-RFLP

En Espana, el consumo de carne procedente de aves de caza ha aumentado de forma notable en los ultimos anos. Por otra parte, cada vez son mas numerosas las explotaciones dedicadas a la produccion intensiva de codorniz, faisan, perdiz y pintada en lo que se conoce como avicultura alternativa (MAPA, 2004). El aumento en el consumo de carne de aves procedentes tanto de la actividad cinegetica como de la avicultura alternativa, unido al incremento del comercio internacional de estos productos, justifica la necesidad de prestar una mayor atencion a la adecuada autentificacion de la carne procedente de estas especies, sobre todo en productos picados, deshuesados, escabechados, etc., donde desaparecen las caracteristicas morfologicas que facilitan su identificacion. La autentificacion es necesaria para evitar practicas fraudulentas derivadas de la sustitucion de las especies mas valoradas por aquellas de menor valor comercial y organoleptico (Partis et al., 2000). Asimismo, la identificacion de especies es importante para verificar el cumplimiento de las prohibiciones y vedas que establece la ley de caza. La caza furtiva, junto con el comercio de especies protegidas, ha contribuido a poner en peligro la supervivencia de un elevado numero de especies de aves (Teletchea et al., 2005).

Autentificación de ciervo (Cervus elaphus), gamo (Dama dama) y corzo (Capreolus capreolus) mediante una técnica de PCR en tiempo real

En Espana, el consumo de carne de caza mayor ha aumentado de forma notable en los ultimos anos debido, en parte, al auge de las explotaciones ganaderas dedicadas a la cria de especies cinegeticas. Las carnes y productos carnicos procedentes de especies de caza mayor son a menudo objeto de un etiquetado fraudulento, dadas las diferencias de precio que se registran entre las distintas especies que se comercializan (Matsunaga y col., 1998). Este hecho justifica la necesidad de disponer de tecnicas analiticas rapidas que permitan la adecuada identificacion de estos productos y la verificacion del cumplimiento de las normas de etiquetado (Partis y col., 2000).