Propidium Monoazide Integrated with qPCR Enables the Detection and Enumeration of Infectious Enteric RNA and DNA Viruses in Clam and Fermented Sausages

The increase of foodborne viral outbreaks highlights the need for a rapid and sensitive method for the prediction of viral infectivity in food samples. This study assesses the use of propidium monoazide (PMA) coupled with real-time PCR methods (RT-qPCR or qPCR for RNA or DNA viruses, respectively) in the determination of viral infectivity in complex animal-related food matrices. Clam and Spanish fermented sausage (“chorizo”) samples were spiked with infectious and heat-inactivated human adenovirus-2 (HAdV-2) and mengovirus (vMC0). PMA-qPCR/RT-qPCR discriminated infective virus particles, with significant reductions (>2.7 log10 or 99.7%). Additionally, infectious HAdV-2 and vMC0 were quantified by plaque assay (in plaque forming units, PFU), and compared with those in virus genomes copies (GCs) quantified by PMA-qPCR/RT-qPCR. A consistent correlation (R2 > 0.92) was showed between PFU and GCs along serial 10-fold dilutions in both DNA and RNA virus and in both food matrices. This study shows the use of PMA coupled to qPCR/RT-qPCR as a promising alternative for prediction of viral infectivity in food samples in comparison to more expensive and time-consuming methods and for those viruses that are not able to grow under available cell culture techniques.ThisstudywasfinanciallysupportedbytheRTA2014-00024- C04-01 fromtheSpanishMinistryofEconomyandInnovation and theBrazilianCNPqProjectnumber472804/2013-8,andby CAPES/PNPD andCAPES/PDSE

Molecular detection of fungi in paprika, chili powder and black pepper

Paprika powder, chili powder, and black pepper are among the most frequently used spices in the world. The Internal Transcribed Spacer (ITS) regions were identified by high sequence similarity with the ITS regions of many microscopic fungi, especially representatives of the phylum Ascomycota, from 18 different spice samples that were examined. However, supplied quality certificates indicated that 10 of the 18 samples had no contamination present and were safe for human consumption. The various genera of fungi that were identified from the spices are considered to be a food safety concern as they are able to produce mycotoxins. Qualitative detection was supplemented by positive detection of viable fungal DNA using qPCR for the genera Aspergillus/Penicillium in two paprika powder and black pepper samples. These results concurred with the control analysis using axenic cultures. The described methods can be used for routine testing of spices to provide safe spices and other products to consumers.O

Estudio de la producción de exopolisacáridos por bacterias lácticas y su aplicación en el desarrollo de alimentos funcionales

Trabajo presentado en la 4º Reunión de la Red Temática (Participación de las bacterias lácticas en la salud humana y en la calidad alimentaria), celebrada en Granada (España) del 12 al 14 de noviembre de 2009

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

Evaluation of yoghurt as a carrier of ß-D-glucan producing lactic acid and bacteria

Trabajo presentado en el 3rd Congress of European Microbiologists (FEMS 2009), celebrado en Gotemburgo (Suecia), del 28 de junio al 2 de julio de 200

Development of Simple Multiplex Real-Time PCR Assays for Foodborne Pathogens Detection and Identification On Lightcycler

Most acute intestinal diseases are caused by food-borne pathogens. A fast and simple real-time PCR-based procedure for simultaneous detection of food contamination by any of the five food-borne pathogens: Campylobacter jejuni, Mycobacterium bovis, Enterobacter sakazaki, Shigella boydii, Clostridium perfrigens using multiplex EvaGreen real-time PCR for LightCycler was developed and evaluated. Real-time qPCR showed excellent sensitivity. Tm calling and Melting Curve Genotyping (MCG) were used for analysis of PCR product melting curves. The Melting Curve Genotyping option showed good performance for discrimination of positive samples containing DNA of single pathogen or pathogen mixtures from negative samples

Potential microbial risk factors related to soil amendments and irrigation water of potato crops

8 pages, 2 tables, 3 figures.[Aims]: This study assesses the potential microbial risk factors related to the use of soil amendments and irrigation water on potato crops, cultivated in one traditional and two intensive farms during two harvest seasons.[Methods and Results]: The natural microbiota and potentially pathogenic micro-organisms were evaluated in the soil amendment, irrigation water, soil and produce. Uncomposted amendments and residual and creek water samples showed the highest microbial counts. The microbial load of potatoes harvested in spring was similar among the tested farms despite the diverse microbial levels of Listeria spp. and faecal coliforms in the potential risk sources. However, differences in total coliform load of potato were found between farms cultivated in the autumn. Immunochromatographic rapid tests and the BAM's reference method (Bacteriological Analytical Manual; AOAC International) were used to detect Escherichia coli O157:H7 from the potential risk sources and produce. Confirmation of the positive results by polymerase chain reaction procedures showed that the immunochromatographic assay was not reliable as it led to false-positive results.[Conclusions]: The potentially pathogenic micro-organisms of soil amendment, irrigation water and soil samples changed with the harvest seasons and the use of different agricultural practices. However, the microbial load of the produce was not always influenced by these risk sources. Improvements in environmental sample preparation are needed to avoid interferences in the use of immunochromatographic rapid tests.[Significance and Impact of the Study]: The potential microbial risk sources of fresh produce should be regularly controlled using reliable detection methods to guarantee their microbial safety.The authors are grateful to CICYT (project AGL2004-03060) and to European Commission IRRIQUAL (FP6-2004-FOOD-3B) for financial support. A. Allende is indebted to the MEC (Juan de la Cierva contract, JCI-2004-000718) and M.V. Selma to CSIC (I3P contract, I3PDR-7-01).Peer reviewe

A real-time PCR assay for detection and quantification of 2-branched (1,3)-β-D–glucan producing lactic acid bacteria in cider

28 p.-1 fig.-4 tab.Ropiness in natural cider is a relatively frequent alteration, mainly found after bottling, leading to consumer rejection. It is derived from the production of exopolysaccharides (EPS) by some lactic acid bacteria most of which synthesize a 2-branched (1,3)-β-D-glucan and belong to the genera Pediococcus, Lactobacillus and Oenococcus. This polysaccharide synthesis is controlled by a single transmembrane glycosyltransferase (GTF). In this work, a method based on quantitative PCR (qPCR) and targeting the gtf gene was developed for detection and quantification of these bacteria in cider. The newly designed primers GTF3/GTF4 delimit a 151 bp fragment within the 417 bp amplicon previously designed for conventional PCR. The inclusivity and exclusivity of the qPCR assay were assessed with 33 cider isolates belonging to genus Lactobacillus, Oenoccocus and Pedioccocus, together with reference strains of 16 species and five genera including β-glucan,α-glucan and heteropolysaccharide (HePS) producing strains and non-EPS producers. The qPCR assay,followed by the melting curve analysis, confirmed the generation of a single PCR product from the β-glucan producers with a Tm of 74.28± 0.08 and CT values (10 ng DNA) ranging between 8.46 and 16.88 (average 12.67± 3.5). Some EPS− LAB strains rendered CT values ranging from 28.04 to 37.75 but they were significantly higher (P(CTb28.54)= 0.05) than those of the β-glucan producers. The assay showed a wide quantification range of 5 log units using calibrated cell suspensions of Pediococcus parvulus 2.6 and Oenococcus oeni I4. The linearity was extended over 7 log orders when calibration curves were obtained from DNA. The detection limit for β-glucan producing LAB in artificially contaminated cider was about 3× 102 CFU per ml. The newly developed qPCR assay was successfully applied to monitor the cidermaking process, in 13 tanks from two cider factories, revealing a decrease in CT values derived from an increase in β-glucan producing LAB populations. In addition, 8 naturally spoiled bottled cider were tested for the quantification of these organisms using the five standard curves constructed: P. parvulus 2.6 genomic DNA and gtf amplicon (417 bp), calibrated cell suspensions of Pediococcus parvulus 2.6, Lactobacillus diolivorans G77 and Oenococcus oeni I4 and results were compared to LAB total counts on MRS. Levels obtained from the different approaches were within a log range and showed no significant differences. Therefore, the amplicon-derived standard curve is proposed for the routine estimation of gtf+populations in cider.This work was supported by the Ministerio de Educación y Ciencia (projects AGL2006-11932 and CSD2007-00063), Universidad del País Vasco (UPV/EHU) (EHU08/37) and the Diputación Foral de Gipuzkoa and the Generalitat Valenciana (GVACOMP2009-257). Programa Red Gipuzkoana de Ciencia, Tecnología e Innovación (co-financed by the European Union).Peer reviewe

Evaluation of yogurt and various beverages as carriers of lactic acid bacteria producing 2-branched (1,3)-β-D-glucan

8 páginas, 2 figuras, 2 tablas -- PAGS nros. 3271-3278Probiotic cultures are increasingly being incorporated into a wide variety of food products. Although lactobacilli and bifidobacteria are the most frequently used, other lactic acid bacteria (LAB) have been reported to be potential probiotics. Of these, the cider isolates Pediococccus parvulus (strains 2.6 and CUPV22) and Lactobacillus suebicus CUPV221 produce a 2-branched (1,3)-β-d-glucan exopolysaccharide that decreases serum cholesterol levels and affects the activation of human macrophages. For this reason, these 3 strains were incorporated into yogurt, orange juice, and 2 juice-milk beverages to evaluate the effect of the food matrix on the resistance of these strains to simulated gastrointestinal tract conditions. Our results showed that incorporation of the LAB did not significantly affect the physical and rheological properties of the food matrices tested. When incorporated in yogurt, LAB strains population decreased by 2 to 3 log orders of magnitude during the shelf life of the product (28 d). However, no significant decrease was observed in the juice and juice-milk beverages during the same storage period, except for Lb. suebicus, whose viability decreased by 3 log orders of magnitude. When strains were subjected to gastrointestinal tract conditions, a decrease in the survival was observed at the lower pH (1.8). However, incorporation of these LAB strains into orange juice increases their resistance to lower pH conditions, thus improving survival to gastrointestinal stress. Moreover, a protective effect was observed for P. parvulus CUPV22 and 2.6 to gastric stress in juice-milk beverages and to gastrointestinal stress in yogurt. Lactobacillus suebicus CUPV221 did not survive when incorporated into yogurt and juice-milk beverageThis study was supported by grants AGL2006-11932-C05-03/ALI, AGL2009-12998-C03-03 and CSD2007-00063 from the Spanish Ministry of Science and Innovation, and ACOMP/2009/257 from the Generalitat Valenciana. G. Sánchez is the recipient of a JAE doctor grant from the “Consejo Superior de Investigaciones Científicas” (CSIC). We thank Jordi Cerveró and Soledad Benlloch (Department of Microbiology and Ecology, University of Valencia, Spain) for technical support. We also thank Stephen Elson for the critical reading of the manuscriptPeer reviewe