Biomedical subjects

Virginia García-Cañas

Publications and source records attributed to Virginia García-Cañas.

7 recordsLinked to original sources

PubMed2004

Simultaneous and sensitive detection of three foodborne pathogens by multiplex PCR, capillary gel electrophoresis, and laser-induced fluorescence.

The simultaneous detection of Staphylococcus aureus, Listeria monocytogenes, and Salmonella spp. has been approached by a new multiplex PCR-based procedure followed by capillary gel electrophoresis with laser-induced fluorescence detection (multiplex-PCR-CGE-LIF). As compared to slab gel electrophoresis, the use of CGE-LIF improved from 10- to 1000-fold the sensitivity of the multiplex PCR analysis, allowing the detection of 2.6 x 10(3) cfu mL(-1) of S. aureus, 570 cfu mL(-1) of L. monocytogenes, and 790 cfu mL(-1) of Salmonella in artificially inoculated food, without enrichment. Following 6 h of enrichment, as low as 260, 79, and 57 cfu mL(-1) of S. aureus, L. monocytogenes, and Salmonella, respectively, were detected. The CGE-LIF method is shown to be reproducible, providing relative standard deviation (RSD) values lower than 0.8% for analysis time and lower than 5.8% for peak areas. The multiplex-PCR-CGE-LIF proved a powerful analytical tool to detect various food pathogens simultaneously in a fast, reproducible, and sensitive way.

Electrophoresis, Capillary↗

PubMed2004

Detection and differentiation of several food-spoilage lactic acid bacteria by multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescence.

In this work, a complete analytical procedure is investigated to differentiate several food-spoilage lactic acid bacteria. To do that, a method involving multiplex Polymerase Chain Reaction (PCR), capillary gel electrophoresis (CGE), and laser-induced fluorescence (LIF) is developed. The PCR-CGE-LIF protocol allows the simultaneous detection and differentiation of the genera Leuconostoc and Carnobacterium, the nonmotile group of species within the genus Carnobacterium, and the three species of the group individually (C. divergens, C. gallinarum, and C. maltaromicum). The capability of this approach is clearly illustrated through the sensitive and efficient analysis of the two closest amplicons, with sizes equal to 397 and 412 bp, showing very different yields in all of the amplification reactions tested. These two fragments, which could not be resolved by agarose gel electrophoresis (AGE), are clearly distinguishable by CGE-LIF even when very different areas for both peaks are obtained. The PCR-CGE-LIF method also allows the sensitive detection of these bacteria, demonstrating both a significant resolution improvement compared with traditional AGE and the usefulness of this approach to solve real-life analytical challenges. Good reproducibility of the CGE-LIF procedure is shown for the analysis of multiplex PCR samples with percent relative standard deviation values for migration times and corrected peak areas as low as 0.80 and 6.50 for the same sample and three different days (n = 12), respectively.

Bacteria↗

PubMed2004

Quantitation of transgenic Bt event-176 maize using double quantitative competitive polymerase chain reaction and capillary gel electrophoresis laser-induced fluorescence.

In this work, a new procedure useful to quantitatively analyze genetically modified organisms (GMOs) in foods is described and applied to analyze transgenic Bt Event-176 maize. The method developed consists of coamplifications of specific DNA maize sequences with internal standards using quantitative competitive PCR (QC-PCR). The QC-PCR products are quantitatively analyzed using a capillary gel electrophoresis (CGE) with laser-induced fluorescence detection (LIF) method developed at our laboratory that utilizes a physically adsorbed coating. The CGE-LIF procedure allows the use of internal standards differing by only 10 bp from the original target fragments, to our knowledge, the smallest size difference that can be found in the bibliography for QC-PCR of GMOs. A spectrofluorometric procedure using ROX reference dye is proposed to solve calibration problems of input DNA concentration. It is demonstrated that the use of ROX drastically enhances the accuracy of the quantitative analysis by QC-PCR. Reproducibility of analysis times and corrected peak areas (measured as target/competitor PCR products ratio) for the CGE-LIF separations are determined to be better than 0.91 and 1.93% (RSD, n = 15) respectively, for three different days. It is shown that CGE-LIF provides better resolution and a signal/noise ratio improvement of approximately 700-fold compared to slab gel electrophoresis. The good possibilities in terms of quantitative analysis of GMOs provided by this new method are confirmed by determining the Bt Event-176 maize content in certified reference maize powder and food samples of known composition. This procedure opens the possibility for accurate quantitation of multiple GMOs in a single run.

Bacterial Proteins↗

PubMed2004

Sensitive and simultaneous analysis of five transgenic maizes using multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescence.

The benefits of using multiplex polymerase chain reaction (PCR) followed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) for the simultaneous detection of five transgenic maizes (Bt11, T25, MON810, GA21, and Bt176) are demonstrated. The method uses a hexaplex PCR protocol to amplify the five mentioned transgenic amplicons plus the zein gene used as reference, followed by a CGE-LIF method to analyze the six DNA fragments. CGE-LIF was demonstrated very useful and informative for optimizing multiplex PCR parameters such as time extension, PCR buffer concentration and primers concentration. The method developed is highly sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 0.054% of Bt11, 0.057% of T25, 0.036% of MON810, 0.064% of GA21, and 0.018% of Bt176 in flour obtaining signals still far from the detection limit (namely, the signal/noise ratios for the corresponding DNA peaks were 41, 124, 98, 250, 252, and 473, respectively). These percentages are well below the minimum threshold marked by the European Regulation for transgenic food labeling (i.e., 0.5-0.9%). A study on the reproducibility of the multiplex PCR-CGE-LIF procedure was also performed. Thus, values of RSD lower than 0.67 and 6.80% were obtained for migration times and corrected peak areas, respectively, for the same sample and three different days (n = 12). On the other hand, the reproducibility of the whole procedure, including four different multiplex PCR amplifications, was determined to be better than 0.66 and 23.3% for migration times and corrected peak areas, respectively. Agarose gel electrophoresis (AGE) and CGE-LIF were compared in terms of resolution and sensitivity for detecting PCR products, demonstrating that CGE-LIF can solve false positives induced by artifacts from the multiplex PCR reaction that could not be addressed by AGE. Moreover, CGE-LIF provides better resolution and sensitivity. To our knowledge, these results demonstrate for the first time that multiplex PCR-CGE-LIF is a solid alternative to determine multiple genetically modified organisms in maize flours in a single run.

DNA Primers↗

PubMed2004

Detection of genetically modified organisms in foods by DNA amplification techniques.

In this article, the different DNA amplification techniques that are being used for detecting genetically modified organisms (GMOs) in foods are examined. This study intends to provide an updated overview (including works published till June 2002) on the principal applications of such techniques together with their main advantages and drawbacks in GMO detection in foods. Some relevant facts on sampling, DNA isolation, and DNA amplification methods are discussed. Moreover; these analytical protocols are discuissed from a quantitative point of view, including the newest investigations on multiplex detection of GMOs in foods and validation of methods.

DNA, Plant↗

PubMed2002

Ultrasensitive detection of genetically modified maize DNA by capillary gel electrophoresis with laser-induced fluorescence using different fluorescent intercalating dyes.

In this work, four different fluorescent intercalating dyes are compared for the ultrasensitive CGE-LIF detection of DNA from transgenic maize in flours. The fluorescent intercalating dyes compared are YOPRO-1, SYBR-Green-I, Ethidium bromide (EthBr), and EnhanCE. For all the four dyes optimum concentrations are established, and efficient separations of DNA fragments ranging in size from 80 to 1000 bp are obtained. The comparative study demonstrates that SYBR-Green-I and YOPRO-1 provide better limits of detection (LODs) than EnhanCE or EthBr (i.e., LODs are, respectively, 700, 1000, 11300, and 97400 zmol, calculated for a 200-bp DNA fragment). Separations using YOPRO-1 are faster than those using SYBR-Green-I (30 min vs 47 min for the analysis of the 80-1000 bp DNA fragments). Also, separations using YOPRO-1 are more efficient than those using SYBR-Green-I (e.g., 2.4 x 10(6) plates/m vs 1.6 x 10(6) plates/m, respectively, calculated for the 200-bp fragment). Also, buffer depletion and cost per analysis are worse with SYBR-Green-I than with YOPRO-1. Therefore, YOPRO-1 was selected as the preferred intercalating dye. Using this fluorescent compound, analysis time reproducibility for the CGE-LIF separation of the DNA fragments is determined to be better than 1.7% (% RSD, n = 10) within the same day, and better than 1.9% (% RSD, n = 30) for three different days. Moreover, the fluorescence signal obtained using this dye is shown to vary linearly with the DNA concentration in the range studied, i.e., 1-500 ng/microL. It is demonstrated that by using this method 0.01% of transgenic maize can be detected in flour by direct injection of the PCR-amplified sample.

Bacillus thuringiensis Toxins↗

PubMed2002

Detection of genetically modified maize by the polymerase chain reaction and capillary gel electrophoresis with UV detection and laser-induced fluorescence.

In this paper, the possibilities of capillary gel electrophoresis (CGE) to detect transgenic maize in flours are shown. The method is based on the extraction and amplification by the polymerase chain reaction (PCR) of a specific DNA fragment from transgenic maize and its subsequent analysis by CGE with UV detection or laser-induced fluorescence (LIF). Some useful considerations regarding the optimization of DNA extraction and amplification conditions are given. Also, a comparison is established between the two CGE protocols for DNA detection based on ultraviolet absorption (CGE-UV) and LIF (CGE-LIF). The requirements, advantages, and limitations of both CGE methods are discussed. To our knowledge, this is the first paper on the use of CGE-LIF to detect transgenic food.

DNA Primers↗