Processing of raw plant materials causes occurrence of degraded DNA in foods. The effect of DNA degradation on amplification and quantification of transgenic and non-transgenic DNA in raw and experimentally thermally processed foods was studied. The degree of DNA degradation was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). Cetyl trimethyl ammonium bromide method yielded DNA of a better quality, while Genespin and Wizard were less appropriate. Baking at 220°C considerably reduced the size of DNA fragments. In order to measure the length of amplifiable DNA, primers for soybean and maize genes were used. Small DNA fragments ranging from 100 to 200 bp were amplified in all samples. DNA fragments over 1 kbp were amplified only if heating at 220°C lasted less than 30 min. Baking of flour (220°C) reduced the size of extracted DNA fragments so that 1,100 bp amplicon was no longer amplifiable, while the amplicons of 913 and 1,100 bp were obtained from the baked bread. When PCR assays targeting maize high mobility group and zein genes were used under the same conditions, analogous results were achieved. Quantification of genetically modified organism content was not influenced by baking.
Bergerová E., Godálová Z., Siekel P. (2011): Combined effects of temperature, pressure and low pH on the amplification of DNA of plant derived foods. Czech J. Food Sci., 29: 337-345.The effect of food processing on the DNA integrity was studied by means of PCR amplification of soybean, transgenic MON 810 and non-transgenic maize, bean, and pea. The degree of DNA degradation was checked by PCR and visualised by agarose gel electrophoresis. The conditions of technological treatment such as temperature, pH, pressure, and their combination may negatively influence the integrity of DNA in processed foods and hence PCR detection of food components. The DNA over 300 bp was amplifiable when mild processing parameters up to 100°C were performed at approximately neutral or low acidic pH. The autoclaving (12°C; 0.1 MPa) significantly reduced the size of amplifiable DNA in the time dependant manner and that was intensified by acidic pH. The maximum amplicons length achieved for highly processed matrices was 300 bp. The major impact on the DNA integrity was exerted by the combination of pressure, temperature, and low pH.
Aims: The aim of this study was to develop a 5′‐nuclease polymerase chain reaction (PCR) for the rapid detection and quantification of Listeria monocytogenes.
Methods and Results: Specific primers and a fluorogenic probe were designed, which target a specific sequence of the actA gene encoding for a protein involved in the actin filament assembly. The PCR system was highly sensitive and specific for L. monocytogenes (inclusivity, 100%; exclusivity, 100%), which was determined using 46 L. monocytogenes and 28 non‐L. monocytogenes strains. Detection limits of 104 cfu ml−1 after 35 cycles and 102 cfu ml−1 after 45 cycles were achieved by PCR in both real‐time and end‐point fluorescence measurement modes. Linear calibration lines were obtained in the range from 102 to 109 cfu ml−1 for three L. monocytogenes strains in real‐time PCR with 45 cycles.
Conclusions: The developed 5′‐nuclease PCR of the actA gene provides a new target for the rapid detection and quantification of L. monocytogenes.
Significance and Impact of the Study: In conjunction with enrichment or with an appropriate quantitative sample preparation technique, the method is suitable for food safety applications.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.