Determinazione del tasso di recupero del DNA dagli alimenti: Uso di un frammento lineare di DNA esogeno

Recovery rates of methods for isolation of DNA from food were determined using exogenous linear DNA fragments. Linear DNA fragments with an incorporated marker sequence were prepared by cloning a fragment of the fimC geneof Salmonella typhimurium in a plasmid, its linearization using the restrictase HihdIII (to produce a linear DNA fragment of 3.156 bp), and by its subsequent shortening using the restrictase EcoRI (to produce a linear DNA fragment of 470 bp). Three defined amounts of linear DNA fragments were added to soya flour and meat pâte, DNA was isolated using three types of methods - cetyltrimethylammonium bromide solubilization with a subsequent liquid-phase extraction (CTAB-LPE), chaotropic solid-phase extraction (SPE), and non-chaotropic SPE - and the recoveries were determined by real-time PCR. Relatively low recovery rates were determined for all methods, addition levels and matrices, ranging from 5.9 to 51.0% for the linear DNA fragment of 3.156 bp and ranging from 1.6 to 44.5% for the linear DNA fragment of 470 bp. Despite the considerable variability in the results of individual experiments, it could be seen that recovery rates of currently used methods for DNA isolation from food are certainly lower than usually believed.