Application of an Alkaline and Silica Membrane DNA Extraction Method to Detect Mitochondrial DNA in Foods

Abstract

To improve the efficiency of mitochondrial DNA (mtDNA) extraction from food, a simple method, PrepM, was developed by effectively combining alkaline sodium dodecyl sulfate lysis and DNA binding to a silica membrane. PrepM was evaluated using fresh, cold, and autoclaved rat liver samples and compared with commercial total DNA extraction kits. When PrepM was applied to fresh and cold samples, the DNA yield was low, but the OD260/280 ratio was close to 1.8. The cytochrome C oxidase subunit I (COI) copy number, which is the number of mtDNA copies, per DNA weight and the ratio of COI to β-actin, which is the ratio of mitochondrial to genomic DNA, were higher with PrepM than with total DNA extraction kits. When PrepM was applied to autoclaved samples, the DNA purity and yield and the COI copy number per DNA weight were relatively high, but the COI to β-actin ratio was low. PrepM was then applied to shrimp samples, a known allergenic food. Data obtained from cold and autoclaved shrimp samples were similar to data obtained from rat liver samples. When PrepM was applied to food samples spiked with shrimp, the DNA purity was high, but the DNA yield and the mtDNA levels were almost same as those obtained with total DNA extraction kits. We concluded that PrepM has a comparative advantage for sensitive polymerase chain reaction detection of mtDNA sequences in relatively fresh samples, and it has almost the same performance as general total DNA extraction kits with autoclaved and/or complex mixture samples.