Application of magnetic polymethacrylate-based microspheres for the isolation of DNA from raw vegetables and processed foods of plant origin

Abstract

A method for the microextraction of DNA from raw vegetables and highly processed foods of plant origin suitable for PCR analysis was developed. It is based on non-selective binding of DNA in the presence of PEG 6,000/NaCl to hydrophilic magnetic nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) -P(HEMA-co-GMA) microspheres covered by carboxyl groups.The quantitative polymerase chain reaction (qPCR) by in-house designed primers targeting a highly repetitive rDNA locus allowed for detection of plant DNAs in a wide range of concentrations (0.1 pg/lL to 10 ng/lL). The described procedure is fast and simple. We further demonstrate that relatively mild acidic treatment of vegetable at elevated temperatures resulted in a dramatic reduction of PCR efficiency indicating extensive degradation of DNA during pickling. The described method is suitable for the analysis of highly degraded DNA from pickled food products. Practical applicationsDNA-based methods, such as the polymerase chain reaction (PCR), represent an important genetic approach for identification of plant species composition of foods. DNA from processed foods varies in both quality and quantity between the protocols used. Plant samples are generally characterised by a complex composition containing various inhibitors of PCR. Current methods have been tailored for specific samples while no universal protocol is available. Development of a simple universal procedure leading to PCR-ready DNA would be beneficial. Isolation strategies based on solid phase systems have become popular for PCR-ready DNA isolations from complex matrices.Here we applied magnetic hydrophilic P(HEMA-co-GMA) microspheres to extract DNA from raw vegetables and highly processed foods of plant origin. Proposed small scale PCR-ready DNA extraction protocol was comparable with a silica-based DNeasy Plant Mini Kit (Qiagen) and classical CTAB extraction methods in quality and amplificability of DNA while it is less costly and time consuming.

Destabilized microbial networks with distinct performances of abundant and rare biospheres in maintaining networks under increasing salinity stress

“…Then, the mycobiome of 18 freshwater (salinity less than 2.86 with a mean value of 1.66) and 18 seawater (salinity > 14 with a mean value of 18.05) sites were further investigated to verify the microbial ecological patterns observed through the bacteriome. The total genomic DNA of each water sample was extracted from the filter using the cetyltrimethylammonium bromide method [53, 54]. The primer pairs 515F/806R and ITS1‐1F‐F/ITS1‐1F‐R were used to amplify the V4 region of the 16S ribosomal RNA gene for bacteria and the ITS1‐1F region of the internal transcribed spacer (ITS) gene for fungi, respectively.…”

Effects of monobutyrin supplementation on egg production, biochemical indexes, and gut microbiota of broiler breeders

“…Total genome DNA from cecal digesta was extracted using the cetyltrimethylammonium bromide method ( Trojanek et al., 2017 ). Extracted DNA was monitored on 1% agarose gels before being diluted to 1 ng/μL to prepare amplicons for high-throughput sequencing.…”