A new and straightforward protocol for microplastic's DNA extraction from freshwater environments

Microplastics (< 5 mm particles) are ubiquitous environmental contaminants, capable of providing an artificial substrate for the development microbial aggregates distinguished between water column, seston, and sediments distinct in taxonomic composition from those on natural surfaces such as sediment. These complex communities, constituted by prokaryotic eukaryotic microorganisms, represent a peculiar micro-ecosystem, identified only recently "plastisphere". The plastisphere associated with microplastics surface waters is poorly known. Therefore, there need thorough investigation into possible role transport vector biological contaminants hygienic-sanitary interest. Several studies have described molecular techniques used to extract nucleic acids aquatic environments matrices. However, little known about DNA extraction methods plastic. This study's main objective develop protocol microplastic's extraction, improving genetic material’s quantity quality necessary future applications gene amplification sequencing. was extracted microplastic samples freshwater ecosystem. Two different were analyzed: a) particles collected Italian river (Ofanto river) composed prevalently black transparent fragments 5-1mm size, b) virgin purchased local industry controls PE pellets 5mm green 100µm. also performed superficial water, suspended organic matter (OM), sediment taking care process three replicates each type. Starting commercial DNeasy PowerSoil Kit, method optimized testing minimum amount, incubation parameters, time cell lysis, final elution volumes. We material forceps glass containers OM, placing 250mg replicate 2-mL microcentrifuge tubes. separated L 500 mL aliquots filtered one Whatman 0.2-μm filters. filters cut sterilized placed tubes (Debeljak et al., 2017). modifications made extractive been alternative lysis including preliminary at 60 °C 20 minutes reduction volume 70 µl order concentrate DNA. obtained respectively through use Qubit™ dsDNA HS Assay Kit (ThermoFisher), agarose gel electrophoresis. Our results showed that amount useful obtain valid genomic 120 mg. modifies applied kit resulted significantly more than eight times. applying improved ranged 7.97 14.1 ng -1 mean value 10.5 ± 3.2 respect medium 1 0.1 same using unmodified kit. As expected, did not show measurable 0.2 ). Concentrations aqueous 19.53 6.47 , while attached OM revealed concentration 35.1 2.19ng above detection limit 100 respectively.