17:15 〜 19:15
[AHW27-P09] Comparison of eDNA Extraction Methods from Autoclaved sul1 Bacterial Suspension: Concentration and Gene Abundance Analysis
キーワード：eDNA抽出、薬剤耐性遺伝子、DNA濃度、遺伝子存在量
Extracellular DNA (eDNA) refers to DNA found outside cells in water, soil, and sediment. eDNA exists in three forms: free DNA, cell-associated DNA, and adsorbed DNA. The spread of antibiotic resistance genes (ARGs) poses significant threats to human health and ecosystems. However, little is known about extracellular ARGs (eARGs) carried by eDNA. Therefore, this study aims to compare eDNA extraction methods for determining the behavior of eARGs in the environment.
A sul1 bacterial in the LB buffer solution (Alcaligenes aquatilis, OD600=0.1) was autoclaved (121°C, 30 min) to obtain eARGs. eDNA was extracted using three methods: 1) Precipitation: 10-mL solution was mixed with 200 µL precipitation buffer (Buffer PREC, Macherey-Nagel), and extracted with NucleoSpin eDNA Water Kit (Macherey-Nagel), 2) Centrifugation: 10-mL solution was centrifuged at 10,000 rpm for 10 minutes; the pellet was extracted with DNeasy PowerWater Kit (QIAGEN), and the supernatant was extracted using the precipitation method, 3) Filtration: 40-mL solution was filtered through a Glass Fiber Filter (45 mm, EO-treated, Macherey-Nagel), the filtered solid was extracted with either DNeasy PowerWater Kit or NucleoSpin eDNA Water Kit, and 10 mL of the filtrate was extracted using the precipitation method.
For all samples, eDNA recovery and sul1 detection were higher by the precipitation method (precipitation-only treatment: 288 ng/mL and 2.6×106 copies/mL, centrifuged supernatant: 253 ng/mL and 1.1×106 copies/mL, filtrate: 160-336 ng/mL and 0.3-1.2×106 copies/mL). When comparing centrifugation with the filtration method, the PowerWater Kit for centrifuged pellets showed a higher DNA concentration but lower sul1 abundance (54 ng/mL and 0.1×106 copies/mL) than the filtered solids (20 ng/mL and 0.2×106 copies/mL). These findings suggest that in autoclaved sul1 bacterial suspension, most of eDNA was present as free DNA, and the best method for eDNA extraction was the precipitation method.
A sul1 bacterial in the LB buffer solution (Alcaligenes aquatilis, OD600=0.1) was autoclaved (121°C, 30 min) to obtain eARGs. eDNA was extracted using three methods: 1) Precipitation: 10-mL solution was mixed with 200 µL precipitation buffer (Buffer PREC, Macherey-Nagel), and extracted with NucleoSpin eDNA Water Kit (Macherey-Nagel), 2) Centrifugation: 10-mL solution was centrifuged at 10,000 rpm for 10 minutes; the pellet was extracted with DNeasy PowerWater Kit (QIAGEN), and the supernatant was extracted using the precipitation method, 3) Filtration: 40-mL solution was filtered through a Glass Fiber Filter (45 mm, EO-treated, Macherey-Nagel), the filtered solid was extracted with either DNeasy PowerWater Kit or NucleoSpin eDNA Water Kit, and 10 mL of the filtrate was extracted using the precipitation method.
For all samples, eDNA recovery and sul1 detection were higher by the precipitation method (precipitation-only treatment: 288 ng/mL and 2.6×106 copies/mL, centrifuged supernatant: 253 ng/mL and 1.1×106 copies/mL, filtrate: 160-336 ng/mL and 0.3-1.2×106 copies/mL). When comparing centrifugation with the filtration method, the PowerWater Kit for centrifuged pellets showed a higher DNA concentration but lower sul1 abundance (54 ng/mL and 0.1×106 copies/mL) than the filtered solids (20 ng/mL and 0.2×106 copies/mL). These findings suggest that in autoclaved sul1 bacterial suspension, most of eDNA was present as free DNA, and the best method for eDNA extraction was the precipitation method.