Data From: Microbial Source Tracking For Antibiotic Resistance Genes In Southwest Wisconsin Private Wells

Groundwater was collected by dead-end ultrafiltration and small-volume grab sampling from 138 wells in southwest Wisconsin across Grant, Iowa, and Lafayette Counties. Samples were collected to assess occurrence of antibiotic resistance genes in private wells and investigate their association with microbial source tracking markers. For ultrafiltration samples, microbes were backflushed, desiccated beef extract was added to the eluate, and samples were concentrated by polyethylene glycol precipitation; concentrate was frozen at -80 degrees Celcius (C). Small-volume grab samples were concentrated on 0.45-micron mixed cellulose ester filters, filters were eluted, and eluate was frozen at -80 degrees C following addition of beef extract. Nucleic acids were extracted from both sample types using a QIAcube and QIAamp DNA mini kit with viral lysis buffer (AVL) and carrier RNA (Qiagen). Nucleic acids were extracted from 280 microliter (µL) of sample concentrate and eluted into 140 µL AE Buffer (Qiagen). Nucleic acids were analyzed in duplicate using quantitative polymerase chain reaction (qPCR) on a Roche LightCycler 480 II using hydrolysis probes. Inhibition was assessed for every sample using Sketa DNA as inhibition control and mitigated by dilution with AE buffer as necessary. No-template negative controls were performed for all analysis steps: secondary concentration, nucleic acid extraction, and qPCR. For each assay with amplification in negative controls, the cycle of quantification (Cq) in unknown samples must be below the censoring threshold to be accepted as positive. Censoring thresholds were calculated as the mean Cq of negative controls - 3 standard deviations; censoring thresholds for each assay and sample type are reported in a separate file (Censoring thresholds.csv). Positive controls (bovine herpes virus vaccine) for extraction were included with each analysis batch and evaluated qualitatively. Positive controls were run in duplicate reactions for all targets and had to be within 0.5 cycles of the expected Cq. Data are expressed as genomic copies per liter of groundwater sampled. Dataset consists of 1 spreadsheet file (qPCR data results.csv). Variables in this file are described in the included data dictionary.
Organization: Department of Agriculture
Last updated: 2025-09-02T23:43:06.777618
Tags: antibiotic-resistance-genes, dead-end-ultrafiltration, groundwater, microbial-source-tracking, private-wells, small-volume-grab-sampling