NEHA December 2022 Journal of Environmental Health

10 Volume 85 • Number 5 A D VANC EME N T O F T H E SCIENCE 10 μl of Fast SYBR Green Master Mix (ThermoFisher 4309155) and 500 nmol/l of each primer. All reactions were performed in triplicate in MicroAmp optical 96-well plates with optical adhesive film. Cycling parameters included a 2-min start at 94 °C followed by 40 cycles of 15 s at 94 °C and 32 s at 60 °C. Cycle threshold for each run was determined by the instrument software. Standards were constructed for each plate using synthetic plasmids consisting of sequences corresponding to the selected markers (Supplemental Table 1, Standards were diluted from a range of 105 to 101 markers per reaction and used to construct calibration curves for quantification for each run. Troubleshooting to achieve amplification of the two SYBR Green assays was performed to reach specific and reliable amplification. These optimization steps included variations in melting temperature, magnesium chloride, and PCR additives such as bovine serum albumin. Quality Assurance and Controls For each round of sampling, one negative control filtration blank (i.e., sterile water known to contain no FIB or genetic markers) was processed following the same protocol as described above to detect contamination in the collection and processing steps. To ensure e•ective DNA extraction and detect inhibitors that could interfere with amplification, sample process controls consisting of salmon DNA were added into the extraction bu•er as previously described (Shanks et al., 2016). Inhibition was measured by comparing the amplification e˜ciency (i.e., cycle threshold) of the blanks compared with the samples. Internal amplification controls (Supplemental Table 1) were used to detect inhibition by comparing the cycle threshold of no-template List of Primers and Probes Used Assay Host Primer and Probe Group Reference TaqMan GenBac3 General Bacteroidetes F: GGGGTTCTGAGAGGAAGGT R: CCGTCATCCTTCACGCTACT P: [FAM]-CAATATTCCTCACTGCTGCCTCCCGTA-[TAMRA] Dick & Field, 2004; Siefring et al., 2008 HF183 Human F: ATCATGAGTTCACATGTCCG R: CTTCCTCTCAGAACCCCTATCC P1: [FAM]-CTAATGGAACGCATCCC-[MGB] P2: [VIC]-AACACGCCGTTGCTACA-[MGB] Bernhard & Field, 2000; Seurinck et al., 2005 HumM2 Human F: CGTCAGGTTTGTTTCGGTATTG R: TCATCACGTAACTTATTTATATGCATTAGC P1: [FAM]-TATCGAAAATCTCACGGATTAACTCTTGTGTACGC-[TAMRA] P2: [VIC]-CCTGCCGTCTCGTGCTCCTCA-[TAMRA] Shanks et al., 2009 Rum2Bac Ruminant F: ACAGCCCGCGATTGATACTGGTAA R: CAATCGGAGTTCTTCGTGAT P: [FAM]-ATGAGGTGGATGGAATTCGTGGTGT-[BHQ-1] Mieszkin et al., 2010 CowM2 Cattle F: CGGCCAAATACTCCTGATCGT R: GCTTGTTGCGTTCCTTGAGATAAT P: [FAM]-AGGCACCTATGTCCTTTACCTCATCAACTACAGACA-[TAMRA] Shanks et al., 2009 LA35 Poultry F: ACCGGATACGACCATCTGC R: TCCCCAGTGTCAGTCACAGC P: [FAM]-CAGCAGGGAAGAAGCCTTC GGGTGACGGTA-[BHQ-1] Nayak et al., 2015 DogBact Canine F: CGCTTGTATGTACCGGTACG R: CAATCGGAGTTCTTCGTG P: [6-FAM]-ATTCGTGGTGTAGC GGTGAAATGCTTAG-[BHQ-1] Schriewer et al., 2015 Sketa22 Quality assurance F: GGTTTCCGCAGCTGGG R: CCGAGCCGTCCTGGTCTA P: [FAM]-AGTCGCAGGCGGCCACCGT-[TAMRA] Haugland et al., 2005 SYBR Green GFD * General avian F: TCGGCTGAGCACTCTAGGG R: GCGTCTCTTTGTACATCCCA Green et al., 2012 GFC * Gull F: CCCTTGTCGTTAGTTGCCATCATTC R: GCCCTCGCGAGTTCGCTGC Green et al., 2012 * Assays were not successfully optimized. TABLE 1