Pokai Bay, Maunalua Bay, Kualoa Regional Park, West Loch Community Shoreline Park, Kahala Beach, and the beach parks of Ala Moana, Diamond Head, Maili, Waialae, Kaiaka Bay, Kahana Bay, Ko Olina, Bellows Field, and Punalu’u. Freshwater sites include Wahiawa Reservoir, Manoa Stream, and Kaelepulu Stream. The sample collected from Ala Wai Canal was brackish. All sampling locations receive, to varying Detection of Enterovirus from Environmental Water occurred. A 50-bp DNA ladder was used for indication of PCR product fragment size. The Molecular Imager Gel Doc XRsystem was used to visualize results under UV light. PCR conditions for all primer sets that successfully detected EnV from untreated wastewater were then adjusted for optimal sensitivity in preparation for environmental detection. Optimiza- tion brackets included annealing temperature, MgCl2 concentration, primer concentration, and the presence or absence of 0.1 mg/mL molecular biology grade, protease/nuclease-free, fraction V BSA . Using the final optimized conditions, primer set detection limits were determined by PCR using 10-fold serial dilutions of influent sewage cDNA template. Detection ” limits were denoted by the highest dilution yielding Primer Detection of Enterovirus from Environmental Water E. Coli Amplification as Internal Control It is well known that environmental water and shellfish samples contain high levels of inhibitory compounds that, if inefficiently removed during sampling processing, can negatively affect downstream molecular analysis. In order to assess nucleic acid extraction efficiency and inhibitor removal during sample processing, DNA extracted from “
17092993“all water and shellfish samples was tested for the presence of E. coli, which is known to grow naturally in the Hawaiian environment and is expected to be readily detectable in all samples. In each 25 mL reaction, 3 mL sample DNA were added to 22 mL PCR Crenolanib web mixture containing 1X Taq reaction buffer, 2.5 mM MgCl2 solution, 200 nM dNTP mixture, 0.1 mg/mL BSA, 400 nM of each primer , and 2 units of Taq polymerase. The amplification cycle consisted of an initial 5 min. denaturation at 94uC, followed by 35 cycles of 30 sec. denaturation at 94uC, 30 sec. annealing at 60uC, and 30 sec. extension at 72uC, completed by a final 5 min. extension at 72uC. Gel electrophoresis was performed as described above. Condition 1. Tanneal 2. 3. 4. BSA a Test Range 5060uCa, 2u increments 1.5, 2.0, 3.0, 4.0 mM 200, 400, 600, 800, 1000 nM Presence/Absence 40-50uC was included if reported Tanneal in literature ” was,50uC. Validation through Screening of Sewage and Environmental Waters The primer set exhibiting the highest sensitivity, EQ-1/EQ-2, was confirmed using cDNA obtained from the three sewage stages described earlier. This set was selected as the optimal candidate for surveillance of EnV presence in the environment; the twenty-two environmental water samples were then tested for EnV contamination using the newly-optimized PCR conditions. PCR Product Sequencing and Analysis In order to confirm true EnV detection and identify enteroviral strains present in 
the Hawaiian environment, selected positive DNA fragments amplified by primer set EQ-1/EQ-2 from sewage, water, and shellfish samples were subjected to DNA sequencing. DNA bands were excised from the 2% agarose gel and recovered using the QIAquick Gel Extraction kit, according to the manufacturer’s instructions. Recovered DNA samples from sewage and water were eluted using 30 mL EB