qPCR sample processing, DNA extraction and calibrator standards

Details of sample processing and the DNA extraction procedure have been previously described [1, 2]. Fifty milliliters of water samples, collected from Edgewater and Fairhope Beach and 100 mL from Goddard Beach were filtered through a 0.4 μm pore size (47 mm in diameter) polycarbonate membrane filter (GE Osmonics, Minnetonka, MA), and the sides of the funnels were rinsed twice with 20 ml of sterile, PCR-grade water. Using sterile forceps, each filter was folded into a cylinder with the sample side facing inward, and then inserted into a 2 ml semiconical screwcap microcentrifuge tube (extraction tube) (catalog #506-636, PGC Scientific, Gaithersburg, MD) containing 0.3 g of acid-washed glass beads (catalog #G-1277, Sigma, St. Louis, MO). The filters were held at -20◦Cuntil overnight shipment to EMSL Analytical (Westmount NJ) on dry ice where DNA extraction and qPCR analysis for Enterococcus, Bacteroidales and fecal Bacteroides were conducted as described below. Frozen DNA extracts were sent from EMSL Analytical to the US EPA in Cincinnati where qPCR analyses were conducted for Clostridium spp. DNA was recovered from the organisms retained on the filters by addition of 600 μl of AE buffer (Qiagen, Valencia, CA) containing 0.2 μg/ml salmon testes DNA (#D-1626, Sigma, St. Louis, MO), added as an exogenous, internal positive control and reference, to each extraction tube and bead milling in an eight position mini bead beater (Biospec Corp., Bartlesville, OK) for 60 s at maximum rate. The tubes were then centrifuged at 12,000 x g for 1 min to pellet the glass beads and debris. Resulting supernatants were transferred to sterile 1.6 ml low retention microcentrifuge tubes (GENE MATE, #C-3228-1) and, if not analyzed immediately, stored at -20 ◦C. Negative controls consisted of two filtrates of 40 ml PCR-grade water, prepared at the same time as the sample filtrates, and six blank filters prepared in the PCR analytical laboratory, that were extracted in the same manner with each batch of samples arriving weekly at the laboratory. Calibrator samples (six replicates), consisting of clean polycarbonate filters amended with known cell quantities of Enterococcus faecalis (ATCC# 29212), Bacteroides thetaiotaomicron (ATCC# 29741), and Clostridium perfringens (ATCC# 13124)and negative control samples (six replicates), consisting of clean filters only, were extracted in the same manner with each batch of test samples. Cells used in the calibrator samples originated from laboratory grown cultures and were enumerated as previously described [2, 3, 1]. Following DNA extraction, polymerase chain reaction (PCR) amplification was carried out on 5-fold dilutions of the DNA extracts in AE buffer using the TaqMan PCR product detection system. The reactions were performed in a thermal cycling instrument (Smart-Cycler System, Cepheid, Sunnyvale, CA ) except for Clostridium spp. which was performed on a Model 7900 DNA thermal cycler (Applied Biosystems, Foster City, CA). Both instruments automated the detection and quantitative measurement of the fluorescent signals