Realplex ep

Melt Curve analysis was performed using Eppendorf RealPlex EP (Eppendorf, Hauppauge, New York, NY, USA) with temperature ramped from 55 to 99 °C in one-degree increments.

All qPCR reactions, except total bacteria qPCR, were run using Eppendorf Realplex EP (Eppendorf, Hauppauge, New York, NY, USA). The total bacteria qPCR was described in previous work [17 (link)]. All primers and probes used in this study were listed in Table 1. The qPCR master mixture and qPCR condition for all real-time PCR assays were followed according to Tsai [18 ] and Wang [19 ]. The minimum detection limit of all three 16s rRNA qPCR assay was 5000 copies per reaction. For Paracoccus spp., Pseudomonas spp.-like, and Thauera spp. quantification, the average efficiency of qPCR runs was 0.99, 0.98, and 0.98, respectively, the mean coefficient of variation (R²) was 0.998, 0.994, and 0.991, respectively, and the average slope was −3.31, −3.37, and −3.47, respectively. For real time quantification of narG-like and napA genes using SYBR Green, the average efficiency was 0.99. The mean R² was 0.998 and 0.996 with a slope of −3.34 and −3.40 for narG-like and napA, respectively. The minimum detection limit of the functional gene assays was 1000 copies per reaction. Melting curves were also conducted after the completion of the PCR cycle sequence. In all qPCR experiments, negative controls containing no template DNA were subjected to the same procedure. For PCR assays, the same temperature profile and master mixture without probe addition were used for each primer set.