title: Preoperative Vaginal Microbiome as a Predictor of Postoperative Urinary Tract Infection Following Pelvic Organ Prolapse Surgery
design: The microbial 16S rRNA gene was amplified through a polymerase chain reaction (PCR) in the following working solution: 10L of 2x Taqman Universal Master  Mix II (AmpliTaq Gold DNA polymerase, deoxynucleotide triphosphates, ROXTM Passive Reference dye, optimized buffer components; ThermoFisher Scientific, Grand Island, NY), 2L (9M) forward primer, 2L (9M) reverse primer, 2L (2.5M) probe, 2L sample DNA, and 2uL molecular grade water up to a final volume of 50L per reaction. V3_357F and V5_926R primers modified with Nextera adaptors were developed in collaboration with the University of Minnesota Genomic Center in Minneapolis, MN. The RT-PCR standard universal thermal cycling protocol was performed using the  QuantStudioTM 6 Flex (ThermoFisher Scientific) as follows: activation of DNA polymerase at 50C for 2 minutes then increased to 95C for 10 minutes followed by 40 cycles of denaturation at 95C for 15 seconds followed by annealing/extension phase at 60C for 1 minute. Data collection occurred during the annealing/extension phase. Samples were plated in 384 well plates and covered with optical adhesive. Plates centrifuged at 2000 RPM for 2 minutes to remove bubble from bottom of wells. All samples were run in triplicate with positive and negative controls.