Allprep bacterial dna kit

The vaginal lavage was diluted tenfold serially with sterile saline and then plated on Columbia blood agar (CBA, Sigma-Aldrich, St. Louis, MO, USA) containing GV-selective supplement (Oxoid, Basingstoke, UK) and 10% horse blood. After incubating the plate under anaerobic conditions at 37 °C for 48 h, the colonies were counted to measure the number of viable GV, and the results were presented as CFU per 1 mL of vaginal fluid. The abundance of GV in the vagina was measured by quantitative polymerase chain reaction (qPCR) analysis of the ratio of GV-specific DNA to the total bacterial DNA in the vaginal fluid (Happel et al., 2020 (link); Naicker et al., 2021 ). After determining the number of viable GV, total DNA was isolated (AllPrep Bacterial DNA Kit, Qiagen, Germantown, MD, USA) from the remaining vaginal lavage and qPCR was performed using the QuantStudio 6 Real-Time PCR System (Applied Biosystems, Foster, CA, USA) with GV-specific primer Ba04646236_s1 (Applied Biosystems) and Pan-bacterial primer Ba04230899_s1 (Applied Biosystems).

We extracted total DNA from Enterobacter E_clo_Niagara and from the putative E. coli transconjugants that had acquired ß-lactam resistance using the AllPrep Bacterial DNA Kit from Qiagen (Hilden, Germany and Waltham, MA) according to the manufacturer's instructions. Purified DNA was sent to ID Genomics, Seattle, WA, for PCR detection of the extended spectrum ß-lactamase genes. PCR assays using broad-range primers initially identified the ß-lactamase as a member of CTX-M family, and then specific primers indicated the ß-lactamase was a CTX-M27. This “head start” from ID Genomics allowed us to then conduct our own PCR assays.