This strategy was then applied to the samples of the healthy women and as a result DNA extraction methods differed between the two groups of women. An aliquot of 250 μL eluate of the specimens collected from the healthy population was processed

using the easyMag (BioMérieux, Marcy l’Etoile, France) BX-795 research buy after an initial lysing step with mutanolysin (Sigma Aldrich, Bornem, Belgium) and proteinase K (PK)(Qiagen, Venlo, the Netherlands). Briefly, the aliquot was centrifuged for 10 min at 12500 rpm, and 250 μL mutanolysin/PK buffer was added to the pellet. After vortexing 2.5 μL mutanolysin (25U/μL) was added and buy Dinaciclib incubated for 15 min at 37 °C. Thereafter, a volume of 12.5 μL PK (25 mg/mL) was added and incubated for 15 min at 55 °C including vortexing every 5 minutes. Finally, 1750 μL of Nuclisens Easymag buffer was added prior to the extraction, following the manufacturer’s instructions. For the specimens collected from the clinic population, an aliquot of 500 μL was processed according

to the Boom extraction using the miniMAG system (BioMérieux, Marcy l’Etoile, France) and according to the manufacturer’s instructions. Quantitative PCR PF299 datasheet Quantitative PCR for total Lactobacillus species, L. crispatus, L. iners, L. jensenii, L. gasseri, L. vaginalis, G. vaginalis, and A. vaginae were performed with the primers as described in Table 1. The primers were synthesized by Eurogentec, Seraing, Belgium. The 25 μL PCR mixture contained QuantiTect SYBR Green PCR (Qiagen, Venlo, the Netherlands) with the exception of the PCR mixture for L. vaginalis which contained Thermo Scientific Absolute SYBR Green Mix (ABgene, Epsom, UK), 5 μL DNA mafosfamide extract,

primers, and Milli-Q water. The amplification reactions were performed using the Corbett Life Science Rotor-Gene™ 6000 (Qiagen, Venlo, the Netherlands) and the amplification programs as described in Table 1. Each sample was run in duplicate. For each of the organisms standard curves were constructed and included in each run. A total of 6 standards were prepared by a tenfold dilution and within a range of 102 copies/5 μL to 107 copies/5 μL. Reference strains (L. crispatus (LMG 9479T), L. jensenii (LMG 6414T), L.iners (LMG 18914 T), L. gasseri (LMG 9203T), L. vaginalis (LMGT 12891), G. vaginalis (LMG 7832 T), A. vaginae (CCUG 38953)) were cultured on Columbia agar (Beckton Dickinson, Le pont de Claix, France) supplemented with 5% Defibrinated Horse Blood (E&O laboratories Ltd, Burnhouse, Bonnybridge, Scotland) and incubated in an anaerobic atmosphere (Anaerocult A, Merck Chemicals, Darmstadt, Germany) for 24 hours at 35°C. A suspension was made in 400 μL molecular biology water and DNA was extracted as described above. The DNA concentration was determined by using the Nanodrop ND-1000 (Nanodrop Technnologies, Wilmington, USA).