The quantitative result obtained with the qPCR was expressed in number of copies/5 μL and was back calculated taking into account the total specimen elute volume, the volume extracted, the DNA extract volume obtained, and find more volume of DNA amplified. Table 1 Primers for Quantitative PCR PCR Reference Primers Target gene Cycling conditions Concentration L. species Zariffard MR [28] F-LBF: 5′- ATGGAAGAACACCAGTGGCG-3′ 16 S r RNA 15 min 95 °C, (15 sec 95 °C, 45 sec 50 °C, 45 sec 72 °C) x37 150 nM R- LBR: 5′- CAGCACTGAGAGGCGGAAAC-3′ L. crispatus Byun R [29] LcrisF: 5′-AGCGAGCGGAACTAACAGATTTAC-3′ 16 S r RNA 15 min, 95 °C,

(15 sec 95 °C, 60 sec 60 °C, 20 sec 72 °C) x40 100 nM LcrisR : 5′-AGCTGATCATGCGATCTGCTT-3′ L. gasseri Tamrakar R [30] LgassF: 5′-AGCGAGCTTGCCTAGATGAATTTG-3′ 16 S r RNA 15 min 95 °C, (15 sec 95 °C, 60 sec 57 °C, 60 sec 65 °C) x40 200 nM LgassR: 5′-TCTTTTAAACTCTAGACATGCGTC-3′ L. iners De Backer E [31] InersFw:

5′-GTCTGCCTTGAAGATCGG-3′ 16 S r RNA 15 min 95 °C, (15 sec 95 °C, 55 sec 60 °C, 60 sec 65 °C) x35 200 nM InersRev: 5′-ACAGTTGATAGGCATCATC-3′ L. jensenii Tamrakar R [30] LjensF: 5′-AAGTCGAGCGAGCTTGCCTATAGA-3′ 16 S r RNA 15 min 95 °C, (15 sec 95 °C, 55 sec 60 °C, 60 sec 72 °C) x40 300 nM LjensR: 5′-CTTCTTTCATGCGAAAGTAGC-3′ L. vaginalis In-house designed primers LV16s_23s_F: 5′-GCCTAACCATTTGGAGGG-3′ 16 S-23 S r RNA 15 min 95 °C, (15 sec 95 °C, 30 sec 56 °C, 30 sec 72 °C)x37 GS-9973 200 nM LV16s_23s_R3: 5′-CGATGTGTAGGTTTCCG-3′ G. vaginalis Zariffard MR [28] C59 supplier F-GV1:

5′-TTACTGGTGTATCACTGTAAGG-3′ 16 S r RNA 15 min 95 °C, (45 sec 95 °C, 45 sec 55 °C, 45 sec 72 °C) x50 260 nM R-GV3: 5′-CCGTCACAGGCTGAACAGT-3′ A. vaginae De Backer E [31] ATOVAGRT3Fw: 5′-GGTGAAGCAGTGGAAACACT-3′ 16 S r RNA 15 min 95 °C, (20 sec 95 °C, 45 sec 60 °C, 45 sec 72 °C) x45 300 nM ATOVAGRT3Rev: 5′-ATTCGCTTCTGCTCGCGCA-3′ Prostate specific antigen The PSA testing was performed using the Seratec® PSA semiquant assay (Seratec Diagnostica, Göttingen, Germany). A volume of 500 μL of PSA buffer was added to the thawed swab and was shaken for 2 hours. After centrifugation of 300 μL for 1 min at 13000 g, 200 μL of https://www.selleckchem.com/HSP-90.html supernatant was used for testing, following the manufacturer’s instructions. Data analysis Baseline characteristics were described using means (ranges) and proportions. We analyzed changes in the profile of the Lactobacillus species in the healthy population by defining groups of women based on the consistent presence (present in samples in at least 4 out of 5 visits) or absence of each Lactobacillus species. We looked for any predictors of “consistently having a particular species” using logistic regression and predictors of the Lactobacillus counts in these women using linear mixed effects models. We compared the presence of individual microbiome species at the baseline visit between ‘healthy population (HP)’ women and ‘clinic population (CP)’ using logistic regression models.