Premix taq polymerase

Gardnerella vaginalis clades were detected by the amplification of Gv1-fucosidase S and Gv1-fucosidase-AS primers, Gv2-hyp-S and Gv2-hyp-AS primers, Gv3-thi-S and Gv3-thi-AS primers and Gv4-cic-S and Gv4-cic-AS primers (Balashov et al., 2014 (link)). Bacterial genomic DNA was extracted from cultures with a Wizard Genomic DNA Purification Kit (Promega, Madison, United States) according to the manufacturer’s instructions. The reactions were performed in a final volume of 25 μl, containing 1.0 μl of each primer, 2.0 μl of DNA template and 12.5 μl of Premix Taq polymerase (Takara). The reaction mixture was subjected to 38 cycles of denaturation at 95°C for 30 s, primer annealing at 60°C for 30 s and extension at 72°C for 30 s. The last cycle included a 7-min extension step. PCR products were separated on 3.0% agarose gels stained with safe stain (Greenview Plus, Andy GoldTM, United States).

The genomic DNA was extracted with an AxyPrep DNAGel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer’s instructions, and quantified using the NanoDrop 2000 UV–Vis spectrophotometer (Thermo Scientific, Wilmington, USA). The endophytic and epiphytic fungal 18ITS1 gene was amplified using primers ITS1F-5′-CTTGGTCATTTAGAGGAAGTAA-3′ and ITS2R-5′-GCTGCGTTCTTCATCGATGC-3′ (Gardes and Bruns, 1993 (link)). The amplicons were purified, quantified, and sequenced using the Illumina Miseq PE250 platform. Primers 338F-5′-ACTCCTACGGGAGGCAGCA-3′ and 806R-5′-GGACTACHVGGGTWTCTAAT-3′ (Mori et al., 2014 (link)) were used to amplify the V3-V4 region of the epiphytic bacterial 16s rRNA gene. Primers 779F-5′- AACMGGATTAGATACCCKG -3′ and 1392R-5′- ACGGGCGGTGTGTRC -3′ were used to amplify the endophytic bacterial gene. The amplicons were purified and sequencing using Illumina Miseq PE 300. The 25 μl PCR system contained 12.5 μl Premix Taq Polymerase (Takara, China), 0.5 μl of each primer, 10 ng genomic DNA. PCR was carried out under the following conditions: 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 45 s, and a final extension step at 72°C for 10 min. Sequencing was performed by Shanghai Majorbio Biopharm Technology Co., Ltd (China).

Template DNA was extracted using an EZ-10 spin column bacterial genomic DNA miniprep kit (Bio Basic Inc., Markham, ON, Canada). Genes encoding AMEs, including aac(6')-Ie-aph(2''), aph(2'')-Ib, aph(2'')-Id, aph(3')-IIIa, ant(3'')-I, ant(4')-Ia, and ant(6')-Ia, along with the virulence genes ace, asa1, cylA, efaA, esp, gelE, and hyl, were examined by PCR. Detection was performed in a final volume of 20 μL, containing 400 nM of each primer (primer sequences are provided in Table 1), 10 µL of premix Taq polymerase (Takara, Otsu, Japan) containing MgCl₂, dNTPs, and reaction buffer, 1 µL of DNA template, and dd H₂O to 20 µL. The PCR conditions consisted of a pre-denaturation step at 94 °C for 4 min, followed by 35 cycles of 94 °C for 40 s, 55 °C for 40 s, and 72 °C for 45 s. A final extension step was performed at 72 °C for 5 min.
The amplified PCR products were analyzed on 1% (w/v) agarose gels. DNA bands were visualized by staining with ethidium bromide and photographed under UV illumination.