Hinfi

Ligation and transformation of amplified 16S rRNA genes were performed as previously described [38 (link)], ultimately resulting in 16 bacterial clone libraries. For each clone library, 450 putative positive transformants were picked randomly and immersed in 30 μL of deionized H₂O, and subjected to three cycles of freezing and thawing for the preparation of plasmid templates. Cloned 16S rRNA genes were re-amplified using the primer pair T7 and SP6. PCR reactions were performed in a 20 μL mixture with 0.4 μM of each primer and 1 μL of template DNA using a Taq PCR Kit (Tiangen Biotech, China) with the same PCR conditions as amplification of community DNA, with the exception that only 30 cycles were performed. Restriction fragment length polymorphism (RFLP) analysis was used to distinguish and classify cloned 16S rRNA gene sequences. A total of 6753 positive PCR products were restricted using the enzymes HinfI and Csp6 (Fermentas, Vilnius, Lithuania) at 37°C for 3.5h. Restriction digests (10μL) were examined on 3.0% (w/v) agarose gels, and unique restriction patterns were identified visually. Representatives of each restriction pattern were chosen for sequencing using the vector primer pair T7 and SP6 by the Major Biotech Co., Ltd (Shanghai, China).

TLR4 A896G (Asp299Gly, rs4986790) and TLR4 C1196T (Thr399Ile, rs4986791) polymorphisms were performed according to Folwaczny et al. [29 (link)] with modifications. The polymerase chain reaction (PCR) was performed in 10 μL final volume with 10 ng of DNA, 1.0 ng of each primer, 3.33 mM of MgCl₂, 0.16 mM of dNTP (Invitrogen®, Frederick, MD, USA), 1X PCR buffer (5X Green GoTaq® Flexi Buffer, Promega, USA), and 0.75 U of Taq Polymerase (GoTaq® DNA Polymerase, Promega, USA). Cycles were performed in the Veriti thermal cycler (Applied Biosystems): one minute at 95°C; 35 cycles of 30 seconds at 95°C, 45 seconds at 60°C (to TLR4 A896G) or 62° (to TLR4 C1196T), and one minute at 72°C; at the end, 10 minutes at 72°C. PCR products were digested for 10 minutes at 37°C with NcoI (Fermentas, Canada) for TLR4 A896G and for 60 minutes at 37°C with HinfI (Fermentas, Canada) for TLR4 C1196T. The results were observed by electrophoresis on 3.5% agarose gel with SYBR Safe (Invitrogen Life Technologies, Grand Island, NY, USA) and visualized under UV light.

Briefly, 10 μg of genomic DNA was digested with RsaI and HinfI (Fermentas, Thermo Scientific) and loaded onto a 0.4% agarose gel61 . Electrophoresis was carried out in 1 × TBE at 1 V cm⁻¹ for 12 h at room temperature. The lane containing DNA was excised from the gel and the gel buffer was exchanged with 1 × TBE with 0.3 μg ml⁻¹ ethidium bromide (EB) (Sigma). The gel slice was placed and cast with 1% agarose gel in 1 × TBE containing 0.3 μg ml⁻¹ EB. The gel was run at 4 °C for 6 h at 3 V cm⁻¹ (refs 62 (link), 63 (link)). The hybridization was performed as above. For the ExoI experiment, genomic DNA was treated with 20 units of Exo I (NEB) for 2 h prior to 2D gel electrophoresis.

Total DNA was extracted by GUTC (Guanidinium-Tris-CDTA buffer with celite) method (Terefework et al., 2001 (link)) from purified bacteria. 16S rDNA and intergenic spacer region (IGS) of the strains were amplified for restriction fragment length polymorphism analysis. Primer pairs P1, P6 and pHr(F), p23SR01(R) (Table 1) were used for polymerase chain reaction (PCR) amplification. Amplification products (5 μl) were digested separately by four restriction enzymes HinfI, TaqI, MspI, and HaeIII following the manufacturer's instructions (Fermentas, EU). The fragments were separated by gel electrophoreses in 2% agarose with 0.5 μg ml⁻¹ ethidium bromide at 80 V for 3 h and photographed. Amplified ribosomal DNA restriction analysis (ARDRA) and IGS-RFLP were done by combining the results from the four restrictions. Clustered analysis of combined ARDRA and IGS-RFLP (CACAI) was conducted by UPGM clustering algorithm in the NTSYS program (Rohlf, 1990 ).

The ITS1, 5.8S, and ITS2 regions were amplified using the conserved primers ITS1 (5’-TCCGTAGTGGAACCTGCGG-3’) and ITS4 (5’ TCCTCCGCTTATTGATATGC-3’)^{33 (link)}. Each PCR mixture (30 μL) contained 1 μL of genomic DNA, 1 μL of 50 pmol of each primer, 12 μL of distilled water and 15 μL of Taq PCR Master Mix Kit (Qiagen). Reaction mixtures were preheated to 95 °C for 15 min, and then 30 PCR cycles were performed under the following conditions: 95 °C for 1 min; 56 °C for 1 min; and 72 °C for 1 min. The thermal cycles were finalized by polymerization at 72 °C for 10 min^{34 (link)}. Detection of PCR products was performed by electrophoresis in a 1% agarose gel stained with ethidium bromide and visualized by UV light. Ninety-nine PCR products of the analysed keratinophilic fungi and reference strains obtained using the ITS1 and ITS4 sets of primers were digested with the HinfI (Fermentas) restriction enzyme at 37 °C for 2 h, according to the manufacturer’s instructions. Digested fragments were separated by electrophoresis in an 8% polyacrylamide gel, stained with ethidium bromide and visualized by UV light. Additionally, traditional identification was confirmed by sequencing selected PCR products (ITS1-5.8S rDNA-ITS4) of A. keratinophilus strains. The results obtained were compared with GenBank data.

The genotyping of MTHFR C677T was performed with slight changes to the protocol of Reljic et al. [26 (link)].
The primers set used for MTHFR C677T genotyping were:
Forward: 5′- TGAAGGAGAAGGTGTCTGGGGGA-3′
Reverse: 5′- AGGACGGTGCGGTGAGAGTG-3′.
The PCR conditions were 30 s at 94 °C, 30 s at 61 °C and 1 min at 72 °C, for 30 cycles. The final extension was at 72 °C for 2 min. The amplified DNA fragment had 198 bp. The PCR product was digested by 1 U of HinfI (Fermentas, St. Leon-Rot, Germany) restriction endonuclease for 16 h. Digested fragments were electrophoresed through 3% agarose gels stained with GreenSafe Premium (NZYTech, Lisbon, Portugal). Homozygous wild type CC genotype was identified by the non-digested fragment of 198 bp; homozygous TT genotype was identified by two digested fragments of 175 bp and 23 bp; heterozygous CT genotype was defined by presence of all fragments 198 bp, 175 bp and 23 bp.

DNA was extracted from purified endophytic bacterial strains, following the procedure by Chen et al. (2018 (link)). Then, full length 16S rDNA was PCR amplified by the primer pairs 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGAC TT-3′) in a 30 μL PCR reaction mixture by the following procedure: one cycle at 95°C for 5 min, followed by 30 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 2 min, and a final extension at 72°C for 10 min. The PCR products (~1,500 bp) were checked in 1% agarose gel and purified for restriction fragment length polymorphism (RFLP) analysis. Three restriction enzymes (HaeIII, Hinf I, and TaqI) were used for the digestion of 16S rDNA, following the manufacturer's instructions (Fermentas, EU). Fragments from the digestion were separated by gel electrophoresis in 2% agarose at 80 V for 3 h and photographed. The 16S rDNA-RFLP analysis was carried out by combining the results of three restriction enzymes. Then, the cluster analysis of 16S rDNA-RFLP was conducted by the UPGM clustering algorithm in a NTSYS program (Chen et al., 2018 (link)).