Sequence detection software v 1

Quantitative real-time PCR was performed with the ABI PRISM 7300 detection system (Applied Biosystems, Foster City, CA, USA) using SYBRGreen dye. Relative RNA abundance was calculated using the ΔΔCT formula and normalized to the transcript levels of the housekeeping gene β-actin with the help of the Sequence Detection Software v.1.4 (Applied Biosystems). Assays were performed in duplicate. TaqMan primers for IL23 and IL17 were purchased from Life Technologies (Mulgrave, Victoria, Australia). Primer sequences used for SybrGreen RT-qPCR were as follows: β-actin/for, 5′-AGCCAGGTCCAGACGCAGG-3′; β-actin/rev, 5′-ACCCACACTGTGCCCATCTAC-3′; β-catenin/for, 5′-GCTGACCTGATGGAGTTGGA-3′; β-catenin/rev, 5′-GCTACTTGCTCTTGCGTGAA-3′; CD44/for, 5′-GTCTGCATCGCGGTCAATAG-3′; CD44/rev, 5′-GGTCTCTGATGGTTCCTTGTTC-3′; Cmyc/for, 5′-TAGTGCTGCATGAGGAGACA-3′; Cmyc/rev, 5′-GGTTTGCCTCTTCTCCACAG-3′; Cox2/for, 5′-ACACACTCTATCACTGGCACC-3′; Cox2/rev, 5′-TTCAGGGAGAAGCGTTTGC-3′; Cryptdin1/for, 5′-AAGAGACTAAAACTGAGGAGCAGC-3′; Cryptdin1/rev, 5′-CGACAGCAGAGCGTGTA-3′; CyclinD1/for, 5′-GCACAACGCACTTTCTTTCCA-3′; CyclinD1/rev, 5′-CGCAGGCTTGACTCCAGAAG-3′; ΔN-Bcat/for, 5′-GGATTACAAAGACGATGATGACAAGTTG-3′; ΔN-Bcat/rev, 5′-GTCAGCTCAGGAATTGCACGTG-3′; MMP7/for, 5′-GAGATGTGAGCGCACATCAGTG-3′; and MMP7/rev, 5′-GATGTAGGGGGAGAGTTTTCCAGT-3′.

The quantitative PCR of M. oxyfera bacteria 16S rRNA gene were performed on LightCycler480 with Sequence Detection Software v1.4 (Applied Biosystems, Life Technologies Corporation, USA). The abundance of 16S rRNA gene was determined using the primers qp1R-qp1F (Ettwig et al., 2009 (link)) with 10 µL of Power SYBR Green PCR Master Mix, 1 µL of template DNA (5–20 ng µL⁻¹), 0.4 µL of each primer and 8.2 µL of ddH₂O. Detailed information is exhibited in Table S1. Negative-control reactions in which the DNA template was replaced by nuclease-free water were also performed. The whole process was performed under sterile conditions on ice and away from light. Triplicate qPCR analyses were performed for each sample. The standard curve was constructed from purified plasmid DNA with the concentrations ranging from 1.0 × 10¹ to 1.0 × 10⁷ copies µL⁻¹, and it showed correlation between the DNA template concentration and the crossing point with coefficients of determination (R² > 0.97). The qPCR amplification efficiency of the standard curve and reactions were both greater than 85%.

The oligonucleotides used in real-time PCR assays are listed in Table 1. The vanA gene assay has previously been described by Fang et al. [20 (link)] and the enterococcus-specific 23S rRNA gene by Ludwig and Schleiferl. [21 (link)]. Primers and probes for vanB detection were designed by importing a partial vanB consensus sequence into the software Primer Express 3 (Applied Biosystem, Foster City, CA, USA). Primer and probe sequences were subjected to In Silico analysis using the custom-designed plugin Assay Validation to CLC Main Workbench (version 8.0) providing a detailed evaluation of primer and probe binding to all available sequences on Genbank^®.
The vanA and vanB reactions were run as duplex real-time PCR and 23S as a separate mono-plex real-time PCR. The 25 µL total reactions contained 12.5 µL of Taqman FAST Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 1000 nM of each primer, 200 nM of the probes, and 5 µL of template DNA. The real-time PCR reactions were carried out as single determinations using 7500 FAST real-time PCR System (Applied Biosystems). The cycling parameters were as follows: 95 °C for 20 s, and 45 cycles of 95 °C for 3 sec, and 60 °C for 30 s. The results were analyzed using Sequence Detection Software v.1.4 (Applied Biosystems) and samples were regarded as positive if Ct-values were less than 40 with exponential real-time curves.