Biometra tone 96g thermal cycler

Seven enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, and cytK-2) and one emetic toxin-producing gene (cesB) were detected by PCR using the primers listed in Supplementary Table S2 (Hansen and Hendriksen, 2001 (link); Ehling-Schulz et al., 2005 (link); Oltuszak-Walczak and Walczak, 2013 (link)). Genomic DNA was extracted using a HiPure Bacterial DNA extraction kit (Magen, Guangzhou, China) under the instruction of the manufacturer. Concentration and purity of the DNA were measured by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, United States). The amplification reactions were performed using ExTaq Mix kit (Takara, China) in a Biometra TOne 96G thermal cycler (Analytik Jena, Jena, Germany). The PCR reaction mixture (25 μL) contained 50 ng of genomic DNA, 1.0 μM of each primer and 12.5 μL ExTaq Mix. Amplification was performed according to the instruction of the manufacturer (ExTaq Mix, Takara, China).

All semen samples were cultured on tryptone soy agar (Oxoid, UK) with 5% sheep blood and MacConkey agar (Oxoid, UK) incubated at 37 °C for 18-24 h. All different colonies were identified using standard biochemical tests followed by 16S rRNA sequencing and stored in Brain Heart Infusion (BHI) (Oxoid, UK) with 20% glycerol at -80 °C. Genomic DNA of all isolates was performed using G-spin^TM genomic DNA extraction kit (iNtRON, Republic of Korea) and amplified 16S rRNA by PCR with a BiometraTOne96G thermal cycler (AnalytikJena, Germany) using UFUL (5’- GCCTAACACATGCAAGTCGA-3’) and 800R (5’-TACCAGGGTATCTAATCC-3’) primers. The PCR was performed with the following protocol: initial denaturation at 94 °C for 3 min followed by 30 cycles of denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 72 °C for 45 sec, with a final extension step at 72 °C for 5 min. The PCR products were purified by MEGAquick-spin^TM Plus Total Fragment DNA purification kit (iNtRON, Republic of Korea) and sequenced with an Applied Biosystems 3730XL DNA Analyzer (Bionics, Republic of Korea). Each 16S rRNA sequences was blasted against the NCBI nucleotide database (https://blast.ncbi.nlm.nih.gov) to identify all isolates.