Hipure soil dna extraction kit

DNA extraction, bacterial 16S rRNA gene amplification, and sequencing were performed as previously described (Shen et al., 2013 (link); Burns et al., 2016 (link)). Microbial DNA was extracted using the HiPure Soil DNA extraction Kit (Magen, Guangzhou, China). The PCR amplification conditions of the V3–V4 region of the 16s rRNA gene were 94°C for 2 min; 30 cycles of 98°C for 10 s, 62°C for 30 s and 68°C for 30 s; and 68°C for 5 min. The V3–V4 highly variable regions of the bacterial 16S rRNA genes were amplified by Illumina sequencing (Illumina NovaSeq 6000) using the following primers, namely, 341F, CCTACGGGNGGCWGCAG; and 806R, GGACTACHVGGGTATCTAAT. The PCR assay was performed in triplicate. The amplification system (a total of 50 μl) included 5 μl of 10 × KOD buffer, 5 μl of 2 mM dNTPs, 3 μl of 25 mM MgSO₄, 1.5 μl of upstream and downstream primers (10 μM), 1 μl of KOD polymerase, and 100 ng of template DNA. The amplified fragments were separated on a 2% agarose gel, extracted, and purified using AxyPrep DNA gel extraction kits (Axygen Biosciences, Union City, CA, USA) according to the instructions of the manufacturer, and the fragments were quantified using ABI StepOnePlus real-time PCR systems (Life Technologies, Foster City, USA). According to the standard operation, the purified fragments were sequenced by double-terminal sequencing (PE250) on the Illumina platform.

Intestinal microbiota total DNA was extracted by using the HiPure Soil DNA Extraction Kit (Magen, Guangzhou, China) according to the instructions, followed by a quality test with a UV spectrophotometer (Thermo, Waltham, MA, USA). Subsequently, the V3-V4 region of the 16SrRNA gene was amplified using a universal primer pair (341F/806R, CCTACGGGNGGCWGCAG/GGACTACHVGGGTATCTAAT) and a commercially available kit (New England Biolabs, MA, USA). The amplification conditions were set as follows: pre-denaturation at 95 °C for 5 min, followed by denaturation at 95 °C for 1 min, annealing at 60 °C for 1 min, extension at 72 °C for 1 min, and incubation at 72 °C for 7 min after performing 30 cycles. PCR amplification was performed using a 50 μL reaction system containing 10 μL of 5 × Q5@ Reaction Buffer, 10 μL 5 × Q5@ High GC Enhancer, 1.5 μL of 2.5 mM dNTPs, 1.5 μL of forward and reverse primers (10 μM), 0.2 μL of Q5@ High-Fidelity DNA Polymerase, and 50 ng of template DNA. Afterward, the amplification products are purified and quantified, and then a library is constructed before initiating the sequencing work (Illumina platform, Illumina, San Diego, CA, USA). Intestinal microbiome analysis was entrusted to Guangzhou Genedenovo Biotechnology Co., Ltd. (Guangzhou, China).

Tilapia hindgut samples were sent to Guangzhou Genedenovo Biotechnology Co., Ltd. (Guangzhou, China). Intestinal microbiota total DNA was extracted by using the HiPure Soil DNA Extraction Kit (Magen, Guangzhou China). After quality testing using an ultraviolet (UV)-spectrophotometer (Thermo Fisher Scientific, United States). Subsequently, the V₃-V₄ region of the bacterial 16S rRNA gene fragment was amplified using universal primers, and the procedure was referred to Liu et al. (43 (link)).