Tiannamp soil dna kit

According to the manufacturer’s instructions, 0.5 g of soil material was weighed to extract total DNA from the soil using a TIANnamp Soil DNA Kit (TIANGEN, China). The highly variable V3–V4 region of the bacterial 16S rRNA gene was amplified using primers 338 F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806 R (5′-GGACTACHVGGGTWTCTAAT-3′; Dennis et al., 2013 (link)). Each sample was subjected to the following amplification process: initial denaturation at 95°C for 3 min, followed by 27 rounds of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, and a final extension at 72°C for 10 min (Yu et al., 2018 (link)). The PCR products were purified, and an Illumina MiSeq PE300 was used to conduct high-throughput paired-end sequencing (Allwegene Co., Ltd., Beijing, China).

Total DNA was extracted from 64 soil samples using the TIANNAMP Soil DNA Kit (DP336, TIANGEN, China) according to the manufacturer’s instructions. The concentration and purity of DNA were checked using ultraviolet absorbance (Quawell Q3000, NanoDrop, America).
The 16S rRNA gene and ARGs were quantified using a StepOnePlus^™ real-time fluorescent quantitative PCR (Thermo, United States) (Zhu et al., 2017 (link); Zhao et al., 2018 (link)). The ARGs were quantified using a StepOnePlus^™ real time PCR system (Wcgene Biotech, Inc., Shanghai, China). The primer sequences in the PCR reaction system and PCR amplification plots of total 28 tetracyclines, β-lactams, and sulfonamides ARGs are shown in Supplementary Table 2 and Supplementary Figure 1. The reaction mixture consisted of 5.0 μl of TB Green Premix Ex Taq II (Tli RNaseH Plus) (2×), 0.4 μl of each primer, 0.2 μl of ROX Reference Dye (50×), 1.0 μl of template DNA, and 3.0 μl of dd H₂O. The temperature program was as follows: 30 s at 95°C, followed by 40 cycles of 5 s at 95°C, 30 s at the annealing temperature, and 72°C for 30 s. The specificity of Q-PCR products was also checked by melting curve analysis and the relative abundance of ARGs was calculated by the 2^−ΔΔCt method, where △Ct = Ct (target gene)—Ct (internal reference gene).

Five grams of soil from each bottle/pot were sampled for analysis. A TIANNAMP Soil DNA Kit (TIANGEN, Beijing, China) was used to extract soil DNA. The quantity and quality of DNA were evaluated, and the DNA was then stored at −80 °C before use. Sample integrity was tested by agarose gel electrophoresis. The 16S rRNA gene of bacteria (in the V3–V4 region) was amplified using the primers 515F and 806R, and 528F and 706R. The rRNA gene of fungi was amplified using the ITS1-F, ITS2-2043R, and ITS5-1737F primers. The sequencing of bacteria and fungi was performed using the Illumina HiSeq 2500 platform (BGI Co., Ltd., Shenzhen, China). Sequence analyses were conducted according to the methods of Yan et al. (2022) [16 (link)].
The 16 S rRNA gene and the ITS gene were quantified using a StepOnePlus™ Real-Time PCR System (Thermo Fisher, Waltham, MA, USA) and TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) kit (Takara, Kyoto Japan). The qPCR conditions, primer sequences targeting these genes, and PCR protocol were based on previously reported methods [17 (link)]. Each reaction contained 0.8 µL of primers, 1 µL of DNA templates, 5 µL of TB Green Premix Ex Taq II (Takara, Japan), 0.2 µL of ROX Reference Dye (Bio-Rad), and 3 µL of ddH₂O (Bio-Rad). Each PCR reaction was conducted in triplicate.