Rox reference dye

Swine wastewater (2 mL) collected from each container on storage days 0, 60, 120, and 180 was extracted using the TIANNAMP DNA Kit (TIANGEN, Beijing, China), according to the abovementioned procedure, and then submitted for quantitative PCR and quantification of ARGs.The wastewater sample volume was in accordance with the standard qPCR procedure at the analysis lab. The 16S rRNA gene and the ITS gene were quantified using a StepOnePlus™ RealTime 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 [24 (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 qPCR reaction was conducted in triplicate. Quantification of ARGs were conducted based on the methods of Pu et al. (2018) [7 (link)]. As only sulfonamides were used in the swine farm, only the sulfonamide-resistant genes Sul1, Sul2, Sul3, and SulA were assessed in this study.

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.