Tiannamp dna kit

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.

The DNA was extracted using rapid DNA extraction kit (TIANNAMP DNA Kit, TIANGEN, China) according to the manufacturer’s instructions. A total of 20 ARGs (tetA, tetB, tetC, tetG, tetM, tetO, tetQ, tetW, tetX, tetZ, sul1, sul2, gyrA, qnrS, ermB, ermC, ermF, ermT, mefA, mphA) and two MGEs (intI1, intI2) were quantified by PCR using StepOnePlus™ real-time PCR system (Thermo, United States). The primer sequences used for the amplification of ARGs are presented in Supplementary Table S2. The thermal cycling steps for quantitative PCR (qPCR) amplification were employed as described previously (Yin et al., 2017 (link)), and the absolute abundance of ARGs and MGEs was defined as the copy number of ARG or MGE per unit of the composting sample (copies/g).

Swine wastewater (30 mL) was collected from each container on storage days 0, 60, 120, and 180 (SWW₀, SWW₆₀, SWW₁₂₀, and SWW₁₈₀). The TIANNAMP DNA Kit (TIANGEN, Beijing, China) was used to extract DNA. Before DNA extraction, the sample was centrifugated at 300× g for 5 min to precipitate large particles. The supernatant was then removed and centrifuged at 19,000× g for 5 min to precipitate cells. Moreover, the pellet was washed twice with 3.0 mL of sterile distilled water to reduce inhibitors for Taq polymerase. The quantity and quality of DNA were evaluated via 1% gel, NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and Qubit 3.0 (Thermo Fisher Scientific, Waltham, MA, USA), 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 primer pairs 515F and 806R, and 528F and 706R [3 (link)]. The rRNA gene of fungi was amplified using the ITS1-F, ITS2-2043R, and ITS5-1737F primers [23 (link)]. 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 Wang et al. [3 (link)] and Shi et al. (2022) [23 (link)].