*2.6. DNA Extraction, PCR Detection, and ARGs Quantification*

The surface-sterilized edible pakchoi portions were cut into pieces and ground with liquid nitrogen before extraction under sterile conditions. Total DNA was extracted using PowerPlant DNA Isolation Kit (MoBio Laboratories, San Diego, CA, USA) following the manufacturer's instructions [37]. The concentrations and qualities of the extracted DNA samples were determined using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively.

PCR detection assays were used to screen for the presence or absence of 23 types of ARGs in the antibiotic-treated samples, including 12 tetracyclines-resistant genes (*tet*A, *tet*C, *tet*G, *tet*K, *tet*L, *tet*M, *tet*O, *tet*Q, *tet*T, *tet*W, *tet*B/P, and *tet*X), 5 sulfonamides-resistant genes (*sul*1, *sul*2, *sul*3, *dfr*A1, and *dfr*A7), and 6 β-lactams-resistant genes (*blaamp*C, *bla*VIM, *bla*CTX-M, *bla*TEM, *bla*SHV, and *bla*Z). PCR detection assays were performed as previously described [39]. Primers and annealing temperatures are described in Table S1.

The positive ARGs and eubacterial 16S rRNA gene were quantified by fluorescence quantitative PCR (qPCR) using a LightCycler real-time PCR system (Roche, Basel, Switzerland) with SYBR Green I. Details of the primers are listed in Table S2. Plasmids carrying target genes in the pMD19-T vector (TaKaRa, Ostu Shiga, Japan) were constructed to produce the standard curves [40], which consisted of at least five orders of magnitude (R2 > 0.99) (Table S3). The 20 μL reactions contained 10 μL of SYBR Premix Ex Taq (TaKaRa), 0.2 μM of each primer, 2 μL of template DNA, and 7.2 μL of ddH2O. The reaction program was set as follows: initial denaturation at 95 ◦C for 30 s, 40 cycles at 95 ◦C for 5 s, annealing temperature for 30 s and 72 ◦C for 30 s, then a melt curve stage with temperature ramping from 60 ◦C to 95 ◦C.
