Nano drop nd 1000uvevis spectrophotometer

Genomic DNA was extracted from L. monocytogenes using Bacterial Genomic DNA Purification Kit (Dongsheng Biotech. Inc., Guangzhou, China) according to the manufacturer’s instruction. DNA concentration was determined at O.D. 260 nm using Nano Drop^®ND-1000UVeVis Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Serogroup analysis of 177 isolates was performed using a multiplex PCR as previously described by Doumith et al. (2004) (link),13 serotypes of L. monocytogenes were categorized into five distinct phylogenetic groups, viz. I.1 (1/2a-3a), I.2 (1/2c-3c), II.1 (4b-4d-4e), II.2 (1/2b-3b-7), and III (4a-4c). The PCR mixture (50-μL) contained 1.5 unit GoTaq^®Hotstart polymerase (Promega, Madison, WI, USA), 1 μM for lmo0737, ORF2819, and ORF2110; 1.5 μM for lmo1118; and 0.2 μM for prs, 2.5 mM MgCl2, 0.2 mM each dNTP, and 40 ng of template genomic DNA. PCR was performed with the following thermal cycle: initial denaturation step at 94°C for 3 min; 35 cycles of 94°C for 35 s, 53°C for 50 s, and 72°C for 60 s; and a final cycle of 72°C for 7 min in a thermocycler (Applied Biosystems, Foster City, CA, USA). Five microliters of the reaction mixture was mixed with 5 μL of loading buffer and separated on a 2% agarose gel in TAE buffer. The PCR product was visualized by Goldview^®staining (0.005%, v/v). The primers are shown in Supplementary Table S1.

Total DNA was extracted from 0.25 g soil in each subsample using a MOBIO PowerSoil DNA Isolation Kit (Carlsbad, CA, USA) according to the manufacturers’ protocol with modifications (Fierer et al., 2012). Briefly, six successive replicate extractions were taken from each subsample and fixed together as one DNA template to provide enough total DNA (Zhou et al., 2016). DNA purification followed, and then, DNA concentration and quality (A₂₆₀/A₂₈₀) of the extracts were estimated visually using a NanoDrop ND‐1000 UVevis spectrophotometer (Thermo Scientific, Rockwood, TN, USA).

All isolates that exhibited a positive phenotype in ESBL screening tests were evaluated by PCR for the presence of genes encoding TEM, SHV, OXA, and CTX-M as previously described, with minor modifications. Genomic DNA was extracted from ESBL-producing isolates using a Bacterial Genomic DNA Purification Kit (Dongsheng Biotech, Guangzhou, China) according to the manufacturer's instruction. Genomic DNA concentration was determined at 260 nm using a Nano Drop^®;ND-1000UVeVis Spectrophotometer (Thermo Fisher Scientific, MA, USA). The primer sequences and their positions, PCR conditions, and references are summarized in Table S1.