PCR Detection and Sequencing

A 25-μL reaction volume containing the following components was used for all the PCRs: 12.5 μL of PCR Master Mix reagent (Tiangen Biotech Co., Ltd., Beijing, China), 9.5 μL of double-distilled water, 0.5 μL of 10 μM NDM-F primer (5′-ATGGAATTGCCCAATATTAT-3′) and NDM-R primer (5′-TCAGCGCAGCTTGTCGGCCA-3′), and 2 μL of DNA template. The PCR cycling conditions were 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s. The final extension step was 72°C for 10 min. PCR products were electrophoretically separated on 1.5% agarose gels and stained with ethidium bromide. Images were documented on the Gel Doc EQ imaging system (Bio-Rad). PCR products were sequenced at Sangon Biotech. The resultant sequences were entered into DNAStar software (DNASTAR Inc., Madison, WI, United States), and sequence alignments were performed by the ClustalW method.