Pcr master mix reagent

Each 25-μL PCR mixture contained the following components: 12.5 μL PCR master mix reagent (Tiangen Biotech Co., Ltd., Beijing, China), 9.5 μL DNase-free water, 0.5 μL of 10 μM forward primer 1 (F1) and reverse primer 1 (R1), and 2 μL DNA template. PCR cycling conditions were 94°C for 10 min; 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 45 s; and a final extension at 72°C for 10 min. The primer sets peg344-F1 and peg344-R1, and rmpA-F1 and rmpA-R1, were predicted to produce amplicons of 224 bp and 180 bp, respectively. PCR products were separated by electrophoresis in 1.5% agarose gels, then stained with GeneGreen. Images were obtained using a Gel Doc EQ imaging system. Amplified products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. Sequences were analyzed with BLAST software.

The class D OXA carbapenemases of Acinetobacter sp. are represented by 4 main phylogenetic subgroups: OXA-23-like, OXA-24-like, OXA-51-like, and OXA-58. The primers used for detecting these genes by PCR are listed in Table 2. PCR was performed in a 25-μl reaction mixtures containing 12.5 μl PCR Master Mix Reagent (Tiangen Biotech Co., Ltd., Beijing, China); 1 μl of forward primer (10 pmol), 1 μl of reverse primer (10 pmol), and 1 μl of DNA template. Reaction mixtures were initially heated to 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 90 s. The final extension step was performed at 72°C for 7 min. The PCR-amplified products were analyzed by 1% agarose gel electrophoresis and stained with ethidium bromide. Images were acquired using a Bio-Rad Gel Doc EQ imaging system.

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.