Position Size (bp) aac(3)-II F:AGGTGACACTATAGAATAACTGTGATGGGATACGCGTC DQ449578.1 87359–87378 274 R:GTACGACTCACTATAGGGACTCCGTCAGCGTTTCAGCYA 87595–87576 aac(6’)-Ib F:AGGTGACACTATAGAATACTGTTCAATGATCCCGAGGT JN861072.1 101468–101487 188 R:GTACGACTCACTATAGGGATGGCGTGTTTGAACCATGTA PI3K inhibitor 101619–101600 aac(6’)-II F:AGGTGACACTATAGAATATTCATGTCCGCGAGCACCCC GU944731.1 1307–1326 215 R:GTACGACTCACTATAGGGAGACTCTTCCGCCATCGCTCT 1485–1466 ant(3″)-I F:AGGTGACACTATAGAATATGATTTGCTGGTTACGGTGAC HM106456.1 2207–2229 321 R:GTACGACTCACTATAGGGACGCTATGTTCTCTTGCTTTTG 2490–2470 aph(3’)-VI F:AGGTGACACTATAGAATACGGAAACAGCGTTTTAGAGC

JF949760.1 727–746 288 R:GTACGACTCACTATAGGGAGGTTTTGCATTGATCGCTTT 975–956 armA F:AGGTGACACTATAGAATATGCATCAAATATGGGGGTCT FJ410928.1 3953–3972 247 R:GTACGACTCACTATAGGGATGAAGCCACAACCAAAATCT 4162–4143 rmtB F:AGGTGACACTATAGAATAGCTGTGATATCCACCAGGGA FJ410927.1 RG7112 manufacturer 5326–5345 177 R:GTACGACTCACTATAGGGAAAGCTTAAAAATCAGCGCCA 5465–5446 Cy5-labled Tag F:AGGTGACACTATAGAATA       R:GTACGACTCACTATAGGGA   *Universal tag sequences are underlined. Evaluation of the specificity of the GeXP assay The DNA templates were extracted bacterial genomic DNAs of the 8 reference strains, 5 positive

control isolates, 2 negative controls and 7 recombinant plasmids harboring each of the 7 resistance genes, respectively. The mono GeXP assay and GeXP assay were developed using single template and each pair of gene-specific primers (for mono GeXP assay) or using single template in a multiplex primer format (for GeXP Cetuximab manufacturer assay), respectively, to ascertain the actual amplicon size of each target region. The PCR assays were performed

with QIAGEN Multiplex PCR kit (Qiagen, Hilden, Germany) in a 25 μl volume containing 12.5 μl of 2× QIAGEN Multiplex PCR Master Mix (HotStarTaq® DNA Polymerase, Multiplex PCR Buffer, dNTP Mix) and 1 μl of DNA templates. The mono GeXP assay contained 50 nM of each pair of gene-specific chimeric primers individually while the GeXP assay contained 50 nM of each of 7 pairs of gene-specific chimeric primers and 500 nM of the universal Tag primers as the final concentrations, nuclease-free water was added to 25 μl reaction volume. The PCR was performed under the following conditions: 95°C for 10 min, followed by three steps of amplification procedures reaction according to the temperature switch PCR (TSP) strategy [29]: step 1, 10 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; step 2, 10 cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s; step 3, 20 cycles of 95°C for 30 s, 48°C for 30 s, and 72°C for 30 s (Figure 1). Figure 1 Diagram of the analysis procedure of GeXP assay. The analysis procedure of GeXP assay consists of chimeric primer-based multiplex PCR amplification and capillary electrophoresis separation.