TABLE 1

Target genes, primer sequences, amplicon lengths, mutations and affected antibiotics, and multiplex combinations for the real-time PCR platform

TargetPrimer (oligonucleotide sequence)aAmplicon length (bp)Associated target (purpose or antibiotic affected)Multiplex type
opaopa_F (5′-GTTCATCCGCCATATTGTGTTGA-3′)56opa (species identification)Triplex
opa_R (5′-AAGGGCGGATTATATCGGGTTCC-3′)
porAporA_F (5′-CAGCAATTTGTTCCGAGTCA-3′)44porA (species identification)Triplex
porA_R (5′-GGCGTATAGGCGGACTTG-3′)
penA Gly545Ser545_F (5′-CCCGCCCCCGCCGACTGCAAACGGTTACTA-3′)61Mosaic penA (decreased susceptibility/resistance to ESCs)Triplex
545_R (5′-CCCGCCCCCGCGGCCCTGCCACTACACC-3′)
penA Ala501501_F (5′-CCCGCCCCCGCCGTCGGCGCAAAAACCGGTACG-3′)79Mosaic penA (decreased susceptibility/resistance to ESCs)Duplex I
501_R (5′-CCCGCCCCCGCCAATCGACGTAACGACCGTTAACCAACTTACG-3′)
23S rRNA C2611TC2611_F (5′-ACGTCGTGAGACAGTTTGGTC-3′)4923S rRNA C2611T (moderate AZM resistance)bDuplex I
C2611_R (5′-CAAACTTCCAACGCCACTGC-3′)
23S rRNA A2059GA2059_F (5′-CTACCCGCTGCTAGACGGA-3′)14223S rRNA A2059G (high-level AZM resistance)bDuplex II
A2059_R (5′-CAGGGTGGTATTTCAAGGACGA-3′)
gyrA Ser91PhegyrA_S91_F (5′-TAAATACCACCCCCACGGCGATT-3′)47GyrA Ser91Phe (CIP resistance)Duplex II
gyrA_S91_R (5′-ATACGGACGATGGTGTCGTAAACT-3′)
rpsE Thr24ProS5_T24_F (5′-ATGGTCGCAGTTAACCGTGTA-3′)56RPS5 Thr24Pro (SPC resistance)Duplex III
S5_T24_R (5′-AAAGCCATAATGCGACCACC-3′)
16S rRNA C1192T16S_1192_F (5′-CCGCCCCCCGGAGGAAGGTGGGGATGA-3′)6416S rRNA C1192T (SPC resistance)Duplex III
16S_1192_R (5′-CCGCCCCCCTGGTCATAAGGGCCATGAG-3′)
  • a GC clamps, which were added to the 5′ ends of some primers to allow multiplexing, are shown in italic type.

  • b Confers moderate- to high-level resistance to AZM (i.e., MIC, >2 μg/ml) when at least 3 out of 4 copies are mutated (9).