TABLE 1.

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

Target	Primer (oligonucleotide sequence)a	Amplicon length (bp)	Associated target (purpose or antibiotic affected)	Multiplex type
opa	opa_F (5′-GTTCATCCGCCATATTGTGTTGA-3′)	56	opa (species identification)	Triplex
	opa_R (5′-AAGGGCGGATTATATCGGGTTCC-3′)
porA	porA_F (5′-CAGCAATTTGTTCCGAGTCA-3′)	44	porA (species identification)	Triplex
	porA_R (5′-GGCGTATAGGCGGACTTG-3′)
penA Gly545Ser	545_F (5′-CCCGCCCCCGCCGACTGCAAACGGTTACTA-3′)	61	Mosaic penA (decreased susceptibility/resistance to ESCs)	Triplex
	545_R (5′-CCCGCCCCCGCGGCCCTGCCACTACACC-3′)
penA Ala501	501_F (5′-CCCGCCCCCGCCGTCGGCGCAAAAACCGGTACG-3′)	79	Mosaic penA (decreased susceptibility/resistance to ESCs)	Duplex I
	501_R (5′-CCCGCCCCCGCCAATCGACGTAACGACCGTTAACCAACTTACG-3′)
23S rRNA C2611T	C2611_F (5′-ACGTCGTGAGACAGTTTGGTC-3′)	49	23S rRNA C2611T (moderate AZM resistance)b	Duplex I
	C2611_R (5′-CAAACTTCCAACGCCACTGC-3′)
23S rRNA A2059G	A2059_F (5′-CTACCCGCTGCTAGACGGA-3′)	142	23S rRNA A2059G (high-level AZM resistance)b	Duplex II
	A2059_R (5′-CAGGGTGGTATTTCAAGGACGA-3′)
gyrA Ser91Phe	gyrA_S91_F (5′-TAAATACCACCCCCACGGCGATT-3′)	47	GyrA Ser91Phe (CIP resistance)	Duplex II
	gyrA_S91_R (5′-ATACGGACGATGGTGTCGTAAACT-3′)
rpsE Thr24Pro	S5_T24_F (5′-ATGGTCGCAGTTAACCGTGTA-3′)	56	RPS5 Thr24Pro (SPC resistance)	Duplex III
	S5_T24_R (5′-AAAGCCATAATGCGACCACC-3′)
16S rRNA C1192T	16S_1192_F (5′-CCGCCCCCCGGAGGAAGGTGGGGATGA-3′)	64	16S rRNA C1192T (SPC resistance)	Duplex III
	16S_1192_R (5′-CCGCCCCCCTGGTCATAAGGGCCATGAG-3′)

^aGC clamps, which were added to the 5′ ends of some primers to allow multiplexing, are shown in italic type.

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