Volume 19, Number 6—June 2013
New Delhi Metallo-β-Lactamase–producing Enterobacteriaceae, United States
|Assay||Primers and probes||Sequence, 5′ → 3′|
|Real-time PCR:NDM/KPC screen
||NDM, forward primer||GAC CGC CCA GAT CCT CAA|
|NDM, Reverse primer||CGC GAC CGG CAG GTT|
|NDM, probe (HEX)†||TG GAT CAA GCA GGA GAT|
|KPC, forward primer||GGC CGC CGT GCA ATA C|
|KPC, reverse primer||GCC GCC CAA CTC CTT CA|
|KPC, probe (FAM)†||TG ATA ACG CCG CCG CCA ATT TGT|
|16S, forward primer||TGG AGC ATG TGG TTT AAT TCG A|
|16S, reverse primer||TGC GGG ACT TAA CCC AAC A|
|16S, probe (CY5)†
||CA CGA GCT GAC GAC AR‡C CAT GCA
|DNA sequence analysis§
||NDM-1 forward||ACT CGT CGC AAA GCC CAG|
||CTC ATG TTT GAA TTC GCC C
|Internal DNA sequencing primers
||NDM-2F||ACA AGA TGG GCG GTA TGG A|
||CGT CCA TAC CGC CCA TCT
|DIG-labeled probe synthesis||NDM-F1||GAA TTG CCC AAT ATT ATG CAC C|
|NDM-R1||AGC GCA GCT TGT CGG CCA TG|
*NDM, New Delhi metallo-β-lactamase; KPC, Klebsiella pneumoniae carbapenemase; DIG, digoxigenin.
†NDM probes were labeled with HEX, KPC probes were labeled with FAM, and 16S probes were labeled with CY5 at their 5′ ends. Each contained a black hole quencher at the 3′ end.
‡R, A or G (International Union of Biochemistry codes for DNA bases).
§Amplification using primers NDM-1 forward and NDM-1 reverse, both located outside the coding region of blaNDM, results in a 1,013-bp product.