Volume 19, Number 6—June 2013
Synopsis
New Delhi Metallo-β-Lactamase–producing Enterobacteriaceae, United States
Table 2
Sequences of primers and probes used for identification of NDM–producing isolates, United States, April 2009–March 2011*
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 |
NDM-1 reverse |
CTC ATG TTT GAA TTC GCC C |
|
Internal DNA sequencing primers |
NDM-2F | ACA AGA TGG GCG GTA TGG A |
NDM-2R |
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.