New Delhi Metallo-β-Lactamase–Producing Enterobacteriaceae

United States

J. Kamile Rasheed; Brandon Kitchel; Wenming Zhu; Karen F. Anderson; Nancye C. Clark; Mary Jane Ferraro; Patrice Savard; Romney M. Humphries; Alexander J. Kallen; Brandi M. Limbago

Disclosures

Emerging Infectious Diseases. 2013;19(6) 

In This Article

Detection of bla NDM and bla KPC by Real-time PCR

A multiplexed Taqman-based real-time PCR for blaNDM and blaKPC, as well as the universal bacterial 16S rRNA–encoding gene[18] as an endogenous control for DNA amplification, was performed on the 7500 Fast system (Applied Biosystems, Carlsbad, CA, USA). Cell lysates were prepared as described.[19] Each PCR (20-μL volume) included 1× QuantiFast Probe PCR Master Mix (QIAGEN, Valencia, CA, USA), a combined primer/probe solution with final concentrations of 500 nmol/L for each primer and 250 nmol/L for each probe (Table 2), and 2 μL of template. Included in each assay were a bla NDM-positive control (K. pneumoniae ATCC BAA-2146), a bla KPC-positive control (K. pneumoniae ATCC BAA-1705), a carbapenemase-negative control (K. pneumoniae ATCC BAA-1706), and a no template control. Cycling conditions were a 3-min enzyme activation step at 95°C, followed by 40 cycles for 3 s at 95°C and 30 s at 60°C.

Reactions with 16S cycle threshold (Ct) values of 10–30 were considered valid, those with NDM or KPC Ct values of 10–30 were considered NDM positive or KPC negative, and those with NDM or KPC Ct values ≥40 were considered NDM positive or KPC negative (www.cdc.gov/HAI/settings/lab/kpc-ndm1-lab-protocol.html).

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