Development of a TaqMan® Multiplex PCR Assay for Detection of Plasmid-Mediated Amp-C β-lactamase (pmAmpC) Genes
Geyer, Chelsie N.
Hanson, Nancy D.
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Background: Isolates that produce pmAmpCs, such as K. pneumoniae (Kp), E. coli (Ec), and Salmonella spp. (Sal), can be resistant to most β-lactams. These enzymes can phenotypically interfere with the detection of ESBL and KPC producing isolates and false susceptible results can lead to inappropriate treatment of infected patients. Therefore, the purpose of this study was to design an easy to use, rapid, multiplex, real-time (RT) PCR assay to identify isolates carrying pmAmpC genes. Methods: Test isolates evaluated included Ec, Kp, and Sal within a panel of 26 characterized AmpC producing isolates, 109 non-AmpC isolates, and 120 randomly selected Gram-negative isolates from the clinical laboratory. DNA template was isolated using the Qiagen DNeasy® kit. Primer sets and TaqMan®(TM) probes were designed using Beacon Designer 7® software for blaCMY-2, blaFOX, blaACT, blaACC, blaMOX, blaDHA, and 16S ribosomal (r) DNA as an internal control for all three genera. Amplicon sizes ranged from 55-148 bp. Primer specificity was evaluated using melt curve analysis. TM probes for each ampC product and rDNA were labeled 5′ with FAM or HEX fluorescent reporter dyes respectively. RT-PCR was performed using a Rotor-Gene Q. E. coli clones containing pmAmpC gene fragments served as positive controls. Results: TM probe binding was specific for each primer combination. All amplifications demonstrated fluorescent signal above background between 9-13 cycles. The sensitivity and specificity of the ampC primer/probe sets and the rDNA control were 100%. Randomly selected clinical isolates (5/120) were positive for a pmAmpC gene using this assay but were reported as susceptible to 3rd generation cephalosporins by conventional susceptibility testing. Conclusions: The ampC multiplex assay is a fast (≤2.5 hours), single step procedure easily monitored for quality by internal controls. Thus, this assay holds promise as a molecular diagnostic increasing the ability of clinical laboratories to identify resistance mechanisms with potentially major therapeutic impact but difficult to detect phenotypically.