Heptaplex PCR melting curve analysis for rapid detection of plasmid-mediated AmpC β-lactamase genes.

Antimicrobial resistance mediated by plasmid-borne AmpC β-lactamase in Gram-negative bacteria is an emerging event of significant clinical importance. Rapid and reliable detection of ampC is in urgent need for appropriate infection control. We described the development and evaluation of a heptaplex PCR melting curve analysis that could identify six groups of ampC, i.e., CIT, EBC, DHA, ACC, MOX and FOX, through predefined melting temperatures. The entire analysis could be finished within 2h for 96 samples after template DNA was prepared. We first evaluated the assay with 176 AmpC-producing isolates of Escherichia coli and Klebsiella pneumoniae, and the results showed that 36 isolates were positive for ampC, including 18 positive for DHA, 12 for CIT, 5 for EBC, and one for both DHA and EBC. These results were fully concordant with sequencing analysis whereas the comparison method, an electrophoresis-based singleplex PCR assay, missed four isolates. The assay was also used to analyze 429 randomly selected clinically relevant Gram-negative isolates involving 22 different species, and 34 isolates were found to be ampC-positive. The results again fully agreed with the sequencing analysis. We conclude that the established assay could be used for rapid and reliable detection of ampC.