Real-time detection of blaKPC in clinical samples and surveillance specimens

Abstract

A real-time PCR assay was developed targeting the bla(KPC) responsible for Klebsiella pneumoniae carbapenemase (KPC)-mediated carbapenem resistance and was validated for testing colonies or enrichment broth cultures. The assay accurately detects KPC-containing strains with high analytical specificity and sensitivity.

Fig. 1..

Amplification curves for analytical validation specimens after color compensation calculations have been applied. The top panel shows the fluorescence measured at 530 nm to detect FAM, indicating the presence of the bla_KPC gene. Note that only the bacteria with KPC activity show positive amplification curves; all bacteria lacking any carbapenemase activity show no fluorescence similar to the water blank reaction. The bottom panel shows fluorescence measured at 610 nm to detect Texas Red, indicating amplification of the 16S rRNA gene as an internal control. The curves in this panel that do not have the typical sigmoid curve and lower 610-nm fluorescence are the KPC-positive bacteria that have strong FAM fluorescence at 530 nm, affecting the amount of Texas Red fluorescence measured for these samples. All bacterial specimens show positive amplification curves, indicating acceptable amplification. The water blank has no fluorescence, indicating no background amplification.