A rapid low-cost real-time PCR for the detection of klebsiella pneumonia carbapenemase genes

Real-time PCR assay based on SYBR GreenIwas designed to amplify a 106 bp product of the blaKPC gene from the159 clinical Gram-negative isolates resistant to several classes of β-lactam antibiotics through antimicrobial susceptibility testing. We confirmed the results of real-time PCR assay by the conventional PCR-sequencing. At the same time, KPCs of these clinical isolates were detected by the modified Hodge test (MHT). Then we compared the results of real-time PCR assay with those of MHT from the sensitivity and specificity. Moreover, we evaluated the sensitivity of the real-time PCR assay.The sensitivity and specificity of the results of the real-time PCR assay compared with those of MHT was 29/29(100 %) and 130/130(100 %), respectively. The results of the real-time PCR and the MHT were strongly consistent (Exact Sig. (2-tailed) =1. 000; McNemar test). The real-time PCR detection limit was about 0.8 cfu using clinical isolates.The real-time PCR assay could rapidly and accurately detect KPCs -harboring strains with high analytical sensitivity and specificity.Carbapenems are widely used to treat serious infections caused by multi-resistant Gram-negative bacteria. However, beginning with the initial description of a novel KPC from an isolate of K. pneumoniae in 2001 [1], carbapenem resistance in Enterobacteriaceae has been rapidly increasing. KPCs are able to hydrolyze the carbapenems, and cause resistance to multiple classes of antibiotics. Treatment of KPC-producing bacterial infection is thus a considerable challenge for clinicians. KPCs have been reported worldwide, such as North America, South America, Greece, Israel, Puerto Rico, China and so on [2-6]. The expanding geographic spread of KPCs underscores the importance of clinical recognition of these enzymes. In addition, KPCs have been found in bacteria other than K. pneumoniae, including K. oxytoca[7,8], P. mirabilis[9], Acinetobacter spp[6], P. aeruginosa , C. freundii[10], S. marcescens and E. coli[11]. Th