ABSTRACT
ObjectiveThe nucleic acid technology for detecting drug-resistant genes has become one of the powerful tools for monitoring and controlling the spreading of drug-resistant bacteria. This study was to establish a method for rapid detection of the drug-resistant genes KPC and NDM and provide some guidance in clinical drug use and monitoring the prevalence of drug-resistant bacteria in the hospital.MethodsAccording to the conserved regions of Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM), we designed the primers of duplex PCR, optimized the amplification system and established a method for simultaneous detection of the drug-resistant genes KPC and NDM. Then, we analyzed the sensitivity and specificity of the method and applied it to the detection of Pseudomonas aeruginosa and Klebsiella pneumoniae.ResultsThe sequences of KPC and NDM exhibited a 100% consistency with those of the original ones. Target fragments of the desired size of 151 bp were detected in the KPC-2 positive standard and Klebsiella pneumoniae ATCC BAA 1705 standard strains, and those of the desired size of 261 bp were observed in the NDM-2 positive standard strain and NDM-positive pneumococcal bacteria, neither with non-specific amplification. Sequencing of the PCR products showed a 100% consistency between the sequences of the products and those of the drug-resistant genes KPC-2 and NDM-1. The detectable limits of KPC and NDM for duplex PCR were 7×102 and 5×102 copies per reaction respectively. Drug-resistant genes were detected in 12 (92.3%) of the 13 carbapenems-resistant strains, including 10 KPC-positive (83.3%) and 2 NDM positive ones (16.7%), but neither KPC nor NDM in the other 10 carbapenems-sensitive strains. In the 13 strains of Pseudomonas aeruginosa, KPC was detected in 2 (33.3%) of the 6 carbapenems-resistant ones, but neither KPC nor NDM in the other 7.ConclusionThe duplex PCR method can be used for rapid and effective detection of the drug-resistance genes KPC and NDM, with the advantages of high sensitivity and specificity, and is therefore of great significance for guiding clinical drug use and monitoring the spreading of carbapenems-resistant bacteria in the hospital.