Rapid 65-min SYBR-Green PCR Assay for Carbapenem Resistant Klebsiella and Acinetobacter Detection

This study developed a rapid and reliable SYBR-Green semiplex PCR assay for simulta-neous detection of major carbapenem resistance genes in <i>Klebsiella pneumoniae</i> and <i>Acinetobacter baumannii</i>. Two primer sets were used: one to detect <i>bla</i>KPC, <i>bla</i>NDM-1, and <i>bla</i>OXA-48 genes in <i>K. pneumoniae</i> and <i>bla</i>OXA-23 in <i>A. baumannii</i>, and another to amplify conserved 16S rRNA gene regions as internal controls. The intra- and inter-assay coeffi-cient of variation ranged from 0.03% to 3.8%. Standard curves exhibited excellent linearity across six logarithmic scales (10¹-10⁶ DNA copies/µL), with detection limits of 10-10² DNA copies/mL. Melting temperatures (Tm) were: 88.85 °C (KPIC), 90.65 °C (NDM-1), 89.45 °C (KPC), 84.23 °C (OXA-48), 87.81 °C (OXA-23), and 80.67 °C (ABIC). The SYBR-Green Semiplex PCR assay offers a rapid (65 min turnaround), cost-effective, and sensitive method for early detection of carbapenem-resistant pathogens, enabling timely targeted therapy and improved infection control by potentially reducing empirical antibiotic use before culture confirmation.