XÂY DỰNG VÀ TỐI ƯU HÓA QUY TRÌNH PCR ĐA MỒI PHÁT HIỆN ĐỒNG THỜI CÁC GEN BLAOXA TRÊN ACINETOBACTER BAUMANNII KHÁNG CARBAPENEM

Abstract

Acinetobacter baumannii is a clinically significant nosocomial pathogen, characterized by its remarkable multidrug resistance, particularly to carbapenems, predominantly mediated via OXA-type β-lactamases. The escalating prevalence of multidrug-resistant strains underscores the urgent need for rapid, accurate, and cost-effective diagnostic tools. This study developed and optimized a multiplex polymerase chain reaction (PCR) assay for the simultaneous detection of four target genes—16S-rRNA, bla_OXA-23, bla_OXA-51, and bla_OXA-58—aimed at species identification and antimicrobial resistance profiling of A. baumannii. Systematic optimization included evaluation of annealing temperature, primer concentration, dNTP concentration, Taq DNA polymerase dosage, detection limit, and primer specificity. The optimized assay employed an annealing temperature of 58°C, primer concentration of 0.15 µM, dNTP concentration of 0.25 mM, and Taq DNA polymerase at 1.25 U, achieving a detection limit of 10 CFU/ml with no false positives. Sequence verification and BLAST analysis confirmed 100% specificity for all primer sets. This multiplex PCR protocol represents a rapid, highly specific, and resource-efficient molecular diagnostic approach, with strong potential for integration into clinical microbiology workflows to facilitate early detection and surveillance of carbapenem-resistant A. baumannii, thereby enhancing infection control and antimicrobial stewardship.