A high throughput multiplex PCR assay for simultaneous detection of seven aminoglycoside-resistance genes in Enterobacteriaceae

BACKGROUND: The aminoglycoside-resistance genes encoding aminoglycoside modifying enzymes and 16S rRNA methyltransferases are main factors contributing to increasing resistance to aminoglycosides. Characterization and distribution of antimicrobial resistance gene profiles provide important information on the potential difficulty of treatment of bacteria. Several molecular methods have been developed to investigate the prevalence of aminoglycoside-resistance genes. These existing methods are time-consuming, labor-intensive, expensive or limited sensitivity in the epidemiological investigation. Therefore, it is necessary to develop a rapid, less-costly and high throughput and sensitive method to investigate the distribution of antimicrobial resistance gene in clinical isolates. RESULTS: In this study, we developed a GeXP analyzer-based multiplex PCR assay to simultaneously detect seven aminoglycoside-resistance genes, including aac(3)-II, aac(6′)-Ib, aac(6′)-II, ant(3″)-I,aph(3′)-VI,armA and rmtB, and to analyze the distribution of these genes in clinical Enterobacteriaceae isolates. Under optimized conditions, this assay achieved a limit-of-detection as low as 10 copies of each of the seven genes. The presented method was applied to analyze the distribution of aminoglycoside-resistance genes in 56 clinical Enterobacteriaceae isolates, and the results were compared with that of the conventional single PCR assay. Kappa values of the two methods for detecting each of the seven resistance genes were 0.831, 0.846, 0.810, 0.909, 0.887, 0.810 and 0.825, respectively. CONCLUSION: This GeXP assay is demonstrated to be a rapid, cost-effective and high throughput method with high sensitivity and specificity for simultaneously detecting seven common aminoglycoside-resistance genes.