Development of multiplex PCR assays for detection of antimicrobial resistance genes in Escherichia coli and enterococci.

Two multiplex polymerase chain reaction (mPCR) assays were developed for simultaneous detection of antimicrobial resistance genes in Escherichia coli and enterococci. Oligonucleotide primer sets were designed and formulated. Primer set I was used to detect resistance genes including tetracycline (tetM and tetA), chloramphenicol (cat1 and cmlA) and sulfonamide (sul1), in 20 E. coli isolates. Primer set II was designed to identify three target genes including tetM, tetL and ermB of tetracycline and erythromycin resistance genes, respectively. In the final volume of 25 µL, optimal concentrations of mPCR primer for investigation of E. coli and enterococci resistance genes were 0.1, 0.4, 0.2, 0.4 and 0.2 µM of tetA, tetM, cat1, cmlA and sul1, and 0.2, 0.2 and 0.1 µM of tetM, tetL and ermB, respectively. The optimal annealing temperatures were 51 and 57C to detect all expected resistance genes of E. coli and enterococci, respectively. The amplicon sizes ranged from 171 to 847 bp for E. coli and 171 to 505 bp for enterococci, differing by at least 83 bp to simplify gel electrophoretic separation. The correlation between dot blot hybridization and mPCR results were investigated. Two of the 20 E. coli isolates showed positive results by mPCR and negative results by hybridization assay. The same results were observed with two different methods among 20 enterococci isolates indicating that the targets were amplified efficiently. The developed mPCR assay is a simple, rapid and useful method for genotyping detection of multiple resistance genes in single reaction. PRACTICAL APPLICATIONS Multiplex polymerase chain reaction (mPCR) can allow the detection of various antimicrobial resistance genes in one PCR reaction tube. The technique is fast and reliable for rapid screening of multiple resistance genes. In this study, we focused on the simultaneous detection of five and three most commonly found resistance genes in Escherichia coli and enterococci isolates, respectively. Our research has shown that the mPCR method could be expanded to include the determination of other antibiotic resistance genes of interest. The system described here can decrease PCR reagent cost by multiple resistance genes detection in each reaction tube. The high throughput and cost-effective mPCR system developed in this study could provide a powerful tool for more accurate detection of various antimicrobial resistance genes associated with E. coli and enterococci isolates.