Discriminatory detection of extended-spectrum β-lactamases by restriction fragment length dimorphism-polymerase chain reaction

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Plasmid-mediated resistance mechanisms to β-lactams, comprising mostly extended-spectrum β-lactamase (ESBL) production, lead to resistance against even the most recently developed β-lactams in enterobacteria, which is now a serious threat to antibiotic therapy. In this work, the diagnostic ability of the restriction fragment length dimorphism (RFLD)-polymerase chain reaction (PCR) method in clinical samples was evaluated. Nine newly designed primer pairs were used to differentiate the genes encoding TEM-1a, SHV-12, MOX-1, MIR-1 and Toho-1 β-lactamases. The RFLD-PCR was carried out successfully and these genes were differentiated by the sizes of their PCR product. This discriminatory detection of the genes was also confirmed by digestion with unique restriction enzyme sites and sequencing of the PCR products. The fragment sizes of PCR products digested with the enzymes were identical to the sizes calculated from nucleotide sequences of five β-lactamase genes deposited in EMBL, GenBank and/or DDBJ databases and the sequences were also identical. In conclusion, the method and newly designed primers applied in this work can differentiate the ESBLs rapidly and effectively.

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