A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of and Mutations Associated With Fluoroquinolone Resistance in .

BACKGROUND

Mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase () and topoisomerase IV () are linked to fluoroquinolone (FQ) resistance. We developed a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect mutations in the and QRDRs associated with FQ resistance in .

METHODS

Based on the conserved sequences of and , two primer sets were designed for mismatched PCR-RFLP to detect mutations in (codons 83 and 87) and (codons 80 and 84) by introducing an artificial restriction enzyme cleavage site into the PCR products. This assay was evaluated using 58 strains and 37 other Acinetobacter strains that have been identified by RNA polymerase β-subunit gene sequence analysis.

RESULTS

PCR amplification of and was successful for all strains. In 11 FQ -susceptible strains, the and PCR products were digested by the selected restriction enzymes at the site containing (codons 83 and 87) and (codons 80 and 84). PCR products from 47 FQ-resistant strains containing mutations in and were not digested by the restriction enzymes at the site containing the mutation. As for the non- strains, although amplification products for were obtained for 28 strains, no amplification product was obtained for any strain.

CONCLUSIONS

This assay specifically amplified and from and detected and mutations with FQ resistance.