Rapid discriminatory detection of genes coding for SHV β-lactamases by ligase chain reaction

Ligase chain reaction (LCR) is a recently developed technique that employs a thermostable ligase and allows for the discrimination of DNA sequences differing in only a single base pair. The method has been adapted and applied to differentiation of bla(SHV) genes. We have developed an LCR typing method to characterize point mutations in genes for SHV-derived extended-spectrum beta-lactamases,vith four different sets of biotinylated LCR primers. To evaluate the applicability of the current technique, we tested seven Escherichia coli strains producing SHV-1, SHV-2, SHV-2a, SHV-3, SHV-4, SHV-5, and SHV-12, With the LCR typing, seven SHV genes can be distinguished according to their incorporating point mutations. In an attempt to characterize SPIV beta-lactamases by LCR typing in clinical isolates, 46 strains carrying bla(SHV) genes (32 Klebsiella pneumoniae, 10 Enterobacter cloacae, and 4 E, coli) were subjected to antibiotic susceptibility testing, isoelectric focusing, and LCR typing, LCR typing allowed the characterization of beta-lactamases, and genotypes obtained by LCR typing were in accordance with phenotypes such as antibiotic resistance profile and pI value of beta-lactamase, Therefore, we concluded that LCR typing may permit defining the SEN families with simplicity and reliability and can be applied to the detailed characterization and molecular epidemiology of SHV-type beta-lactamases.