The aim of this study was to detect point mutations in extended-spectrum beta-lactamase (ESBL) genes in a background of wild-type (non-ESBL-producing) bacteria using a highly sensitive and specific method developed for this purpose. The ligase detection reaction-polymerase chain reaction (LDR-PCR) method was used to test different ESBL-producing strains and clinical isolates for a specific point mutation in the bla SHV-ESBL gene (glycine to serine mutation at position 238) and was compared with the commercially available E test ESBL (AB Biodisk, Sweden). Nine of the 40 clinical isolates tested were positive for the bla SHV-ESBL point mutation when tested by the LDR-PCR method but negative when tested by the E test. In contrast to the E test or other molecular genetic tests, the LDR-PCR method is able to identify a single bacterium with a point mutation in a background of 100,000 wild-type (non-ESBL-producing) bacteria.