Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP)

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFL P has been developed to extend the identification of SHV beta-lactamases pr eviously characterised by PCR-single strand conformational polymorphism (PC R-SSCP) analysis alone. Eight bacteria, each producing a different SHV beta -lactamase, were used in this study. These bacteria harbour bla(SHV-1), bla (SHV-2a), bla(SHV-3), bla(SHV-4,) bla(SHV-5) (two strains), bla(SHV-11) and bla(SHV-12). All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion. By a combination of these techniques, the genes e ncoding these beta-lactamases could be differentiated from each other. In a ddition. the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from th ose encoding other SHV variants. We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisatio n of known SHV beta-lactamases in a combination with the previously publish ed PCR-SSCP analysis. (C) 2000 Federation of European Microbiological Socie ties. Published by Elsevier Science B.V. All rights reserved.