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-RFLP) has been developed to extend the identification of SHV beta-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-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 encoding these beta-lactamases could be differentiated from each other. In addition, 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 those encoding other SHV variants. We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV beta-lactamases in a combination with the previously published PCR-SSCP analysis.
Chanawong
A
A
Department of Microbiology, University of Leeds, Leeds, UK.
M'Zali
F H
FH
Heritage
J
J
Lulitanond
A
A
Hawkey
P M
PM
D004269:DNA, Bacterial / Q000235:genetics; D004754:Enterobacter / Q000201:enzymology / Q000235:genetics; D004755:Enterobacteriaceae / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007709:Klebsiella / Q000201:enzymology / Q000235:genetics; D008826:Microbial Sensitivity Tests; D009154:Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational; D001618:beta-Lactamases / Q000235:genetics*
D004269:DNA, Bacterial; D001618:beta-Lactamases