Discrimination of SHV beta-lactamase genes by restriction site insertion-PCR

Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for d etection of point mutations. This technique exploits primers with one to th ree base mismatches near the 3' end to modulate a restriction site. We have developed this technique to identify described mutations of the bla(SHV) g enes for differentiation of SHV variants that cannot be distinguished easil y by other techniques. To validate this method, eight standard strains were used, each producing a different SHV beta -lactamase: SHV-1 SHV-2, SHV-3, SHV-4, SHV-5, SHV 6, SHV-8, and SHV-18. Mismatch primers were designed to d etect mutations affecting amino acids at positions 8 (SspI), 179 (HinfI), 2 05 (PstI), 238 (Gly --> Ala) (BsrI), and 240 (NruI) of bla(SHV) genes. All amplimers of the bla(SHV) genes used in this study yielded the predicted re striction endonuclease digestion products. In addition, this study also mak es theoretical identification of bla(SHV-6), bla(SHV-8), and 12 novel bla(S HV) variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible. By using a combination of PC-RRFLP and RSI-PCR techniqu es, up to 27 SHV variants can now be distinguished rapidly and reliably. Th ese simple techniques are readily applied to epidemiological studies of the SHV beta -lactamases and may be extended to the characterisation of other resistance determinants.