Discrimination of SHV β-Lactamase Genes by Restriction Site Insertion-PCR

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Abstract
Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for detection of point mutations. This technique exploits primers with one to three base mismatches near the 3′ end to modulate a restriction site. We have developed this technique to identify described mutations of the bla SHV genes for differentiation of SHV variants that cannot be distinguished easily by other techniques. To validate this method, eight standard strains were used, each producing a different SHV β-lactamase: SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, SHV-6, SHV-8, and SHV-18. Mismatch primers were designed to detect mutations affecting amino acids at positions 8 ( Ssp I), 179 ( Hin fI), 205 ( Pst I), 238 (Gly→Ala) ( Bsr I), and 240 ( Nru I) of bla SHV genes. All amplimers of the bla SHV genes used in this study yielded the predicted restriction endonuclease digestion products. In addition, this study also makes theoretical identification of bla SHV-6 , bla SHV-8 , and 12 novel bla SHV variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible. By using a combination of PCR-RFLP and RSI-PCR techniques, up to 27 SHV variants can now be distinguished rapidly and reliably. These simple techniques are readily applied to epidemiological studies of the SHV β-lactamases and may be extended to the characterisation of other resistance determinants.

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