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Antimicrobial Agents and Chemotherapy, July 2001, p. 2110-2114, Vol. 45, No. 7
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.7.2110-2114.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Aroonwadee Chanawong,1 Fatima Hannachi M'Zali,1 John Heritage,1 Aroonlug Lulitanond,2 and Peter Michael Hawkey1,*

Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom,1 and Department of Clinical Microbiology, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand2

Received 8 September 2000/Returned for modification 15 January 2001/Accepted 10 April 2001

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 blaSHV 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 beta -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 (SspI), 179 (HinfI), 205 (PstI), 238 (Glyright-arrowAla) (BsrI), and 240 (NruI) of blaSHV genes. All amplimers of the blaSHV genes used in this study yielded the predicted restriction endonuclease digestion products. In addition, this study also makes theoretical identification of blaSHV-6, blaSHV-8, and 12 novel blaSHV 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 beta -lactamases and may be extended to the characterisation of other resistance determinants.

* Corresponding author. Mailing address: Division of Microbiology, University of Leeds, Leeds LS2 9JT, United Kingdom. Phone: 44 113 233 5597. Fax: 44 113 233 5649. E-mail: p.m.hawkey{at}leeds.ac.uk.

Antimicrobial Agents and Chemotherapy, July 2001, p. 2110-2114, Vol. 45, No. 7
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.7.2110-2114.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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