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Journal of Clinical Microbiology, March 2006, p. 909-915, Vol. 44, No. 3
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.3.909-915.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Detection and Genotyping of SHV ß-Lactamase Variants by Mass Spectrometry after Base-Specific Cleavage of In Vitro-Generated RNA Transcripts

Enno Stürenburg,1* Niels Storm,2,{dagger} Ingo Sobottka,1 Matthias A. Horstkotte,1 Stefanie Scherpe,1 Martin Aepfelbacher,1 and Susanne Müller2

Institut für Infektionsmedizin, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany,1 SEQUENOM GmbH, Mendelssohnstrasse 15D, 22761 Hamburg, Germany2

Received 11 August 2005/ Returned for modification 3 October 2005/ Accepted 6 January 2006

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR-amplified and in vitro-transcribed blaSHV genes was used for the identification and genotyping of SHV ß-lactamases. For evaluation, blaSHV stretches of 21 clinical Enterobacteriaceae isolates were PCR amplified using T7 promoter-tagged forward and reverse primers, respectively. In vitro transcripts were generated with T7 RNA and DNA polymerase in the presence of modified analogues replacing either CTP or UTP. Using RNase A, the in vitro transcripts were base-specifically cleaved at every "T" or "C" position. Resulting cleavage products were analyzed by MALDI-TOF MS, generating a characteristic signal pattern based on the fragment masses. All 21 individual SHV genes were identified unambiguously using reference sequences, and the results were in perfect concordance with those obtained by fluorescent dideoxy sequencing, which represents the current standard method. As multiple point mutations can be detected in a single assay and newly emerged mutations which are not yet described in public databases can be identified too, MALDI-TOF MS appears to be an ideal tool for analysis of sequence polymorphisms in resistance-associated gene loci.

* Corresponding author. Present address: LADR GmbH, Labormedizinisches Versorgungszentrum Geesthacht, Lauenburger Straße 67, 21502 Geesthacht, Germany. Phone: 49 4152 803 120. Fax: 49 4152 803 383. E-mail: stuerenburg{at}ladr.de.

{dagger} Present address: BioGlobe GmbH, Grandweg 64, 22529 Hamburg, Germany.

Journal of Clinical Microbiology, March 2006, p. 909-915, Vol. 44, No. 3
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.3.909-915.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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