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Journal of Clinical Microbiology, October 2006, p. 3712-3719, Vol. 44, No. 10
0095-1137/06/$08.00+0     doi:10.1128/JCM.00843-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Staphylococcus aureus Genotyping Using Novel Real-Time PCR Formats

Flavia Huygens,^1, John Inman-Bamber,^1, Graeme R. Nimmo,² Wendy Munckhof,³ Jacqueline Schooneveldt,² Bruce Harrison,⁴ Jennifer A. McMahon,⁴ and Philip M. Giffard^1*

Cooperative Research Centre for Diagnostics, Institute of Health and Biomedical Innovation, QUT, Brisbane, Australia,¹ Queensland Health Pathology Service, Brisbane, Australia,² Princess Alexandra Hospital and District Health Service, Brisbane, Australia,³ Corbett Life Science, Eight Mile Plains, Brisbane, Australia⁴

Received 21 April 2006/ Returned for modification 29 June 2006/ Accepted 29 July 2006

One approach to microbial genotyping is to make use of sets of single-nucleotide polymorphisms (SNPs) in combination with binary markers. Here we report the modification and automation of a SNP-plus-binary-marker-based approach to the genotyping of Staphylococcus aureus and its application to 391 S. aureus isolates from southeast Queensland, Australia. The SNPs used were arcC210, tpi243, arcC162, gmk318, pta294, tpi36, tpi241, and pta383. These provide a Simpson's index of diversity (D) of 0.95 with respect to the S. aureus multilocus sequence typing database and define 61 genotypes and the major clonal complexes. The binary markers used were pvl, cna, sdrE, pT181, and pUB110. Two novel real-time PCR formats for interrogating these markers were compared. One of these makes use of "light upon extension" (LUX) primers and biplexed reactions, while the other is a streamlined modification of kinetic PCR using SYBR green. The latter format proved to be more robust. In addition, automated methods for DNA template preparation, reaction setup, and data analysis were developed. A single SNP-based method for ST-93 (Queensland clone) identification was also devised. The genotyping revealed the numerical importance of the "South West Pacific" and "Queensland" community-acquired methicillin-resistant S. aureus (MRSA) clones and the clonal complex 239 "Aus-1/Aus-2" hospital-associated MRSA. There was a strong association between the community-acquired clones and pvl.

F.H. and J.I.-B. contributed equally to this study.

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