Staphylococcus aureus Genotyping Using Novel Real-Time PCR Formats

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....

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Bibliographic Details
Published in:Journal of Clinical Microbiology 2006-10, Vol.44 (10), p.3712-3719
Main Authors: Huygens, Flavia, Inman-Bamber, John, Nimmo, Graeme R, Munckhof, Wendy, Schooneveldt, Jacqueline, Harrison, Bruce, McMahon, Jennifer A, Giffard, Philip M
Format: Article
Language:English
Subjects:
Bacterial Typing Techniques - methods
Bacteriology
Biological and medical sciences
Fluorescent Dyes
Fundamental and applied biological sciences. Psychology
Genotype
Infectious diseases
Medical sciences
Microbiology
Miscellaneous
Polymerase Chain Reaction - methods
Polymorphism, Single Nucleotide
Staphylococcus aureus
Staphylococcus aureus - genetics
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Summary: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.
ISSN:0095-1137
1098-660X
1098-5530
DOI:10.1128/JCM.00843-06