Evaluation of MLST for Staphylococcus aureus typing using labelled-primers chemistry
Abstract number: P934
Chini V., Anastassiou E.D., Spiliopoulou I.
Objective: Staphylococcal typing is important for studying nosocomial and community outbreaks, defining the relationship between isolates. Multilocus Sequence Typing (MLST) relates staphylococci on the basis of the nucleotide sequences of ~450 bp internal fragments of seven conserved housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, yqiL). For each gene fragment, the different sequences are assigned as distinct alleles at each of the seven housekeeping loci (the allelic profile or sequence type, ST). A major advantage of MLST is the ability to compare results obtained by different studies via Internet, in freely accessible databases. The purpose of the present study was the evaluation of MLST using the labeled-primers chemistry.
Methods: Five representative MRSA were selected according to their phenotypic characteristics, SCCmec and agr types, PFGE, and the presence of toxin genes. These strains were also previously characterised by MLST using the BigDye Terminator chemistry (BigDye Terminator v3.1 cycle sequencing Kit, Appliedbiosystems) and the ABI 3730 XL Capillary Sequencing (Appliedbiosystems) device. Bacterial cultures in Brain Heart Infusion broth (BHI), after overnight incubation at 37°C, were used for DNA extraction applying the phenol:chloroform method. The seven housekeeping genes were first amplified by PCR. PCR products were then purified and sequenced bi-directionally by using labeled-primers chemistry (Cy5.5 for the forward and Cy5.0 for reverse primers) and the OpenGene DNA Sequencing System (Visible Genetics, Inc. Toronto, Ontario, Canada). PCR conditions were optimised in experiments applying gradient of primer concentrations.
Results: Optimised final concentration of primers was achieved at 0.1microM for the downstream and 0.15microM for the upstream in each reaction tube. Results from both MLST techniques concerning the allelic profiles, the Sequence Types (ST) and the Clonal Complexes (CC) agreed and no variations were detected.
Conclusion: The adaptation of MLST using the labeled-primers chemistry and vertical electrophoresis, which is for the first time reported, gave reproducible results, allowing its application in laboratories that do not possess a capillary sequencing device.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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