One platform and multiple assay formats: mass spectrometry for molecular typing of the Mycobacterium tuberculosis complex
Abstract number: P509
Honisch C., Niemann S., Mosko M., Arnold C., Gharbia S.
Objectives: The analysis of nucleic acids by mass spectrometry (MS) has evolved to a user friendly technology for characterising DNA, and RNA in clinical research and molecular medicine. Recently, the technology has become a versatile tool for microbial identification utilising comparative sequence analysis as shown for 16S based typing of mycobacteria .
Here, we present examples for the development of MS specific assays for molecular typing of the M. tuberculosis Complex including spoligotyping and antibiotic resistance identification, which is essential for epidemiological analysis. One technology, the MassARRAY® platform can be used with different assay formats and typing schemes to obtain results from species to strain identification in an automated fashion.
Methods: Nucleic acid analysis by MS is based on PCR amplifications using unique primer sets.
Traditional spoligotyping detects the presence or absence of 43 different spacer sequences. For MS 43 spacer oligonucleotide probes were designed and the presence of a spacer is detected by base extension (TypePLEX).
Resistance regions are amplified by PCR with a tagged primer system in multiplex followed by in vitro transcription of both DNA strands. Subsequent endonuclease digests of the RNA transcripts at the bases cytosine and uracil result in four mixtures of RNA cleavage products (iSEQ). Resistance is identified by correlating acquired spectra with theoretical peak patterns predicted for in silico cleavages of sequences contained in a reference database. Microheterogeneities are identified and deliver new resistance types.
Results: Over 200 characterised strains from different reference centres representing the major M. tuberculosis Complex lineages were run over the established spoligotyping and resistance assays. Results were in concordance with traditional spoligotyping and dideoxy sequencing data.
Advantages of the MS approach are the homogeneous assay formats without any clean-up steps, semi-automated processing, the time-to result with a throughput of 192 samples in 8 hours for spoligotyping and plexing capabilities for comparative sequence analysis of multiple genomic regions in one reaction.
Conclusion: Mass spectrometry specific assay formats for genotyping and comparative sequence analysis generate highly accurate qualitative and quantitative data and provide a toolbox for molecular typing of microbes and viruses.
|Session name:||19th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Helsinki, Finland, 16 - 19 May 2009|
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