European Society of Mycobacteriology - Instituto Nacional de Saúde ...

OP-1
Mass Spectrometry for Molecular Typing of the Mycobacterium
tuberculosis Complex: One Platform and Multiple Assay Formats
C. Honisch 1 , M. Mosko 1 , C. Arnold 2 , S. Gharbia 2 , S. Feuerriegel 3 , S. Niemann 3
1 - SEQUENOM, Inc., San Diego
2 - Health Protection Agency, London
3 - Molecular Mycobacteriology, NRC for Mycobacteria, Forschungszentrum Borstel, Borstel
Objectives
The analysis of nucleic acids by mass spectrometry has evolved to a user friendly technology for characterizing DNA,
and RNA via SNP genotyping and comparative sequencing in clinical research, agricultural applications, molecular medicine
and non-invasive prenatal diagnostics research. Recently, the technology has become a versatile tool for microbial
detection and identification utilizing comparative sequence analysis. An example is the successful application to 16S based
typing of mycobacteria. Here, we adopted the technology to perform high throughput spoligotyping and detection of
resistance conferring SNPs.
Methods
Assays were designed in silico for spoligotyping analysis and detection of key resistance mutations. Both assays were evaluated
by using well characterized reference collections.
Results
For MassARRAY 43 spacer oligonucleotide probes were designed and grouped into two multiplexed assays (TypePLEX TM ).
Over 200 characterized strains from different reference centers representing the major M. tuberculosis complex lineages
were analyzed by the MassARRAY spoligotyping assays. Results were in concordance with classical spoligotyping data.
For detection of resistance mutations, assay were developed based on the MassCLEAVE TM system. Resistance regions
are amplified by PCR with a tagged primer system 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. Resistance is identified by correlating acquired spectra with theoretical peak patterns predicted for in silico
cleavages of sequences contained in a reference database. The first assays have been successfully evaluated in a set of
reference strains, further analyses are in progress.
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. Existing typing
schemes can be translated onto the mass spectrometry platform and new typing schemes can easily be developed.
European Society of Mycobacteriology | 30 th Annual Congress | July 2009 | Porto - Portugal
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