Development of a molecular typing scheme by mass spectrometry

Abstract number: O364

Honisch C., Mosko M., Arnold C., Gharbia S.

Objectives: Comparative sequence analysis in large collections of microbial isolates utilising marker regions provides the framework in molecular typing and a combination of PCR, electronically accessible databases, standardised protocols and automated, high-throughput technologies make the analysis of these regions easily achievable.

We have recently developed a comparative sequence analysis tool based on mass spectrometry, which serves these needs. The technology has successfully been applied to 16S based typing (1) and multi-locus sequence typing (2). Here, we present the workflow, data analysis and statistical measures of the technology for the development of a novel molecular typing scheme for Neisseria gonorrhoeae.

Methods: Marker regions are amplified by PCR with a tagged primer system, which facilitates in vitro transcription of both DNA strands. Subsequent base-specific endonuclease digests of the two RNA transcripts at the bases cytosine and uracil result in four mixtures of RNA cleavage products. The high precision measurement obtained by a mass spectrometer is used to resolve the mixtures. Nucleic acid sequences are identified by correlating the acquired spectra with theoretical peak patterns predicted for in silico cleavages of each sequence contained in a reference sequence database. Microheterogeneities between the best reference and the sample sequence are identified and deliver new reference sequences.

Results: 32 marker regions of potential importance for the development of a molecular typing scheme for N. gonorrhoeae and a set of 267 phenotypically characterised samples were subject to comparative sequence analysis by mass spectrometry. 94% of the marker regions were converted into PCR assays and 83% of all samples resulted in high quality data throughout the set of 30 markers. Data analysis was a result of UPGMA peak pattern clustering for sample grouping, statistical analysis and sequence analysis by a time-efficient SNP Discovery algorithm. Dideoxy sequencing was used to resolve samples of high variance and showed concordance with the mass spectrometry data.

Conclusion: The introduced mass spectrometry based comparative sequence analysis tool provides a rapid alternative to dideoxy sequencing for microbial identification and the development of molecular typing schemes.

References

1. Lefmann, M. et al. (2004). J Clin Microbiol 42(1): 339–46.

2. Honisch, C. et al. (2007). PNAS 104(25): 10649–54.