Automated comparative sequence analysis by base-specific cleavage and mass spectrometry for nucleic acid-based microbial typing

Traditional microbial typing technologies for the characterization of pathogenic microorganisms and monitoring of their global spread are often difficult to standardize and poorly portable, and they lack sufficient ease of use, throughput, and automation. To overcome these problems, we introduce the...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2007-06, Vol.104 (25), p.10649-10654
Main Authors: Honisch, Christiane, Chen, Yong, Mortimer, Chloe, Arnold, Catherine, Schmidt, Oliver, van den Boom, Dirk, Cantor, Charles R, Shah, Haroun N, Gharbia, Saheer E
Format: Article
Language:English
Subjects:
Alleles
Automation
Bacteria
Bacterial Typing Techniques
Bacteriology
Base Sequence
Biological Sciences
Cluster Analysis
Comparative analysis
Computer software
Deoxyribonucleic acid
DNA
DNA, Bacterial - analysis
Genetic loci
Microorganisms
Molecular Sequence Data
Neisseria meningitidis
Neisseria meningitidis - classification
Neisseria meningitidis - genetics
Neisseria meningitidis - pathogenicity
Nucleotides
Pathology
Polymerase chain reaction
Promoter regions
Reproducibility of Results
RNA
Sequence Analysis, DNA - methods
Sequencing
Signatures
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Summary:Traditional microbial typing technologies for the characterization of pathogenic microorganisms and monitoring of their global spread are often difficult to standardize and poorly portable, and they lack sufficient ease of use, throughput, and automation. To overcome these problems, we introduce the use of comparative sequencing by MALDI-TOF MS for automated high-throughput microbial DNA sequence analysis. Data derived from the public multilocus sequence typing (MLST) database (http://pubmlst.org/neisseria) established a reference set of expected peak patterns. A model pathogen, Neisseria meningitidis, was used to validate the technology and explore its applicability as an alternative to dideoxy sequencing. One hundred N. meningitidis samples were typed by comparing MALDI-TOF MS fingerprints of the standard MLST loci to reference sequences available in the public MLST database. Identification results can be obtained in 2 working days. Results were in concordance with classical dideoxy sequencing with 98% correct automatic identification. Sequence types (STs) of 89 samples were represented in the database, seven samples revealed new STs, including three new alleles, and four samples contained mixed populations of multiple STs. The approach shows interlaboratory reproducibility and allows for the exchange of mass spectrometric fingerprints to study the geographic spread of epidemic N. meningitidis strains or other microbes of clinical importance.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0704152104