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 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.
Honisch
Christiane
C
SEQUENOM, Inc., San Diego, CA 92121, USA. chonisch@sequenom.com
Chen
Yong
Y
Mortimer
Chloe
C
Arnold
Catherine
C
Schmidt
Oliver
O
van den Boom
Dirk
D
Cantor
Charles R
CR
Shah
Haroun N
HN
Gharbia
Saheer E
SE
D000483:Alleles; D015373:Bacterial Typing Techniques; D001483:Base Sequence; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000032:analysis*; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004269:DNA, Bacterial