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Abstract
Background
In this study, we compared various methods of taxonomic identification of Bacillus strains: biochemical methods, 16S rRNA gene sequencing, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). We also developed a pathogen-isolate resource database, thus increasing the identification rate when using MALDI-TOF MS.
Methods
Thirty Bacillus strains were obtained from the NCCP (National Culture Collection for Pathogens) and were identified using the VITEK 2 system (bio-Mérieux, France), API kit (bioMérieux, France), 16S rRNA gene sequencing, and MALDI-TOF MS. The pathogenicity of Bacillus cereus was confirmed through the identification of virulent genes using a multiplex PCR, and both protein extraction for protein profiling in MALDI-TOF MS and repetitive-sequence fingerprinting were performed.
Results
The identification rates at the species level were 40%, 80%, and 76.3% for the VITEK 2 system (bioMérieux), 16S rRNA gene sequencing, and MALDI-TOF MS, respectively. When the major spectrum-profiling dendrogram was compared with the phylogenetic tree, which was constructed based on the 16S rRNA gene sequences and rep-PCR fingerprinting, the classifications were confirmed to be effective.
References
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Fig. 1.
Representative spectra from Bacillus strains supplemented in the database by MALDI-TOF MS. The intensity is shown as a percentage of the total intensity on the y-axis, and the mass to charge ration (m/z) is shown on the x-axis. (A) Bacillus cereus NCCP10841 DSMZ32. (B) B. cereus NCCP14796 entFM, CER. (C) B. cereus NCCP15909 entFM, nheA, cytK. (D) B. thuringiensis NCCP11234.(E) B. subtillis NCCP10908. (F) B. licheniformis NCCP11231.
Fig. 3.
Comparison of 16S rRNA gene, repetitive-sequence based PCR fingerprinting and MALDI-TOF MS analysis in Bacillus strains. Phylogenetic tree based by 16S rRNA gene with UPGMA method (A), rep-PCR fingerprinting (B) and MALDI-TOF MS (C).
Table 1.
Bacillus strains used in this study. Pathotypes and serotypes were determined by performing agglutination tests and PCR
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