MALDI-TOF 질량분석기를 이용한 바실러스균의 동정
Taxonomic Identification of Bacillus Species Using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry
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배경: 본 연구에서는 그람양성세균 식중독관련 Bacillus 균주를 대상으로 MALDI-TOF MS를 이용한 동정결과를 VITEK 2 system (bioMérieux, France), 16S rRNA 염기서열 분석결과와 비교하고 MALDI-TOF MS 동정률을 높이기 위한 단백체 기반 국내임상분리주 데이터베이스를 제작하고자 하였다. 방법: 세균의 동정은 VITEK 2 system (bioMérieux), API kit (bioMérieux, France), 16S rRNA 유전자 염기서열, MALDI-TOF MS 분석을 이용하였다. 병원성확인을 위하여 multiplex PCR 방법을 이용하여 독성 유전자를 확인하였다. 세균의 단백체 프로파일의 추출 및 데이터베이스는 제작은 MALDI BioTyper 3.0 (Bruker Daltonics, Germany)을 이용하여 수행하였고 16S rRNA 유전자 기반 계통수, repetitive-sequence fingerprinting 계통분석결과와 비교분석을 수행하였다. 결과: Bacillus 균주 30주에 대한 생화학적 동정법, 16S rRNA 유전자 염기서열분석 및 MALDI-TOF MS 방법에 의한 종 수준 동정률은 40%, 80%, 76.3%였다. 제작된 단백체 기반 MSP dendrogram을 16S rRNA 유전자기반 계통수 및 rep-PCR fingerprinting 계통수와 비교하였을 때 종별 구분이 효과적으로 이루어졌음을 확인할 수 있었다. 결론: Bacillus 균주들의 동정에서 MALDI-TOF MS는 VITEK 2 system (bioMérieux)보다 효과적인 동정방법이었다. MALDI- TOF MS는 16S rRNA 유전자 기반 동정법과도 비슷한 수준의 동정률을 나타냈으며 단백체 기반 국내임상분리주 데이터베이스를 지속적으로 확보한다면 동정률이 훨씬 높아질 것으로 기대되었다.
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 MALDITOF MS, respectively. When the major spectrumprofiling 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. Conclusion: Identification of Bacillus strains using MALDI-TOF MS was more effective than that using the VITEK 2 system (bioMérieux), but was similar to that using 16S rRNA gene sequencing. Continual addition to a proteome-based database can result in increased identification rates for MALDI-TOF MS.
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