研究目的
To analyze a modified method for extracting proteins from M. bovis, in comparison with conventional method, and to evaluate an updated database with proper reference spectra, for identification of M. bovis in bovine/bubaline clinical samples, cultured in solid medium.
研究成果
The use of the MycoLyser method associated with the expansion of the database with proper reference mass spectra resulted in the successful identification of M. bovis in bovine/bubaline clinical samples cultured in solid medium. MALDI-TOF MS provides a short-term and low-cost method for M. bovis identification and may become a useful tool for bovine tuberculosis diagnosis in the future.
研究不足
The study was unable to obtain isolates from other MTC members in addition to M. bovis and M. tuberculosis, which was a limitation. Also, the study did not evaluate spectra of M. bovis BCG, since the scope of the study was restricted to bovine/bubaline tuberculosis.
1：Experimental Design and Method Selection:
The study used MALDI-TOF mass spectrometry for the identification of Mycobacterium bovis from cattle and buffalo tissue isolates. The protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria.
2：Sample Selection and Data Sources:
Eighty tissue samples from cattle and buffalo, naturally infected with or without lesions suggestive of tuberculosis, were obtained from North and South regions of Brazil.
3：List of Experimental Equipment and Materials:
The study utilized the Autoflex III Smartbeam mass spectrometer (Bruker Daltonics), MagNA Lyser apparatus (Roche Life Science), and other materials for protein extraction and analysis.
4：Experimental Procedures and Operational Workflow:
The protein extraction protocol, MycoLyser, was developed and compared with the conventional MycoEx method. The mass spectra were acquired and processed using FlexControl
5：3 software. Data Analysis Methods:
The mass spectra were processed using the Bruker MALDI Biotyper? 3.1 software, with default settings. The reliability of the identification was evaluated based on the consistency of the three highest scores regarding the identification of the same species/genus.
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