MSACL 2017 US Abstract

Identification of Mycobacterium to Species Level using MALDI-TOF MS and ASTA MycoDB

Hyung Soon Park (Presenter)
ASTA Inc.

Authorship: Jae-Seok Kim (1), Kyu H. Park (3), Sue Shin (2), Taek Soo Kim (2), Eun Gae Lee (1), Eun-kyeong Choi (3), Hyung Soon Park (3), Yangsun Kim (3)
(1) Department of Laboratory Medicine, Hallym University College of Medicine, (2) Seoul National University College of Medicine, (3) ASTA Inc.

Short Abstract

Over 170 species in the Mycobacterium genus confers the limitation of precise identification with a single test. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) based identification has been successfully implemented in clinical laboratories for bacteria identification and might change the standard identification methods of mycobacteria in clinical laboratories. Here we introduce a linear type MALDI-TOF mass spectrometer, Tinkerbell LT (ASTA, Korea), a mycobacteria MALDI-TOF mass spectrum database, MycoDB v.1.10 (ASTA, Korea) and its evaluation for identification of mycobacteria isolates. MycoDB v.1.10 contains more than 90 species-specific spectra covering most of mycobacterial species that frequently isolated in clinical laboratories in Korea.

Long Abstract

The Mycobacterium genus includes pathogens known to cause diseases in humans such as M. tuberculosis, M. leprae, and M. avium-intracellulare (MAI) and in animals including other mammals, birds, and fishes. It has been reported that there are over 170 species in the Mycobacterium genus conferring the limitation of precise identification with a single test. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been introduced in clinical laboratories for the identification of ordinary bacteria and might change the standard identification methods of mycobacteria in clinical laboratories performing various kinds of nucleic acid tests currently. The thick lipid layer composed of mycolic acid hindered the analysis of proteins or nucleic acids in mycobacteria and inactivation of mycobacteria. Here we introduce a linear type MALDI-TOF mass spectrometer, Tinkerbell LT (ASTA, Korea), a mycobacteria MALDI-TOF mass spectrum database, MycoDB v.1.10 (ASTA, Korea) and its evaluation for identification of mycobacteria isolates. A linear type MALDI-TOF mass spectrometer, Tinkerbell LT (ASTA, Suwon, Korea) was applied to analyze mycobacteria which harnesses Nd:YLF UV laser to provide fast and durable laser source. The positive ions in the range of m/z 2,000-20,000 were accelerated at 18 kV and transmitted to a MCP detector showing mass resolving power of 1000 at m/z 12,000 and mass accuracy of 300 ppm by external calibration. The mycobacteria isolates were inactivated by heating (95¡É for 10 min) and 70% ethanol then subjected to bead-beating in formic acid to extract cytoplasmic material. MALDI-TOF MS analysis of the mycobacterial extracts that mixed with α–cyano-4-hydroxycinnamic acid. A mycobacteria MALDI-TOF mass spectrum database, MycoDB v.1.10 was developed using a number of clinical isolates and type cultures obtained from hospitals and governmental institutes in Korea. The species of the isolates were identified by gene sequence analysis of three genes; 16S rRNA, rpoB, and tuf. Each isolate was cultured in various culture media and repeatedly analyzed by MALDI-TOF MS to obtain a reference spectrum of the cultured isolate. The MycoDB v.1.10 contains more than 90 species-specific spectra covering most of mycobacterial species that frequently isolated in clinical laboratories in Korea.


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