Doenças Micobacterianas
Acesso livre

Abstrato

Use of Ultrasonication as a Rapid Pretreatment Method for MALDI-TOF MS of Mycobacterial Samples

Katsunao Niitsuma, Shizuko Koshiba, Miwako Saitou, Tomoko Suzuki, Kinuyo Chikamatsu, Akiko Takagi and Satoshi Mitarai

Background: In Japan, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is used as a simple and accurate method for mycobacterial identification, but existing pretreatment techniques are time-consuming and laborious.
Objective: We characterized a new pretreatment technique that extracts proteins using ultrasonic disruption.
Methods: We compared this new technique with the current pretreatment method and examined the usefulness of ultrasonication, including its use in mycobacterial inactivation. A total of 174 mycobacterial isolates were tested, including 50 Mycobacterium tuberculosis complex strains, 57 Mycobacterium avium strains, 55 Mycobacterium intracellulare strains, and 12 Mycobacterium kansasii strains. The ultrasonic pretreatment method was performed with or without heat-pretreatment at 95°C for 30 minutes, in parallel with the current conventional method. For all tested strains, the mycobacterial identification agreed when comparing the new and conventional methods.
Results: Samples prepared by ultrasonication without heat pretreatment exhibited multiple significant differences when compared with samples prepared by ultrasonication with heat pretreatment or by conventional methods. However, most scores were over 2.0, and the lowest score exceeded 1.7. Furthermore, the new techniques could be performed in only 10 minutes for diagnosis. In addition, we confirmed that 1 minute of ultrasonic pretreatment yielded complete inactivation of M. tuberculosis complex strains.
Conclusion: The more rapid technique of protein extraction/inactivation by ultrasonication without heat pretreatment is expected to be highly useful in clinical laboratory settings.