C-209. Novel Diagnostic Algorithm for Identification of Mycobacterium spp. Using tuf Gene Amplification and Restriction Fragment Length Polymorphism

J-H. Shin, E-J. Cho, J-Y. Lee, J-Y. Yu, Y-H. Kang;
Natl. Inst. of Hlth., Ctr. for Disease Control and Prevention, Seoul, REPUBLIC OF KOREA.

Background: Nontuberculous mycobacteria (NTM) are considered as a major cause of opportunistic infection in those who have immunocompromised patients. The reliable and rapid identification of NTM to the species level is very important for the treatment of patients. However it is not easy to identify and differentiate between closely related NTM species, or novel NTM species, in particular. The aim of the study was to evaluate the usefulness of the tuf gene PCR-restriction fragment length polymorphism (RFLP) analysis for the identification of NTM to the species level. Methods: A total of 44 reference strains of the Mycobacterium genus were used. For PCR, DNA was extracted by a simple boiling method. The 741bp tuf genes were amplified, restricted with restriction enzyme HaeIII, and sequenced. The tuf genes PCR-RFLP patterns were compared with those of the hsp65 (HaeIII) and rpoB (MspI) genes. Fragment band sizes were exactly calculated based on sequence data. Results: The tuf gene PCR-RFLP analysis yielded 44 HaeIII patterns, of which 36 (81.8%) showed unique patterns on the species level, whereas the hsp65 and rpoB genes RFLP analysis showed 27 (61.4%) and 19 (43.2%) sole patterns, respectively. The tuf gene RFLP analysis was able to clearly distinguish closely related NTM species, such as M. abscessus (557-84-58)/M. chelonae (477-84-80-58) and M. kansasii (141-136-80-63-58-54-51)/M. gastri (141-136-117-80-58-51). Mycobacteria species (e. g. M. celatum/M. branderi), that were unable to distinguish by the hsp65 and rpoB genes RFLP, were also correctly identified. Conclusion: The tuf gene targeting PCR-RFLP is a promising novel method with better advantages then previously used hsp65 and rpoB genes based methods for rapid and reliable identification of mycobacteria to the species level.