Abstract：Objective To compare the reversed phase high performance liquid chromatography (RP-HPLC) and 16S rRNA sequence analysis for the identification of Mycobacterium species.
Methods 49 Mycobacterium species which recorded in ‘Bergey’s manual of systematic bacteriology’ were inoculated in Lowenstein cultured medium. Mycolic acids from each culture of Mycobacterium species were extracted, saponified, acided, derivatized and analyzed by the RP-HPLC and a mycobacteria mycolic acids fingerprints library of HPLC patterns was constructed. The DNA of each culture of Mycobacterium species was also amplified by PCR and purified for the identification by 16S rRNA sequencing.
Results Among 49 Mycobacterium mode strains, 7 kinds of strains, which included single-cluster peak of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium gastri, double-cluster peaks of Mycobacteriumaichiense and Mycobacterium rhodesiae, and triple-cluster peaks of Mycobacterium austroafricanum and cMycobacterium vaccae, were difficult to be identified because of the similarity of relative retention time and relative peak height ratio using RP-HPLC. Using analysis of 16S rRNA sequence analysis, 12 kinds of strains, which included Mycobacteriumfarcinogenes andMycobacterium senegalense, Mycobacterium ulcerans and Mycobacterium marinum,Mycobacterium kansasii and Mycobacteriumgastri, Mycobacteriumchelonae subsp. Chelonae and Mycobacteriumchelonae subsp. Abscessus, and Mycobacterium tuberculosis complex(M. tuberculosis, M. bovis, M. microti and M. africanum), were difficult to be identified because of the same gene sequences. Using the two complementary methods, 47 of the 49 Mycobacterium species could be clearly identified except M. tuberculosis and M. bovis.
Conclusion The combination of RP-HPLC and 16S rRNA sequence analysis provides an accurate and efficient technique for Mycobacterium identification. With these two complementary methods, most of the Mycobacterium species can be well distinguished and identified.
陈保文, 都伟欣, 杜蓉, 闫李侠, 郭磊, 杨蕾, 王国治. 反相高效液相色谱法和16S rRNA序列分析法对分枝杆菌分型鉴别的比较研究[J]. 《中国医药生物技术》杂志, 2012, 7(4): 263-269.
CHEN Bao-Wen, DU Wei-Xin, DU Rong, YAN Li-Xia, GUO Lei, YANG Lei, WANG Guo-Zhi. Identification of Mycobacterium species by reversed phase high performance liquid chromatography and 16S rRNA sequence analysis. , 2012, 7(4): 263-269.
2012, Vol. 7 
