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

目的比较反相高效液相色谱法(RP-HPLC)和16S rRNA序列分析法对分枝杆菌分型鉴别的异同。方法将《伯杰细菌鉴定手册》中载入的49种分枝杆菌模式株接种于改良罗氏培养基上,置于最适温度下孵育。挑取生长良好且无污染的培养物,一部分经皂化和酸化提取分枝菌酸并衍生后,采用反相高效液相色谱法进行分枝菌酸指纹图谱构建;另一部分经裂解和PCR扩增,获得DNA,纯化后,采用核酸分析仪进行16S rRNA序列测定。结果 49种分枝杆菌模式株中,采用RP-HPLC分析时,单簇峰的结核分枝杆菌、牛分枝杆菌和胃分枝杆菌,双簇峰的爱知分枝杆菌和罗德岛分枝杆菌,三簇峰的南非分枝杆菌和母牛分枝杆菌共7种因各组内相对保留时间和相对峰高比值相近而难以进行鉴别;采用16S rRNA序列分析法分析时,产鼻疽分枝杆菌和塞内加尔分枝杆菌、溃疡分枝杆菌和海分枝杆菌、堪萨斯分枝杆菌和胃分枝杆菌、龟亚分枝杆菌和龟脓分枝杆菌以及结核分枝杆菌复合群(结核分枝杆菌、牛分枝杆菌、田鼠分枝杆菌和非洲分枝杆菌)共12种因各组内基因序列相似性百分比为100%而难以进行鉴别。通过两种分型鉴别方法的比较,可见除结核分枝杆菌和牛分枝杆菌外,两种分型方法相互补充,可将49种分枝杆菌模式株中的47种进行明确鉴别。结论分枝菌酸RP-HPLC和16S rRNA序列分析法均为分枝杆菌的分型鉴定提供了准确和有效的技术方法。两种方法相互借鉴能准确地将大多数分枝杆菌鉴定到种。

Identification of Mycobacterium species by reversed phase high performance liquid chromatography and 16S rRNA sequence analysis

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 Mycobacterium aichiense 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 Mycobacterium farcinogenes and Mycobacterium senegalense, Mycobacterium ulcerans and Mycobacterium marinum,Mycobacterium kansasii and Mycobacterium gastri,Mycobacterium chelonae subsp.Chelonae and Mycobacterium chelonae 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.