Bull T J, Shanson D C, Archard L C
Medical Microbiology Department, Charing Cross and Westminster Medical School, London W6 8RF.
Clin Mol Pathol. 1995 Jun;48(3):M124-32. doi: 10.1136/mp.48.3.m124.
Aim-Rapid differentiation of mycobacterial species at the genomic level.Methods-The manganese superoxide dismutase (SOD) gene (464 bp) and 16SrRNA (353 bp) from 104 isolates (18 species) of mycobacteria were amplified using polymerase chain reaction (PCR). Products were sequenced and a phenogram of SOD sequences derived. PCR products of SOD gene were digested with HaeIII, and restriction fragment profiles visualised using capillary electrophoresis.Results-Novel SOD sequences were found for M szulgai, M marinum, M phlei, M smegmatis, M chelonei, M paratuberculosis, M malmoense, M intracellulare serotype 7, M intracellulare serotype 18, and M celatum types 1, 2, and 3. Phylogenetic analysis indicated that 18 of 19 species studied had 8-29% interspecies and <6% intraspecies sequence diversity in the SOD gene. No consistent differences were detected between AIDS and non-AIDS isolates. M paratuberculosis showed a unique SOD sequence with a 1.1% (SD 0.5%) diversity from M avium. Capillary electrophoresis profiles were able to differentiate 16 of 18 species within 24 hours.Conclusions-A phenogram of SOD sequences clearly delineated all mycobacterial species and showed two distinct clusters, fast growing species, and the M avium complex (MAC). Within the MAC, M avium (five types), M intracellulare (five types), M scrofulaceum (two types), and M paratuberculosis (one type) could be demonstrated. Phylogenetic diversity of M celatum from MAC, previously suggested by 16SrRNA data, was confirmed. This simple and rapid method for DNA extraction, in conjunction with capillary electrophoresis of SOD restriction fragments, allows rapid identification of mycobacterial isolates.
目的——在基因组水平快速鉴别分枝杆菌菌种。方法——使用聚合酶链反应（PCR）扩增104株（18个菌种）分枝杆菌的锰超氧化物歧化酶（SOD）基因（464bp）和16S rRNA（353bp）。对产物进行测序并得出SOD序列的系统发育树状图。用HaeIII消化SOD基因的PCR产物，并使用毛细管电泳观察限制性片段图谱。结果——发现了堪萨斯分枝杆菌、海分枝杆菌、草分枝杆菌、耻垢分枝杆菌、龟分枝杆菌、副结核分枝杆菌、马尔默分枝杆菌、细胞内分枝杆菌血清型7、细胞内分枝杆菌血清型18以及1型、2型和3型塞拉分枝杆菌的新SOD序列。系统发育分析表明，所研究的19个菌种中有18个在SOD基因上具有8% - 29%的种间序列差异和<6%的种内序列差异。在艾滋病患者和非艾滋病患者分离株之间未检测到一致的差异。副结核分枝杆菌显示出独特的SOD序列，与鸟分枝杆菌有1.1%（标准差0.5%）的差异。毛细管电泳图谱能够在24小时内鉴别出18个菌种中的16个。结论——SOD序列的系统发育树状图清晰地描绘了所有分枝杆菌菌种，并显示出两个不同的簇，即快速生长的菌种和鸟分枝杆菌复合群（MAC）。在MAC内，可以区分鸟分枝杆菌（五种类型）、细胞内分枝杆菌（五种类型）、瘰疬分枝杆菌（两种类型）和副结核分枝杆菌（一种类型）。先前16S rRNA数据表明的MAC中塞拉分枝杆菌的系统发育多样性得到了证实。这种简单快速的DNA提取方法，结合SOD限制性片段的毛细管电泳，能够快速鉴定分枝杆菌分离株。
