Therese K Lily, Bartell John, Deepa P, Mangaiyarkarasi S, Ward Diedra, Dajcs Joseph, Madhavan H N, Stroman David
L & T Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
Indian J Med Res. 2009 Feb;129(2):176-81.
BACKGROUND & OBJECTIVES: Identification of mycobacteria to the species level is of therapeutic significance. Conventional methods are laborious and time consuming so we did 16S rRNA sequencing using a commercial MicroSeq sequencing kit, which includes DNA sequencing with software package for identification and phylogenetic analysis of clinical mycobacterial isolates.
A total of 47 mycobacteria were tested by both conventional and genotypic method using commercially available MicroSeq 500 amplification kit assay. The identification was determined by comparing the 500 bp amplified product of 16S rDNA sequence to the MicroSeq database.
The phenotypic identification was concordant with genotypic identification in 33 (70.2%) isolates of 14 Mycobacterium tuberculosis, 11 M. fortuitum, 7 M. abscessus and 1 M. duvalii. For the discrepant isolates, identification was possible only by DNA sequencing in 14 (29.7%) isolates. The 14 discrepant isolates were 5 M. farcinogenes, 3 M. genavense, 2 M. species. nov and 1 each of M. fortuitum, M. immuogenum, M. simiae and M. wolinskyi. Of these, five were uncommon species that were difficult to identify by phenotypic method.
INTERPRETATION & CONCLUSION: The MicroSeq DNA sequencing is an excellent tool for species identification of mycobacteria, which reduces the turn around time, makes repeat analysis easy as compared to phenotypic identification specially for mycobacterial isolates with ambiguous biochemical profiles.
将分枝杆菌鉴定到种水平具有治疗意义。传统方法费力且耗时，因此我们使用商业性的MicroSeq测序试剂盒进行16S rRNA测序，该试剂盒包括用于临床分枝杆菌分离株鉴定和系统发育分析的DNA测序及软件包。
使用市售的MicroSeq 500扩增试剂盒，通过传统方法和基因分型方法对总共47株分枝杆菌进行检测。通过将16S rDNA序列的500 bp扩增产物与MicroSeq数据库进行比较来确定鉴定结果。
在14株结核分枝杆菌、11株偶然分枝杆菌、7株脓肿分枝杆菌和1株杜氏分枝杆菌的33株（70.2%）分离株中，表型鉴定与基因分型鉴定结果一致。对于不一致的分离株，仅通过DNA测序在14株（29.7%）分离株中实现了鉴定。这14株不一致的分离株为5株产鼻疽分枝杆菌、3株日内瓦分枝杆菌、2株新种分枝杆菌，以及各1株偶然分枝杆菌、免疫原性分枝杆菌、猿分枝杆菌和沃林斯基分枝杆菌。其中，5株是难以通过表型方法鉴定的罕见菌种。
MicroSeq DNA测序是分枝杆菌菌种鉴定的优秀工具，它缩短了周转时间，与表型鉴定相比，重复分析更容易，特别是对于生化特征不明确的分枝杆菌分离株。
