hsp65 Sequencing for Identification of Rapidly Growing Mycobacteria

Partial sequencing of the hsp65 gene was used for the identification of rapidly growing mycobacteria (RGM). A 441-bp fragment (A. Telenti, F. Marchesi, M. Balz, F. Bally, E. Böttger, and T. Bodmer, J. Clin. Microbiol. 31:175–178, 1993) was amplified and sequenced by an automated fluorescence-based method involving capillary electrophoresis. Type strains of 10 RGM species were first studied. Each species had a unique nucleotide sequence, distinguishing it clearly from the other species. A panel of strains from the four main RGM species responsible for human infections, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium peregrinum, was also studied. There were few sequence differences within each of these species (<2% of bases were different from the type strain sequence), and they had no effect on species assignment. hsp65 sequencing unambiguously differentiated M. chelonae and M. abscessus, two species difficult to identify by classical methods and 16S rRNA gene sequencing. The devised procedure is a rapid and reliable tool for the identification of RGM species.

本研究采用hsp65基因（hsp65 gene）部分测序技术，对快速生长分枝杆菌（Rapidly Growing Mycobacteria, RGM）进行鉴定。研究通过自动化荧光检测结合毛细管电泳的方法，扩增并测序了一段441碱基对（bp）的片段（A. Telenti、F. Marchesi、M. Balz、F. Bally、E. Böttger及T. Bodmer，《临床微生物学杂志》，31卷：175–178，1993年）。首先对10种快速生长分枝杆菌的模式菌株展开研究，结果显示各物种均具有独特的核苷酸序列，可与其他物种清晰区分。同时还对导致人类感染的4种主要快速生长分枝杆菌的菌株集进行了分析，包括脓肿分枝杆菌（Mycobacterium abscessus）、龟分枝杆菌（Mycobacterium chelonae）、偶发分枝杆菌（Mycobacterium fortuitum）以及佩里格林分枝杆菌（Mycobacterium peregrinum）。这些物种内部的序列差异极小（与模式菌株序列相比，碱基差异率低于2%），且该差异不会对物种鉴定结果造成影响。hsp65基因测序可明确区分龟分枝杆菌（M. chelonae）与脓肿分枝杆菌（M. abscessus）——这两种物种通过传统鉴定方法及16S rRNA基因测序均难以准确鉴定。本研究所建立的实验流程是一种快速且可靠的快速生长分枝杆菌物种鉴定工具。