Molecular and Phenotypic Methods for Identifying Mycobac

聚合酶链反应（PCR）扩增快速生长的分枝杆菌和诺卡氏菌基因组的一部分，然后对扩增产物进行限制性片段长度多态性（RFLP）分析，已被证明是诊断实验室中有用的技术。与基于生化检测的常规鉴定所需的一个月或更长时间相比，使用该技术可以在微生物分离的几天内获得许多分离株的种水平鉴定。此外，这些分子程序允许更好的物种和亚种之间的区别比可能与生化检测，并促进迄今未描述的物种的检测。我们对诺卡氏菌基因组的两个不同区域（16 S核糖体RNA基因的一部分和热休克蛋白基因的一部分）的研究表明存在几种未被识别的诺卡氏菌;正在进一步研究这些生物体的特征。此外，我们还发现了几种属于兽医诺卡氏菌的临床分离株，这些分离株是新认识到的一个种和一种人类病原体。刚刚发表的一份手稿描述了我们对这种生物的发现，包括将其与另一种新描述的致病物种非洲诺卡氏菌区分开来的问题。即使使用我们的RFLP方法或完整的16 S rDNA测序，也不能精确鉴定诺卡氏菌属的一些分离株。DNA-DNA杂交目前被用来更精确地定义这些分离株。目前正在根据表型和分子生物学特征对几种此类分离株进行表征。我们还希望进一步表征我们发现的几种有趣的诺卡氏菌分离株，这些分离株似乎每个细胞具有几种不同的16 S rRNA基因拷贝。确定这些基因之间的相似程度以及它们中的哪些是功能性的，将具有分类学、生物化学和医学上的意义。此外，我们已经开始与其他拥有广泛微生物收集的人合作，以评估他们的一些分离株可能属于只能使用分子方法可靠鉴定的物种的程度。

Polymerase chain reaction (PCR) amplification of portions of the genome of both rapidly growing mycobacteria and nocardiae, followed by restriction fragment length polymorphism (RFLP) analysis of the amplification products, has proven to be a useful technique in the diagnostic laboratory. Identification of many isolates to the species level can be obtained within a few days of organism isolation using this technique, as compared with the month or more required for conventional identification based on biochemical testing. In addition, these molecular procedures allow better discrimination among species and subspecies than is possible with biochemical testing, and facilitate the detection of hitherto undescribed species. Our work with two different areas of the Nocardia genome (a portion of the gene for 16S ribosomal RNA and a portion of the gene for the heat-shock protein) has suggested the existence of several such unrecognized Nocardia species; work is ongoing to characterize these organisms further. In addition, we have found several clinical isolates belonging to the species Nocardia veterana, newly recognized both as a species and as a human pathogen. A manuscript describing has just been published which describes our findings with regard to this organism, including the problems with distinguishing it from another newly described pathogenic species, Nocardia africana. Some isolates of Nocardia species cannot be precisely identified even with our RFLP procedure or with complete 16S rDNA sequencing. DNA-DNA hybridization is currently being utilized to define these isolates more precisely. Characterization of several such isolates, based based both on phenotypic and molecular biologic features, is currently underway. We also hope to characterize further several interesting Nocardia isolates we have found that appear to possess several different 16S rRNA gene copies per cell. It would be of taxonomic, biochemical, and medical interest to determine the extent of similarity among these genes, and which of them is functional. In addition, we have begun collaborations with others who have extensive organism collections to assess the extent to which some of their isolates may belong to species that can only reliably be identified using molecular methods.