Molecular and Phenotypic Methods for Identifying Mycobac

对快速生长的分枝杆菌和诺卡菌的部分基因组进行聚合酶链反应（PCR）扩增，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。与基于生化测试的常规鉴定需要一个月或更长时间相比，使用该技术可以在生物分离后几天内获得许多物种水平的分离物的鉴定。此外，与生物化学测试相比，这些分子程序可以更好地区分物种和亚种，并有助于检测迄今未描述的物种。我们对诺卡菌基因组的两个不同区域（16S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，存在几种这样未被识别的诺卡菌；进一步鉴定这些生物的工作正在进行中。此外，我们还发现了几个临床分离株，属于诺卡迪亚兽疫，这是一种新认识的物种和人类病原体。刚刚发表的一份手稿描述了我们关于这种生物的发现，包括将它与另一种新描述的致病物种非洲诺卡迪亚区分开来的问题。即使使用RFLP程序或完整的16S rDNA测序，也无法精确鉴定诺卡菌的某些分离株。DNA-DNA杂交目前被用来更精确地定义这些分离物。基于表型和分子生物学特征，目前正在对几个这样的分离物进行表征。我们还希望进一步表征我们发现的几个有趣的诺卡菌分离株，它们似乎每个细胞具有几个不同的16S rRNA基因拷贝。确定这些基因之间的相似程度，以及其中哪些是功能性的，将是分类学、生物化学和医学上的兴趣所在。此外，我们已经开始与其他拥有广泛生物收集的机构合作，以评估他们的一些分离物可能属于只能用分子方法可靠地识别的物种的程度。

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.