Development of new classification system for pathogenic Nocardia based on whole genome sequences and microarray analysis

我们于2004年报道了farcinica诺卡菌IFM 10152的基因组序列。根据该序列，我们发现并分析了该物种特有的基因。选取法氏乳杆菌特有的238 bp的DNA进行进一步研究。该DNA的一部分部分编码了一个假设的蛋白质（nfa 29510），其余部分是位于膜蛋白（nfa 29520）基因旁边的非编码区域。根据这些遗传分析，设计了一种特异性的PCR引物，并证实了其特异性。此外，该引物可用于检测实验性诺卡菌病小鼠血液中的法氏奈瑟菌。该引物也可用于RealTime PCR。以诺卡菌IFM 10152的gyrB基因序列为基础，设计了一种能在诺卡菌中高效扩增该基因的改良PCr引物。分析了所有60种诺卡菌型菌株的gyrB基因序列，并构建了系统发育树。这使得诺卡菌可以与分枝杆菌和红球菌明确区分，均匀性值分别为82%和76%。在细菌分类中，最常用的是16S rDNA序列，诺卡菌也不例外。60种诺卡菌的差异宽度分布在94.5% ~ 100.0%之间。然而，在我们分析的gyrB中，这一差异显着增加到82.4%至99.9%。gyrB序列在诺卡迪亚属的分类中具有一定的应用价值，并作为诺卡迪亚属的一种新的分类系统。虽然在本研究中使用了1200bp的gyrB序列，但开发一个使用小于600bp基因变异域的系统的进一步工作正在进行中。

We reported the genomic sequence of Nocardia farcinica IFM 10152 in 2004. Based on the sequence, we found and analyzed genes which were specific to this species. DNA of size 238 bp specific to N. farcinica was selected for further studies. A section of this DNA partially encoded a hypothetical protein (nfa 29510), and the remaining was a non-coding region located next to a membrane protein (nfa 29520) gene. From these genetic analyses, a specific PCR primer for N. farcinica strains was designed, and their specificity was confirmed. In addition, the primer was found to be useful for the detection of N. farcinica in the blood of mice with experimental nocardiosis. The primer was also found to be useful for RealTime PCR.Based on the sequence of gyrB gene from N. farcinica IFM 10152, a modified PCr primer for the efficient amplification of the gene in Nocardia was designed. The gyrB gene sequences of all 60 Nocardia type species were analyzed, and a phylogenetic tree was constructed. This allowed the Nocardia species to be clearly differentiated from Mycobacterium and Rhodococcus, with homogeneity values of 82% and 76%, respectively.In bacterial classification, sequence of 16S rDNA is most commonly used, with no exception in Nocardia. As for the width of difference among the 60 Nocardia species, their values are distributed from 94.5 to 100.0%. However, in gyrB which we analyzed, this difference markedly increased to 82.4 to 99.9%. Usefulness of gyrB sequence for the classification of these species was confirmed, and we proposed it as a new classification system n Nocardia. Although 1,200 bp sequence of gyrB was used in the present study, further work is in progress for the development of a system using less than 600 bp from variation domains in the gene.