| Co-Investigator(Kenkyū-buntansha) |
HASHIMOTO Koichi Fukushima Medical University, School of Medicine, Assistant professor, 助手 (50322342)
MUNAKATA Mitsuru Fukushima Medical University, School of Medicine, Professor, 教授 (00209991)
SITOH Junpei Fukushima Medical University, School of Medicine, Assistant professor, 助手 (50332929)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2005: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Accurate identification of bacterial species is essential for the diagnosis and treatment of infectious diseases. However, identification of mycobacteria to a species level by traditional techniques based on the analysis of biochemical characteristics of cultured clones is difficult because of their slow replication and the overlapping phenotypic patterns among species. To meet the need for more rapid and accurate identification of mycobacteria, molecular methods utilizing the amplification of DNA by PCR have been developed. In particular, sequence-based methodologies have taken their places as part of routine clinical examination.
For identification of species by sequence, the 16S rRNA gene has been considered to be the "golden standard". However, as there are no differences in the 16S rRNA sequence between M. kansasii and M gastri, M cbelonae and M abscessus, and M.marinum and M ulcerans, an additional gene is required for analysis and various genes have been reported as potential can
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didate target genes. Among these, we chose the rpoB and secA genes for evaluation as potential target genes in this study. The results indicated that the secA gene is more effective than the rpoB gene and 30 reference strains of mycobacteria were accurately identified by sequencing both the 16S rRNA and secA genes. Moreover, 213 of 214 clinical isolates obtained in Fukushima Medical University Hospital during the period from 2001 to 2004 could be identified using the method.
Mycobacterium tuberculosis complex (MTC) consists of M.tuberculosis, M.bovis, M.bovis BCG, M.africanum, and M.microti. There are no difference in gene sequence among members of the MTC ; therefore, identification methods based on gene sequence can not be applied for these species. A new method that can distinguish between species in the MTC through the analysis of deletions or additional genes using PCR was reported. In this study, we analysed two clinical cases using the novel method; one was successfully diagnosed as a case of BCG vertebral osteomyelitis following intravesical BCG therapy, and the other was diagnosed as a case of BCG dermatitis appearing more than 50 years after vaccination. These results indicate that the clinical application of this method is important for the differentiation of BCG from M tuberculosis infections. Less
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