The integrated system with Tm mapping and the FCS-based NF-kB assay for sepsis patients

2014 Fiscal Year Final Research Report

• Project/Area Number: 25670268
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Laboratory medicine
• Research Institution: University of Toyama
• Principal Investigator: KITAJIMA Isao 富山大学, 大学院医学薬学研究部(医学), 教授 (50214797)
• Co-Investigator(Kenkyū-buntansha): NIIMI Hideki 富山大学, 大学病院, 助教 (50401865)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Keywords: 感染症 / 微生物 / 敗血症 / 遺伝子検査 / 転写因子 / 1分子計測

Outline of Final Research Achievements

The earliest possible identification of pathogenic microorganisms is critical for selecting appropriate antimicrobial therapy. We developed a nested-PCR-based assay for detecting Mycoplasma and other bacteria in amniotic fluid samples using eukaryotic, thermostable DNA polymerase. Next, a "melting temperature (Tm) mapping method" was developed for identifying a broad range of bacteria. Employing primer sets for a 16S ribosomal gene, more than 100 bacterial species can be identified. Nuclear transcription factor-kappa B (NF-kB) activation plays a key role in the regulation of immune responses to inflammation. We developed an experimental apparatus for detection of NF-kB activation based on fluorescence correlation spectroscopy (FCS), which permits analysis of DNA-protein binding in the liquid phase. FCS was performed using the single-molecule fluorescence detention system. We have integrated Tm mapping and the FCS based NF-kB assay and are conducting clinical trials in sepsis patients.

Free Research Field

臨床検査