2001 Fiscal Year Final Research Report Summary
Preparation of DNA chip of level 2 and 3 pathogenic bacteria and development of diagnostic sutem of infectious diseases
| Project/Area Number |
12557238
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Laboratory medicine
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| Research Institution | Gifu University |
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Principal Investigator |
EZAKI Takayuki Gifu Univ., Microbiology, Professor, 医学部, 教授 (90151977)
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| Co-Investigator(Kenkyū-buntansha) |
KAWAMURA Yoshiaki Gifu Univ., Microbiology, Assist. Professor, 医学部, 助教授 (80262757)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Lebel 2 / Lebel 3 / detection / DNA chip / Identification / Microarray / Multiplex PR / Diagnosis |
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| Research Abstract |
Human pathogenic bacteria are classified as level 2 ( 350 species ) and level 3 ( 33 species ). We prepared ribosomal RNA microarray for most of these level 2 and 3 pathogens to establish comprehensive identification and detection system of pathogenic bacteria. PCR products of ribosomal DNA of these were quantitatively Spotted on glass slide. Cye3 or Cye5 labeled PCR products from unidentified bacterial colonies were hybridized On DNAs of slides and hybridized DNAs were quantitatively measured by a laser scanner. Ribosomal RNA array could not differentiate some potent pathogens which shared similar ribosomal RNA sequences. Therefore, we prepared pathogenic factprs DNA microarray for them.
Immobilizing these two different sources of PCR products, we could identify and detect most of human pathogenic level 2 and level 3 pathogens. Universal primers were not applicable to amplify contaminated sample such as stool, sputum and urinary smears. So we prepared each specific primer and mixed in a single tube to simplify amplification procedure. This method successfully worked. We mixed more than 50-100 different specific primers but could successfully amplify DNA of pathogens.
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[Publications] Ezaki,T., Kawamura,Y., Li,N., Li,Z-Y., Zhao,L., and Shu,S.: "Propo sal of genera Anaerococcus, gen. nov., Peptoniphilus, gen. nov., and Catonia, gen. nov. for members of Genus Peptostreptococcus."Int. J. Syst. Evol Microbiol. 1528 (2001)
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[Publications] Xu,H.X, Kawamura,Y., Li,N., Zhao,L., Li,T.M., Shu,Z.H., and Ezaki,T.: "Arapid method to determine the G+C ntent of bacterial chromosomes by monitoring fluorescence intensity during DNA denaturation in a capillary tube"Int. J. Syst. Evol Microbiol. 50. 1463-1469 (2000)
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[Publications] Hirose,K., Itoh,Ken-ichiro, Nakajima,Hiroshi,Kurazono,Takayuki, Yamaguchi,Masanori, Moriya,Kazuo, Ezaki,Takayuki, Kawamura,Yoshiaki, Tamura,Kazuyuki, and Watanabe,Haruo: "amplification of tyv ( rfbE ), prt ( rfbS ), viaB and fliC genes by multiplex PCR for identification of Salmonella enterica serovar Typhi, and Paratyphi A"J. Clin Microbiol. 40. 633-636 (2001)
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