Detail Molecular Identification of Bacteria by Fluorescence-Based PCR–Single-Strand Conformation Polymorphism Analysis of the 16S rRNA Gene (Journal of Clinical Microbiology, Vol.33 No.10, October 2006) Bibliografi Author: Widjojoatmodjo, Myra N. ; Fluit, Ad C. ; Verhoef, Jan Topik: PCR–single-strand conformation polymorphism analysis; PCR-SSCP; 16S rRNA Gene; mutations; allelic variants Bahasa: (EN )    Edisi: October 2006     Penerbit: American Society for Microbiology     Tempat Terbit: Washington    Tahun Terbit: 2006     Jenis: Article - diterbitkan di jurnal ilmiah nasional Fulltext: Molecular Identification of bacteria by fluorescence-based-PCR.pdf (224.9KB; 5 download) Abstract PCR–single-strand conformation polymorphism (PCR-SSCP) analysis is a rapid and convenient technique for the detection of mutations and allelic variants. We have adapted this technique for the identification of bacteria by PCR with fluorescein-labeled primers chosen from the conserved regions of the 16S rRNA gene flanking a variable region. The PCR product was denatured, separated on a nondenaturing gel, and detected by an automated DNA sequencer. The mobility of the single-stranded DNA is sequence dependent and allows the identification of a broad panel of bacteria. A single nucleotide difference in the amplified region was sufficient to obtain different PCR-SSCP patterns. The simultaneous amplification of multiple polymorphic regions by multiplex PCR with subsequent multiplex SSCP increased the discriminatory power of PCR-SSCP. A broad range of gram-negative and gram-positive bacteria were tested by PCR-SSCP, including, e.g., Escherichia coli, Enterobacter spp., Klebsiella spp., Haemophilus spp., Neisseria spp., Staphylococcus spp, Streptococcus spp., Enterococcus spp., and Bacillus spp. In total, a panel of 178 strains of bacteria representing 51 species in 21 genera was examined. Although a limited number of strains from each species were tested, the strains tested gave species-specific patterns, with only one exception: Shigella species were indistinguishable from E. coli. PCR is a sensitive technique; as few as 10 CFU of E. coli was sufficient to produce PCR-SSCP patterns suitable for identification. The whole fluorescence PCR-SSCP procedure takes approximately 8 h for the detection and identification of low numbers of bacteria. Fluorescence PCR-SSCP seems to be a promising method for the differentiation of a broad range of pathogens found in usually sterile clinical sites, such as blood and cerebrospinal fluid. Opini Anda Klik untuk menuliskan opini Anda tentang koleksi ini! Lihat Sejarah Pengadaan  Konversi Metadata   Kembali

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