title: Rapid identification of bacteria by PCR-single-strand conformation polymorphism.
abstract: A new molecular biological approach for the identification of bacteria is described. This approach employs PCR of bacterial cell lysates with conserved primers located in the 16S rRNA sequence flanking a variable region, and analysis of the amplified product was based on the principle of single-strand conformation polymorphism (SSCP). The PCR product was denatured and separated on a nondenaturing polyacrylamide gel. SSCP patterns were detected by silver staining the nucleic acids. The mobility of the single-stranded DNA is sequence dependent and could be used to identify the unknown bacteria. Feasibility of the technique was demonstrated for a broad panel of gram-negative and gram-positive bacteria. We tested over 100 strains of bacteria representing 15 genera and 40 species. With the use of only two primer sets, P11P-P13P and ER10-ER11, we were capable to discriminate the tested species at the genus and species levels. Species-specific patterns were obtained for, e.g., Clostridium spp., Listeria spp., Pseudomonas spp., and Enterobacter spp. PCR-SSCP is a sensitive technique; e.g., the sensitivity obtained for Escherichia coli cells was 30 CFU. This technique is a simple and rapid method for the detection and identification of a wide spectrum of bacteria by whole-cell-based PCR amplification with the use of conserved primers and identification by nondenaturing gel electrophoresis.
authors.affiliation: Eijkman Winkler Institute for Medical and Clinical Microbiology, University Hospital Utrecht, The Netherlands.
authors.forename: M N
authors.initials: MN
authors.lastname: Widjojoatmodjo
authors.forename: A C
authors.initials: AC
authors.lastname: Fluit
authors.forename: J
authors.initials: J
authors.lastname: Verhoef
mesh_terms: D001419:Bacteria / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S