Molecular identification of bacteria by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.
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.2+ 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.
Widjojoatmodjo
M N
MN
p4p4man Winkler Institute for Medical and Clinical Microbiology, University Hospital Utrecht, The Netherlands.
Fluit
A C
AC
Verhoef
J
J
D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000472:pathogenicity; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D004275:DNA, Ribosomal / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004795:Enzyme Stability; D005453:Fluorescence; D005798:Genes, Bacterial; D006090:Gram-Negative Bacteria / Q000145:classification / Q000235:genetics; D006094:Gram-Positive Bacteria / Q000145:classification / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
D017931:DNA Primers; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D004259:DNA-Directed DNA Polymerase