Consensus PCR and Microarray for Diagnosis of the Genus Staphylococcus, Species, and Methicillin Resistance

Biotechniques. 2001 Dec;31(6):1364-6, 1368, 1370-2. doi: 10.2144/01316md04.

Abstract

We propose the use of DNA microarray for the discrimination of homologous products after a single PCR amplification with consensus primers. The method was applied to Staphylococcus identification. The femA nucleotide sequences, which are phylogenetically conserved among the staphylococci, were first amplified using a consensus primer pair together with the mecA sequence, a molecular marker for methicillin resistance. Products were then identified on a glass array. The microarray contained five selective DNA capture probes for the simultaneous and differential identification of the five most clinically relevant staphylococcal species (S. aureus, S. epidermidis, S. haemolyticus, S. hominis, and S. saprophyticus), while a consensus capture probe could detect all femA sequences, allowing the identification of the genus Staphylococcus. The mecA sequence hybridized to a specific capture probe. The identification was univocal because only a single capture probe had to be present for each sequence to be identified. The hybridization and identification processes were completed in less than 2 h. Current results demonstrate that low-density microarrays are powerful multigenotypic post-PCR analyzers and could compete with conventional bacteria identification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Carrier Proteins / genetics
  • DNA, Bacterial / analysis
  • Hexosyltransferases*
  • Methicillin Resistance*
  • Muramoylpentapeptide Carboxypeptidase / genetics
  • Oligonucleotide Array Sequence Analysis*
  • Penicillin-Binding Proteins
  • Peptidyl Transferases*
  • Plasmids / genetics
  • Polymerase Chain Reaction*
  • Sensitivity and Specificity
  • Staphylococcus aureus / drug effects
  • Staphylococcus aureus / genetics*
  • Staphylococcus epidermidis / drug effects
  • Staphylococcus epidermidis / genetics

Substances

  • Bacterial Proteins
  • Carrier Proteins
  • DNA, Bacterial
  • FemA protein, Bacteria
  • Penicillin-Binding Proteins
  • Peptidyl Transferases
  • Hexosyltransferases
  • Muramoylpentapeptide Carboxypeptidase