Simultaneous identification of Haemophilus influenzae and Haemophilus haemolyticus using real-time PCR

Future Microbiol. 2017 Jun:12:585-593. doi: 10.2217/fmb-2016-0215. Epub 2017 Jun 12.

Abstract

Aim: To design a highly specific and sensitive multiplex real-time PCR assay for the differentiation of the pathogen Haemophilus influenzae from its nonpathogenic near-neighbor Haemophilus haemolyticus.

Materials & methods: A comparison of 380 Haemophilus spp. genomes was used to identify loci specific for each species. Novel PCR assays targeting H. haemolyticus (hypD) and H. influenzae (siaT) were designed.

Results & discussion: PCR screening across 143 isolates demonstrated 100% specificity for hypD and siaT. These two assays were multiplexed with the recently described fucP assay for further differentiation among H. influenzae.

Conclusion: The triplex assay provides rapid, unambiguous, sensitive and highly specific genotyping results for the simultaneous detection of hypD and siaT, including fucose-positive H. influenzae (fucP), in a single PCR.

Keywords: Haemophilus haemolyticus; Haemophilus influenzae; TaqMan; fucP; genotyping; hypD; multiplex; real-time PCR; siaT; triplex.

MeSH terms

  • Bacterial Proteins / genetics
  • Genome, Bacterial
  • Genotype
  • Haemophilus / classification*
  • Haemophilus / genetics*
  • Haemophilus / isolation & purification
  • Haemophilus influenzae / classification*
  • Haemophilus influenzae / genetics*
  • Haemophilus influenzae / isolation & purification
  • Haemophilus influenzae / pathogenicity
  • Humans
  • Multiplex Polymerase Chain Reaction / methods*
  • Phylogeny
  • Proteins / genetics
  • Real-Time Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Sequence Analysis, DNA

Substances

  • Bacterial Proteins
  • HypD protein, Bacteria
  • Proteins