Simultaneous identification of Haemophilus influenzaeand Haemophilus haemolyticususing real-time PCR

Aim:To design a highly specific and sensitive multiplex real-time PCR assay for the differentiation of the pathogen Haemophilus influenzaefrom 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 hypDand siaT. These two assays were multiplexed with the recently described fucPassay for further differentiation among H. influenzae. Conclusion:The triplex assay provides rapid, unambiguous, sensitive and highly specific genotyping results for the simultaneous detection of hypDand siaT, including fucose-positive H. influenzae(fucP), in a single PCR.