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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.
Acknowledgements
The authors thank A Leach (Menzies School of Health Research), A Keil (PathWest Laboratory Medicine Western Australia) and LA Kirkham (University of Western Australia) for previously providing isolates for genome sequencing. The authors are also grateful to all researchers in the Pasteurellaceae community who have generously made their genome data publicly available.
Financial & competing interests disclosure
This project was funded by the Channel 7 Children's Research Foundation (awards 13699 and 151068). Clinical specimens used in this project were partly funded by the Australian National Health and Medical Research Council (NHMRC; awards 1023781 and 1042601). EP Price is funded by a USC Research Fellowship, TM Harris is funded by a NHMRC Centre of Research Excellence in Lung Health of Aboriginal and Torres Strait Islander Children Fellowship (1040830) and AB Chang is funded by an NHMRC Practitioner Fellowship (1058213). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.