Murdoch University Research Repository

Welcome to the Murdoch University Research Repository

The Murdoch University Research Repository is an open access digital collection of research
created by Murdoch University staff, researchers and postgraduate students.

Learn more

Species-level identification of trypanosomes infecting Australian wildlife by High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR)

Keatley, S., Botero, A., Fosu-Nyarko, J., Pallant, L., Northover, A. and Thompson, R.C.A. (2020) Species-level identification of trypanosomes infecting Australian wildlife by High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR). International Journal for Parasitology: Parasites and Wildlife . In Press.

[img]
Preview
PDF (In Press)
Download (1MB) | Preview
Free to read: https://doi.org/10.1016/j.ijppaw.2020.11.003
*No subscription required

Abstract

Conventional nested PCR and Sanger sequencing methods are currently the gold standards for detecting trypanosomes in wildlife. However, these techniques are time-consuming and can often overlook mixed infections. True trypanosome prevalence can thus be underrepresented. Here, we designed an 18S rDNA-based real-time quantitative PCR (qPCR) assay coupled with High-Resolution Melting Analysis (HRMA) to detect and discriminate three Trypanosoma species (T. copemani, T. noyesi, and T. vegrandis) commonly infecting Australian marsupials. A total of 68 genetically characterised samples from blood and tissue were used to validate the High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR) assay. A further 87 marsupial samples consisting of blood, tissue and in vitro cultures derived from wildlife blood samples, were screened for the first time using this assay, and species identity confirmed using conventional PCR and Sanger sequencing. All three Trypanosoma species were successfully detected in pure cultures using the HRM-qPCR assay, and in samples containing mixed trypanosome infections. Of the 87 marsupial samples screened using the HRM-qPCR assay, 93.1% were positive for trypanosomes, and 8.0% contained more than one trypanosome species. In addition to the three targeted Trypanosoma species, this assay was also able to detect and identify other native and exotic trypanosomes. The turnaround time for this assay, from sample preparation to obtaining results, was less than 2 h, with a detection limit of 10 copies of the amplicon in a reaction for each of the targeted trypanosome species. This more rapid and sensitive diagnostic tool provides a high throughput platform for the detection, identification and quantification of trypanosome infections. It will also improve understanding of host diversity and parasite relationships and facilitate conservation management decisions.

Item Type: Journal Article
Murdoch Affiliation(s): College of Science, Health, Engineering and Education
Western Australian State Agricultural Biotechnology Centre
Publisher: Elsevier Limited
Copyright: © 2020 The Authors.
United Nations SDGs: Goal 15: Life on Land
URI: http://researchrepository.murdoch.edu.au/id/eprint/58831
Item Control Page Item Control Page

Downloads

Downloads per month over past year