A single test approach for accurate and sensitive detection and taxonomic characterization of Trypanosomes by comprehensive analysis of internal transcribed spacer 1 amplicons
AuthorsGaithuma, Alex Kiarie
Permanent link to this recordhttp://hdl.handle.net/10754/631794
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AbstractTo improve our knowledge on the epidemiological status of African trypanosomiasis, better tools are required to monitor Trypanosome genotypes circulating in both mammalian hosts and tsetse fly vectors. This is important in determining the diversity of Trypanosomes and understanding how environmental factors and control efforts affect Trypanosome evolution. We present a single test approach for molecular detection of different Trypanosome species and subspecies using newly designed primers to amplify the Internal Transcribed Spacer 1 region of ribosomal RNA genes, coupled to Illumina sequencing of the amplicons. The protocol is based on Illumina’s widely used 16s bacterial metagenomic analysis procedure that makes use of multiplex PCR and dual indexing. Results from analysis of wild tsetse flies collected from Zambia and Zimbabwe show that conventional methods for Trypanosome species detection based on band size comparisons on gels is not always able to accurately distinguish between T. vivax and T. godfreyi. Additionally, this approach shows increased sensitivity in the detection of Trypanosomes at species level with the exception of the Trypanozoon subgenus. We identified subspecies of T. congolense, T. simiae, T. vivax, and T. godfreyi without the need for additional tests. Results show T. congolense Kilifi subspecies is more closely related to T. simiae than to other T. congolense subspecies. This agrees with previous studies using satellite DNA and 18s RNA analysis. While current classification does not list any subspecies for T. godfreyi, we observed two distinct clusters for these species. Interestingly, sequences matching T. congolense Tsavo (now classified as T. simiae Tsavo) clusters distinctly from other T. simiae Tsavo sequences suggesting the Nannomonas group is more divergent than currently thought thus the need for better classification criteria. This method presents a simple but comprehensive way of identification of Trypanosome species and subspecies-specific using one PCR assay for molecular epidemiology of trypanosomes
CitationGaithuma AK, Yamagishi J, Martinelli A, Hayashida K, Kawai N, et al. (2019) A single test approach for accurate and sensitive detection and taxonomic characterization of Trypanosomes by comprehensive analysis of internal transcribed spacer 1 amplicons. PLOS Neglected Tropical Diseases 13: e0006842. Available: http://dx.doi.org/10.1371/journal.pntd.0006842.
SponsorsThis study was funded by AMED program of the International Collaborative Research Program for Tackling NTD (Neglected Tropical Disease) Challenges in African countries (JP18jm0510001) and supported by Ministry of Education, Culture, Sports, Science and Technology through the Graduate School of Veterinary medicine, Hokkaido University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PublisherPublic Library of Science (PLoS)
JournalPLOS Neglected Tropical Diseases
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