Single nucleotide polymorphisms and copy-number variations in the Trypanosoma brucei repeat (TBR) sequence can be used to enhance amplification and genotyping of Trypanozoon strains

Abstract

The Trypanosoma brucei repeat (TBR) is a tandem repeat sequence present on the Trypanozoon minichromosomes. Here, we report that the TBR sequence is not as homogenous as previously believed. BLAST analysis of the available T. brucei genomes reveals various TBR sequences of 177 bp and 176 bp in length, which can be sorted into two TBR groups based on a few key single nucleotide polymorphisms. Conventional and quantitative PCR with primers matched to consensus sequences that target either TBR group show substantial copy-number variations in the TBR repertoire within a collection of 77 Trypanozoon strains. We developed the qTBR, a novel PCR consisting of three primers and two probes, to simultaneously amplify target sequences from each of the two TBR groups into one single qPCR reaction. This dual probe setup offers increased analytical sensitivity for the molecular detection of all Trypanozoon taxa, in particular for T.b. gambiense and T. evansi, when compared to existing TBR PCRs. By combining the qTBR with 18S rDNA amplification as an internal standard, the relative copy-number of each TBR target sequence can be calculated and plotted, allowing for further classification of strains into TBR genotypes associated with East, West or Central Africa. Thus, the qTBR takes advantage of the single-nucleotide polymorphisms and copy number variations in the TBR sequences to enhance amplification and genotyping of all Trypanozoon strains, making it a promising tool for prevalence studies of African trypanosomiasis in both humans and animals.

SHERLOCK4HAT: a CRISPR-based tool kit for diagnosis of Human African Trypanosomiasis

“…Therefore, the development of a robust diagnostic hinges upon the ability to detect all parasite strains or variants. To demonstrate the robust specificity of our SHERLOCK4HAT diagnostic kit, we analyzed total RNAs from 57 Trypanozoon strains, isolated from their host over the course of 50 years and maintained at the Institute of Tropical Medicine (ITM, Antwerp, Belgium) ( 40 ) (table S4). Using our two-step SHERLOCK assay, all samples were positive for 7SLRNA , confirming 7SLRNA SHERLOCK as a pan- Trypanozoon diagnostic and epidemiological tool (Fig.…”

“…The collection contained 50 T. b. gambiense group 1 (46 bloodstream forms + 4 insect forms), 1 T. b. gambiense group 2, 2 T. b. rhodesiense , 1 T. b. brucei , 1 T. equiperdum and 2 T. evansi strains or clones. They were kindly provided by Nick Van Reet and Philippe Büscher (Institute of Tropical Medicine [ITM], Antwerp, Belgium) ( 40 ). The RNA was kept at −80 and the concentration normalized to 5 ng/µL.…”

Deep kinetoplast genome analyses result in a novel molecular assay for detecting Trypanosoma brucei gambiense-specific minicircles

“…Each qPCR targeting Tbg 1-specific MSCs was multiplexed with the Trypanozoon -specific q18S-assay targeting the multi-copy 18S rRNA gene ( 53 ), the Trypanozoon -specific qGPI-PLC-assay targeting the single-copy glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) gene ( 53 ) and a Tbg 1-specific qTgsGP-assay, designed to target the single-copy TgsGP gene and avoid amplification of TgsGP-like genes ( 21 ). The multi-copy 18S rRNA gene was used as an internal standard for the sensitive detection of Trypanozoon DNA.…”

“…The single-copy TgsGP gene was used as a golden standard for the specific detection of Tbg 1 DNA. The q18S-assay contains a CAL Fluor Orange 56 dye labeled probe paired with BHQ-1 plus ( 53 ). The qGPI-PLC-assay contains a CAL Fluor Red 610 dye labeled probe paired with BHQ-2 plus ( 53 ).…”

“…The q18S-assay contains a CAL Fluor Orange 56 dye labeled probe paired with BHQ-1 plus ( 53 ). The qGPI-PLC-assay contains a CAL Fluor Red 610 dye labeled probe paired with BHQ-2 plus ( 53 ). The qTgsGP-assay contains a Quasar 670 dye labeled probe paired with BHQ-2 plus .…”