The use of ITS1 rDNA PCR in detecting pathogenic African trypanosomes

Abstract

There are 11 different pathogenic trypanosomes in trypanosomiasis endemic regions of Africa. Their detection and characterisation by molecular methods relies on species-specific primers; consequently several PCR tests have to be made on each sample. Primers ITS1 CF and ITS1 BR, previously designed to amplify the internal transcribed spacer (ITS1) of rDNA, have been evaluated for use in a universal diagnostic test for all pathogenic trypanosomes. Blood was collected from 373 cattle and 185 camels. The primers gave constant PCR products with the stocks of each taxon tested. Members of subgenus Trypanozoon (T. brucei brucei, T. evansi, T. b. rhodesiense and T. b. gambiense) gave a constant product of approximately 480 bp; T. congolense, savannah 700 bp, T. congolense kilifi 620 bp and T. congolense forest 710 bp: T. simiae 400 bp, T. simiae tsavo 370 bp, T. godfreyi 300 bp and T. vivax 250 bp. The sensitivity of the test ranged from 10 pg for Trypanozoon, T. congolense clade and T. vivax to 100 pg for T. simiae and T. godfreyi. The primers detected cases of multi-taxa samples, although the sensitivity was reduced with an increase in the combinations. A better detection rate of trypanosome DNA was recorded with buffy coats than from direct blood. With the field samples, the diagnostic sensitivity was close to the sensitivity obtained using single reactions with species-specific primers for Trypanozoon 38/40 (95%) and T. congolense savannah 30/33 (90.9%) but was lower with T. vivax 25/31 (77.4%). The primers offer promise as a routine diagnostic tool through the use of a single PCR; however, further evaluation is recommended.

Bovine trypanosome species prevalence and farmers’ trypanosomiasis control methods in south-western Uganda

“…Eluted DNA samples were screened for different trypanosome species using a single pair of primers (ITS1 CF, 5´CCGGAAGTTCACCGATATTG-3´ and ITS1 BR, 5´TTGCTGCGTTCTTCAACGAA-3´) previously designed to amplify internal transcribed spacer (ITS1) of different trypanosomes ribosomal deoxyribonucleic acid (rDNA) (Njiru et al 2005). The 250, 480 and approximately 700 base pair fragments corresponding to T. vivax, T. brucei s.l.…”

“…The PCR was carried out in 25 μL reaction volume, 20 μL of which was the PCR master mix containing 10x-reaction buffer (670 mM Tris-HCl pH 8.8, 166 µM [NH4] 2 SO 4 , 4.5% Triton X-100, 2 mg/mL gelatine) (Fisher Biotech), 1.0 mM MgCl 2 , 200 µM of each dNTP, 5 µM each of the CF and BR primers, 0.5 U of Taq DNA polymerase (Fisher Biotech), 15.2 μL RNase-free water and 5 μL of extracted test sample DNA or positive control DNA or negative control eluate. PCR was carried out in a DNA Engine Dyad ® Cycler (PTC-0221, Bio-Rad Laboratories Inc.) at cycling conditions including a denaturation step at 95 °C for 5 min, 35 cycles of denaturation at 94 °C for 40 s each cycle, annealing at 58 °C for 40 s, extension at 72 °C for 1.5 min and a final elongation step at 72 °C for 5 min (Njiru et al 2005). PCR products were electrophoresed in 1.5% agarose (Bio Tolls Inc., Japan), stained in GelRed™ (Biotium Inc., Hayward, USA) and visualised on an ultraviolet transilluminator for fragment size determination (also see Muhanguzi et al 2014).…”

Molecular Characterization and Phylogenetic Analysis of Trypanosoma evansi from Local and Imported Camels in Egypt

“…There are 11 different pathogenic trypanosomes known to exist in Africa [2]. The subgenus of Trypanozoon comprises three socio-economically important and highly pathogenic African trypanosomes; Trypanosoma brucei, T. evansi and T. equiperdum [3].…”

Molecular Diagnosis of Natural Infection of Trypanosoma Congolense in a Dog in Abeokuta, Nigeria: A Case Report

“…Two sets of primers were selected for optimization based on the published work. The primer sets include TBR 1 & 2 [9] and ITS1 CF & BR [10].…”