A final extension step occurred at 72 °C for 5 min Each PCR run

A final extension step occurred at 72 °C for 5 min. Each PCR run included a positive control (0.5 ng parasite DNA) and a no-template negative control (5 μl PCR-grade water). PCR products were resolved by electrophoresis at 6 V/cm in 2% (w/v) agarose gels stained with 0.5 μg/ml ethidium bromide, and photographed under ultraviolet light. On all blood specimens that were negative in the ITS1 TD PCR, a PCR for vertebrate cytochrome b was performed (Kocher

et al., 1989 and Fikru et al., 2012). A positive result with vertebrate cytochrome b PCR indicates that a negative ITS1 TD PCR result of the same specimen is not due to poor DNA quality selleck products or presence of inhibitors. The ITS1 TD PCR assay conditions were optimised in order to obtain maximal specificity and sensitivity using parasite-infected mouse blood, and

non-infected blood from mouse, human, bovine, goat, horse and dog. No cross-reactivity was observed with the non-infected blood specimens, while the ITS1 TD PCR allowed detection and differentiation of the trypanosome taxa by amplicon length polymorphism. Indeed, the assay generated amplicons of the expected sizes: 612 bp with T. congolense Savannah type, 165 bp with T. vivax and 391–393 bp with the Trypanozoon subgenus, including T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. evansi, and CH5424802 supplier T. equiperdum. The non-pathogenic T. theileri could be discriminated by a PCR amplicon of approximately 300 bp ( Fig. 1). To assess the lower detection limit

according to the trypanosome taxon, 10-fold serial dilutions of live parasites in 1 ml aliquots of naïve human blood were used. At 200 μl blood per sample, ITS1 TD PCR achieved an analytical sensitivity of 10 parasites/ml blood or 0.2 parasite equivalent/reaction with T. congolense ( Fig. 2), T. brucei and T. evansi (data not shown). The analytical sensitivity of ITS1 TD PCR for T. vivax was 100 parasites/ml blood or 2 parasite equivalent/reaction ( Fig. 3). A total of 246 blood specimens from 57 cattle were examined with ITS1 TD PCR and HCT and results were interpreted with only reference to their infection status with T. congolense Savannah or Kilifi type. Specimens that were negative in ITS1 TD PCR were verified with the vertebrate cytochrome b PCR and were all positive (data not shown). Firstly, specificity and sensitivity of ITS1 TD PCR were evaluated on a collection of 114 reference specimens (69 non-infected and 45 infected specimens) from 57 cattle of known disease status. Specificity of ITS1 TD PCR was 100%, which was in agreement with that of HCT. Sensitivity of ITS1 TD PCR at 14 days post infection was 100%, and that of HCT was 97.8%.

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