PCR-ELISA for diagnosis of Trypanosoma evansi in animals and vector.

A highly sensitive and specific polymerase chain reaction (PCR) based assay for the detection of Trypanosoma evansi present in the blood of different animals and vector was developed. A simple lysis method was used to remove of the red blood cells to facilitate direct input of samples into the PCR reactions. The primer set was designed and synthesized to amplify a single band of 257 bp PCR product that was subsequently examined by enzyme-linked immunosorbent assay (ELISA). The sensitivity limit of PCR-ELISA was 0.01 pg that was corresponded to 1 parasite/ml of blood. No cross-reactivity of the assay was observed against Babesia bovis, B. bigemina, Anaplasma marginale,Theileria sp. and host DNA. The PCR-ELISA was shown to detect 33 samples of T. evansi infected blood of animals and 10 mosquitoes from different geographical area in Thailand. The results were corresponded to those of the PCR and mouse inoculation. This implies that the technique of PCR-ELISA is not only beneficial for diagnosis of the parasite but also useful for epidemiological study and designing rational trypanosomiasis control program.