Study on setting up a nested PCR applying in diagnosis of Trypanosoma evansi in horses

Abstract

Trypanosoma evansi (T. evansi), a haemoparasite causes trypanosomiasis for ruminants, horses, and humans in Viet Nam. Many disagnostic methods, such as: injecting horse blood into the white mice, giemsa staining, ELISA, dot-ELISA, PCR have been developed and applied so far for detecting of T. evansi. However, due to the change of its antigen namely the variant surface glycoprotein (VSG), those developed diagnostic methods have certain limitations. The nested PCR
(nPCR) could overcome the disadvantages of previous methods of T. evansi detection. To establish a nPCR to detect T. evansi, two pairs of specific primers NTE-F1/NTE-R1 and NTE-F2/NTE-R2 (20 nucleotides each) amplifying the VSG gene of T. evansi were designed. These two designed primer
pairs were tested for correct primer binding. The optimal annealing temperature of designed primers were detemined (570C in round 1 PCR; 580C in nested PCR). And the cycle threshold (Ct) of the nPCR was (Ct = 35). The nPCR was thermally programmed. The sensitivity and specificity of the
nPCR were both 100%. The established nPCR reaction was successfully applied to diagnose T.evansi species for 100 horse blood samples. The similarity level of diagnostic results between nPCR and mice innocular diagnostic method was 98% (Kappa = 0.95; p < 0.0001).