title: A comparison of six primer sets for detection of Trypanosoma evansi by polymerase chain reaction in rodents and Thai livestock.
abstract: To face the worldwide threat of Surra caused by Trypanosoma evansi, international organizations have stressed the need to evaluate and standardize diagnostic tools. PCR detection of T. evansi has known a great expansion during the last 20 years, but primer sets are often insufficiently assessed and compared. In this work, we compared the performances of six primer pairs-TBR1/2 (Masiga et al., 1992), ESAG6/7 (Holland et al., 2001a, b), TEPAN1/2 (Panyim et al., 1993), pMUTEC F/R (Wuyts et al., 1994), TRYP1 R/S (Desquesnes et al., 2001) and TRYP4 R/S (Desquesnes et al., unpublished)-tested with purified T. evansi DNA serial dilutions, T. evansi-infected rat blood serial dilutions and Thai dairy cattle samples. TBR1/2 primer set was able to detect 0.01 pg of purified DNA, and a parasitaemia below one parasite per ml in rat blood. They presented the highest sensitivity in cattle samples as well as a high specificity, without non-specific products nor false positive reactions out of 84 negative cattle samples tested. ESAG6/7 showed equivalent results with purified DNA and rat samples but presented non-specific products with Thai dairy cattle samples, leading to non interpretable results. TEPAN1/2 was not able to detect less than 0.1 pg of purified DNA or 50 trypanosomes/ml in rat blood. In cattle, TEPAN1/2 primers detected only 36% of the positives detected by TBR1/2. Given the parasitemic level detected, pMUTEC F/R, TRYP1 R/S and TRYP4 R/S were not more sensitive than classical microscopic examination of the buffy coat. TBR1/2, TEPAN1/2, pMUTEC F/R and TRYP4 R/S did not cross-reacted with Babesia sp., Trypanosoma theileri and Anaplasma marginale. TBR1/2 was the most sensitive primer set to detect T. evansi in purified DNA, rodent blood and cattle blood, and did not show cross reaction with the other pathogens tested: it should be therefore preferred for epidemiological surveys. These results confirmed that TBR1/2 primers remain the reference for the detection of Trypanozoon DNA and should therefore be included in subsequent evaluations of new diagnosis tools based on DNA detection.
authors.affiliation: Centre de Coopération internationale en Recherche Agronomique pour le Développement (CIRAD)-Département Systèmes Biologiques (Bios), UMR177-Trypanosomes, Montpellier, F-34000 France. mpruvot@ucalgary.ca
authors.forename: Mathieu
authors.initials: M
authors.lastname: Pruvot
authors.forename: Ketsarin
authors.initials: K
authors.lastname: Kamyingkird
authors.forename: Marc
authors.initials: M
authors.lastname: Desquesnes
authors.forename: Nachai
authors.initials: N
authors.lastname: Sarataphan
authors.forename: Sathaporn
authors.initials: S
authors.lastname: Jittapalapong
mesh_terms: D000818:Animals; D002417:Cattle; D017931:DNA Primers; D003612:Dairying; D005260:Female; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013785:Thailand / Q000453:epidemiology; D014345:Trypanosoma / Q000145:classification* / Q000302:isolation & purification*; D014352:Trypanosomiasis / Q000175:diagnosis / Q000453:epidemiology / Q000469:parasitology / Q000662:veterinary*
chemical_list: D017931:DNA Primers