A comparison of six primer sets for detection of Trypanosoma evansi by polymerase chain reaction in rodents and Thai livestock

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• 作者：Mathieu Pruvot ; Ketsarin Kamyingkird ; Marc Desquesnes ; Nachai Sarataphan ; Sathaporn Jittapalapong
• 关键词：Trypanosma evansi ; PCR ; Primers ; Standardization ; Rodent ; Livestock
• 刊名：Veterinary Parasitology
• 出版年：2010
• 出版时间：4 August 2010
• 年：2010
• 卷：171
• 期：3-4
• 页码：185-193
• 全文大小：1514 K

文摘

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] and [Holland et al., 2001b]), 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et al., 1993), pMUTEC F/R (Wuyts et al., 1994), TRYP1 R/S (Desquesnes et al., 2001) and TRYP4 R/S (Desquesnes et al., unpublished)–tested with purified T. evansi DNA serial dilutions, T. evansi-infected rat blood serial dilutions and Thai dairy cattle samples.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.