Centre de Coopération internationale en Recherche Agronomique pour le Développement (CIRAD)-Département Systèmes Biologiques (Bios), UMR177-Trypanosomes, Montpellier, F-34000 France.
Vet Parasitol. 2010 Aug 4;171(3-4):185-93. doi: 10.1016/j.vetpar.2010.04.001. Epub 2010 Apr 10.
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et al., 1993), pMUTEC F/R (Wuyts et al., 1994), TRYP1 R/S (Desquesnes et al., 2001) and TRYP4 R/S (Desquesnes et al., unpublished)-tested with purified T. evansi DNA serial dilutions, T. evansi-infected rat blood serial dilutions and Thai dairy cattle samples.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.
为了应对由伊氏锥虫引起的苏拉在全球范围内的威胁，国际组织强调需要评估和标准化诊断工具。聚合酶链反应（PCR）检测伊氏锥虫在过去 20 年中得到了广泛应用，但引物的评估和比较往往不够充分。在这项工作中，我们比较了六种引物对-TBR1/2（Masiga 等人，1992 年）、ESAG6/7（Holland 等人，2001a，b）、TEPAN1/2（Panyim 等人，1993 年）、pMUTEC F/R（Wuyts 等人，1994 年）、TRYP1 R/S（Desquesnes 等人，2001 年）和 TRYP4 R/S（Desquesnes 等人，未发表）-在纯化的伊氏锥虫 DNA 系列稀释液、伊氏锥虫感染大鼠血液系列稀释液和泰国奶牛样本中进行了测试。TBR1/2 引物组能够检测到 0.01 pg 的纯化 DNA，以及大鼠血液中每毫升低于一个寄生虫的寄生虫血症。它们在牛样本中表现出最高的敏感性，并且特异性高，在 84 份测试的阴性牛样本中没有非特异性产物或假阳性反应。ESAG6/7 在纯化 DNA 和大鼠样本中表现出等效的结果，但在泰国奶牛样本中表现出非特异性产物，导致结果无法解释。TEPAN1/2 无法检测到低于 0.1 pg 的纯化 DNA 或大鼠血液中 50 个锥虫/ml。在牛中，TEPAN1/2 引物仅检测到 TBR1/2 检测到的阳性的 36%。鉴于检测到的寄生虫血症水平，pMUTEC F/R、TRYP1 R/S 和 TRYP4 R/S 并不比经典的血涂片显微镜检查更敏感。TBR1/2、TEPAN1/2、pMUTEC F/R 和 TRYP4 R/S 与巴贝斯虫属、泰勒氏锥虫和边缘无浆体无交叉反应。TBR1/2 是检测纯化 DNA、啮齿动物血液和牛血液中伊氏锥虫最敏感的引物组，与测试的其他病原体无交叉反应：因此，它应优先用于流行病学调查。这些结果证实，TBR1/2 引物仍然是检测锥虫 DNA 的参考，因此应包含在基于 DNA 检测的新诊断工具的后续评估中。
