Cabrera Leyda, De Witte Jacob, Victor Björn, Vermeiren Lieve, Zimic Mirko, Brandt Jef, Geysen Dirk
Alexander von Humboldt Institute of Tropical Medicine (IMTAvH), Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, San Martín de Porras, Lima 31, Peru.
Vet Parasitol. 2009 Oct 14;164(2-4):111-7. doi: 10.1016/j.vetpar.2009.06.017. Epub 2009 Jun 24.
The aim of the present study was to develop a PCR-ELISA assay for the detection and differentiation of the main African pathogen trypanosomal species present in peripheral blood of cattle. The proposed methodology allows to specifically differentiate Trypanosoma congolense, Trypanosoma vivax and the subgenus Trypanozoon, by means of a sensitive universal PCR amplifying trypanosome DNA followed by an ELISA-based hybridization with three highly specific probes. The semi-nested PCR had a sensitivity of 15 fg, 15 fg, and 0.15 fg of DNA from T. vivax, T. congolense, and Trypanosoma brucei brucei, respectively that is sufficient to detect parasites in blood during the chronic phase of the disease. Biotinylated second round asymmetric PCR amplification products were used in an ELISA set up using three species-specific probes for the diagnosis of T. congolense (type Riverine, Kilifi or Savannah), T. vivax and T. brucei brucei. A factor O.D. of 0.082 was determined on blood samples from bovines (n=18) from a non-endemic area in Africa. In a pilot study of blood samples of naturally and experimentally Trypanosoma infected cattle previously characterized by PCR-RFLP (n=42), a high rate of concordance (93.3%) was found between PCR-RFLP and PCR-ELISA. There is a good ratio between positive and negative O.D. values (3.00 vs. 0.1) and the technique can also be used to distinguish mixed infections.
本研究的目的是开发一种PCR-ELISA检测方法，用于检测和区分牛外周血中存在的主要非洲致病锥虫物种。所提出的方法能够通过一种敏感的通用PCR扩增锥虫DNA，随后与三种高度特异性探针进行基于ELISA的杂交，从而特异性地区分刚果锥虫、活跃锥虫和布氏锥虫亚属。半巢式PCR对来自活跃锥虫、刚果锥虫和布氏锥虫的DNA的灵敏度分别为15 fg、15 fg和0.15 fg，这足以在疾病的慢性阶段检测血液中的寄生虫。生物素化的第二轮不对称PCR扩增产物用于ELISA检测，该检测使用三种物种特异性探针诊断刚果锥虫（河流型、基利菲型或萨凡纳型）、活跃锥虫和布氏锥虫。在来自非洲非流行地区的牛（n=18）的血液样本中确定了0.082的O.D.因子。在一项对先前通过PCR-RFLP鉴定的自然感染和实验感染锥虫的牛的血液样本（n=42）的初步研究中，发现PCR-RFLP和PCR-ELISA之间的一致性率很高（93.3%）。阳性和阴性O.D.值之间有良好的比例（3.00对0.1）,并且该技术还可用于区分混合感染。
