Division of Veterinary Infection Biology and Immunology, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany.
Parasitol Res. 2012 Feb;110(2):533-8. doi: 10.1007/s00436-011-2518-x. Epub 2011 Jul 9.
A loop-mediated isothermal amplification (LAMP) assay was developed for the diagnosis of Theileria lestoquardi infection. The primers were designed based on the clone-5 sequence of T. lestoquardi. The specificity and sensitivity of the assay were established. Analysis of the specificity showed that the selected LAMP primers amplified the target sequence from T. lestoquardi DNA successfully, while no amplification was seen with DNA from Theileria annulata, Theileria ovis, Babesia ovis, Anaplasma ovis, or ovine genomic DNA. The specificity of the LAMP product was further confirmed by restriction digestion and sequencing. The sensitivity of the LAMP assay was analyzed in comparison to PCR resulting in a detection limit of 10 fg/μl of plasmid DNA containing the clone-5 sequence. The suitability for utilizing the LAMP assay in the field for the diagnosis of T. lestoquardi infection was tested on 100 field samples collected in Sudan and compared with results obtained by PCR. The relative specificity and sensitivity of the established LAMP assay was determined to be 92.1% and 87.5%, respectively, indicating that it may be regarded as an alternative molecular diagnostic tool to PCR which could be used for epidemiological surveys on T. lestoquardi infection.
建立了一种用于诊断马媾疫锥虫感染的环介导等温扩增（LAMP）检测方法。根据马媾疫锥虫克隆-5 序列设计了引物。该检测方法的特异性和敏感性得到了验证。特异性分析表明，所选 LAMP 引物成功扩增了马媾疫锥虫 DNA 的靶序列，而从环形泰勒虫、绵羊泰勒虫、绵羊边缘无浆体、绵羊无形体或绵羊基因组 DNA 中未观察到扩增。LAMP 产物的特异性进一步通过限制性内切酶消化和测序得到证实。与 PCR 相比，分析了 LAMP 检测方法的灵敏度，导致含有克隆-5 序列的质粒 DNA 的检测限为 10 fg/μl。在苏丹采集的 100 个现场样本上测试了该 LAMP 检测方法在现场诊断马媾疫锥虫感染的适用性，并与 PCR 结果进行了比较。建立的 LAMP 检测方法的相对特异性和敏感性分别为 92.1%和 87.5%，表明它可能被视为替代 PCR 的分子诊断工具，可用于马媾疫锥虫感染的流行病学调查。
