Division of Health Sciences, School of Nursing and Midwifery, Murdoch University, Education Drive, Mandurah, WA, Australia.
Exp Parasitol. 2010 Jul;125(3):196-201. doi: 10.1016/j.exppara.2010.01.017. Epub 2010 Jan 28.
Camel Trypanosomiasis (Surra) is mainly caused by Trypanosoma evansi strains that express variable surface glycoprotein (VSG) RoTat 1.2. However, in Kenya a second causative strain that does not express RoTat 1.2 VSG (T. evansi type B) has been identified. The prevalence of T. evansi type B largely remains unknown due to inadequate diagnostic assay. This work reports the development of a sensitive and specific diagnostic assay capable of detecting T. evansi type B based on the strategy of Loop-mediated Isothermal Amplification (LAMP) of DNA. The test is rapid and amplification is achieved within 20-25min at 63 degrees C using a real time PCR machine. Restriction enzyme AluI digestion of the amplicon gave the predicted 83bp and 89bp sized bands and the LAMP product melt curves showed consistent melting temperature (T(m)) of approximately 89 degrees C. The assay analytical sensitivity is approximately 0.1tryps/ml while that of classical PCR test targeting the same gene is approximately 10tryps/ml. There was a 100% agreement in detection of the LAMP amplification product in real time, gel electrophoresis, on addition of SYBR Green I, and when using chromatographic Lateral Flow Dipstick (LFD) format. The use of the LAMP test revealed nine more T. evansi type B DNA samples that were not initially detected through PCR. The robustness and higher sensitivity of the T. evansi type B LAMP assay coupled with the visual detection of the amplification product indicate that the technique has strong potential as a point-of-use test in surra endemic areas.
骆驼锥虫病（苏拉病）主要由表达可变表面糖蛋白（VSG）RoTat 1.2 的伊氏锥虫埃氏亚种引起。然而，在肯尼亚，已经鉴定出第二种不表达 RoTat 1.2 VSG 的致病菌株（伊氏锥虫 B 型）。由于诊断检测方法不足，伊氏锥虫 B 型的流行情况在很大程度上仍不清楚。本研究报告了一种基于 DNA 环介导等温扩增（LAMP）策略的、能够检测伊氏锥虫 B 型的敏感且特异的诊断检测方法的开发。该检测方法快速，在 63°C 下使用实时 PCR 仪 20-25 分钟即可实现扩增。扩增产物的限制性内切酶 AluI 消化产生了预期的 83bp 和 89bp 大小的条带，LAMP 产物的熔解曲线显示出约 89°C 的一致熔点（T(m)）。该检测方法的分析灵敏度约为 0.1 个锥虫/ml，而针对同一基因的经典 PCR 检测方法的灵敏度约为 10 个锥虫/ml。在实时、凝胶电泳、添加 SYBR Green I 和使用层析侧向流动试纸（LFD）格式时，LAMP 扩增产物的检测结果完全一致。LAMP 检测方法显示，在最初通过 PCR 检测不到的情况下，有 9 个样本为伊氏锥虫 B 型 DNA。该伊氏锥虫 B 型 LAMP 检测方法具有较高的灵敏度和稳健性，且扩增产物可目视检测，表明该技术具有作为苏拉病流行地区现场使用检测方法的巨大潜力。
