Salim Bashir, Hayashida Kyoko, Mossaad Ehab, Nakao Ryo, Yamagishi Junya, Sugimoto Chihiro
Department of Parasitology, Faculty of Veterinary Medicine, University of Khartoum, P.O. Box 32, Khartoum North, Sudan; Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Department of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, Sapporo, 001-0020, Japan.
Vet Parasitol. 2018 Aug 30;260:53-57. doi: 10.1016/j.vetpar.2018.08.009. Epub 2018 Aug 23.
Non-tsetse transmitted Trypanosoma evansi infection (Surra) is one of the most important diseases of camels in north and east Africa and of buffalo and cattle in Asia. Early, accurate and feasible diagnosis is a crucial step towards the control of Surra. Dry format of loop-mediated isothermal amplification (LAMP) diagnostics for the detection of T. evansi was developed, where the detection limit was determined as to equivalent to one parasite per reaction. The assay was validated by testing blood from 48 camels clinically diagnosed to have Surra, which all tested negative microscopically and revealed 43 (89.6%) to be positive for T. evansi when tested by the dry-LAMP. Furthermore, DNA extracted from a randomly selected subset of 20 of these blood samples were then subjected to RoTat1.2-PCR (TaKara Ex Taq), with 14 matching results, with six that were positive by dry-LAMP and negative by PCR. The kappa value of dry-LAMP applied to direct blood was 0.4211, indicating moderate agreement to RoTat 1.2-PCR. In addition, 103 genomic DNA extracted from camels' blood were tested by both dry-LAMP and RoTat1.2-PCR revealed 67 matching results and 31 positive by dry-LAMP and negative by PCR and a further five positives by PCR and negative by dry-LAMP. This novel dry-LAMP method is more sensitive than conventional PCR, direct (without DNA extraction step), is user friendly and does not require cold chain or highly trained personnel.
非采采蝇传播的伊氏锥虫感染（苏拉病）是非洲北部和东部骆驼以及亚洲水牛和牛的最重要疾病之一。早期、准确且可行的诊断是控制苏拉病的关键步骤。开发了用于检测伊氏锥虫的环介导等温扩增（LAMP）诊断干试剂，其检测限确定为每个反应相当于一个寄生虫。通过检测48头临床诊断患有苏拉病的骆驼的血液对该检测方法进行了验证，这些骆驼经显微镜检查均为阴性，但用干LAMP检测时发现43头（89.6%）伊氏锥虫呈阳性。此外，从这些血液样本中随机选取的20个样本提取的DNA随后进行RoTat1.2-PCR（TaKara Ex Taq）检测，有14个结果匹配，其中6个干LAMP检测为阳性而PCR检测为阴性。应用于直接血液的干LAMP的kappa值为0.4211，表明与RoTat 1.2-PCR有中度一致性。此外，对从骆驼血液中提取的103份基因组DNA进行干LAMP和RoTat1.2-PCR检测，结果显示67个匹配，31个干LAMP检测为阳性而PCR检测为阴性，另有5个PCR检测为阳性而干LAMP检测为阴性。这种新型干LAMP方法比传统PCR更灵敏，直接（无需DNA提取步骤），用户友好，不需要冷链或高技能人员。
