Development of a Reverse Transcriptase Loop-Mediated Isothermal Amplification (LAMP) Assay for the Sensitive Detection of Leishmania Parasites in Clinical Samples
Development of a Reverse Transcriptase Loop-Mediated Isothermal Amplification (LAMP) Assay for the Sensitive Detection of Leishmania Parasites in Clinical Samples
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DOI：
10.4269/ajtmh.2010.09-0369
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发表时间：
2010-04-01
影响因子：
3.3
通讯作者：
Schallig, Henk D. F. H.
中科院分区：
文献类型：
--
作者：
Adams, Emily R.;Schoone, Gerard J.;Schallig, Henk D. F. H.
在这里，我们描述了一个通用的，逆转录酶环介导的等温扩增（RT-LAMP）测定，从临床样本中鉴定利什曼原虫属。LAMP是最近开发的作为即时诊断工具的等温反应。在18 S核糖体RNA（rRNA）基因的保守区域中设计引物;通过预扩增添加荧光检测试剂（FDR）和简单的UV灯来可视化扩增。通过使用逆转录酶步骤，该系统检测到每毫升10至100个寄生虫的感染。该检测试剂盒对来自利什曼原虫属、来自苏丹的内脏利什曼病（VL）患者和来自苏里南的皮肤利什曼病（CL）患者的一系列核酸提取物进行了测试。与骨髓和淋巴结抽吸物的显微镜检查相比，VL患者血液中RT-LAMP的灵敏度为83%（N = 30）;对于CL患者，观察到的灵敏度为98%（N = 43）。潜在的使用LAMP作为利什曼病的诊断工具进行了讨论。
Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved region of the 18S ribosomal RNA (rRNA) gene; amplification was visualized by the pre-amplification addition of fluorescent detection reagent (FDR) and a simple UV lamp. By using a reverse-transcriptase step, the system detected infections between 10 and 100 parasites per mL. The assay was tested on a range of nucleic acid extracts from Leishmania species, visceral leishmaniasis (VL) patients from Sudan, and cutaneous leishmaniasis (CL) patients from Suriname. The sensitivity of RT-LAMP from the blood of VL patients was 83% (N = 30) compared with microscopy of bone-marrow and lymph-node aspirates; for CL patients the observed sensitivity was 98% (N = 43). The potential to use LAMP as a diagnostic tool for leishmaniasis is discussed.